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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3309-3316
Interferon  Induces Upregulation and Activation of Caspases 1, 3, and 8 to Produce Apoptosis in Human Erythroid Progenitor Cells
By
Chunhua Dai and
Sanford B. Krantz
From the Hematology/Oncology Division, Department of Medicine,
Department of Veterans Affairs Medical Center, Nashville, TN; and the
Vanderbilt Cancer Center, Vanderbilt University School of Medicine,
Nashville, TN.
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ABSTRACT |
Interferon  (IFN) induces apoptosis in purified human
erythroid colony-forming cells (ECFC) and inhibits cell growth. Fas (APO-1; CD95) and Fas ligand (FasL) mediate apoptosis induced by
IFN, because Fas is significantly upregulated by IFN, whereas Fas
ligand is constitutively present in the ECFC and neutralization of FasL
greatly reduces the apoptosis. Because conversion of caspases from
their dormant proenzyme forms to active enzymes has a critical role in
transducing a cascade leading to apoptosis, we performed further
studies of the expression and activation of caspases in normal human
and IFN-treated day-6 ECFC to better understand the mechanism of
IFN action in producing this cell death. RNase protection assays
showed that the caspase-1, -2, -6, -8, and -9 mRNAs were upregulated by
IFN, whereas the caspase-5 and -7 mRNAs were not increased. Western
blots showed that FLICE/caspase-8 was upregulated and activated by 24 hours of incubation with IFN. FADD was not similarly altered by
incubation with IFN. Western blots of ICE/caspase-1, which might be
required for amplification of the initial FLICE activation signal,
showed that pro-ICE expression significantly increased after treatment
with IFN for 24 hours and cleavage of pro-ICE also increased.
CPP32/apopain/caspase-3, responsible for the proteolytic cleavage of
poly (ADP) ribose polymerase (PARP), was also studied and treatment of
ECFC with IFN resulted in an increased concentration of caspase-3 by
24 hours and a clear induction of enzyme activation by 48 hours, which
was identified by the appearance of its p17-kD peptide fragment. The
cleavage of PARP was demonstrated by an obvious increase of the 89-kD
PARP cleavage product, which was observed at almost the same time as
caspase-3 activation in the IFN-treated cells, whereas untreated
ECFC showed little change. Peptide inhibitors of the caspase proteins,
DEVD-fmk, DEVD-cho, YVAD-cho, and IETD-fmk, were incubated with the
ECFC to obtain further evidence for the involvement of caspases in
IFN-induced apoptosis. The activation of FLICE/caspase-8 and
CPP32/caspase-3 and cleavage of PARP clearly were inhibited, but the
reduction of cell growth due to apoptosis, induced by IFN, was only
partially blocked by the presence of the inhibitors. These results
indicate that IFN acts on ECFC not only to upregulate Fas, but also
to selectively upregulate caspases-1, -3, and -8, which are activated
and produce apoptosis, whereas the concentrations of FasL and FADD are
not demonstrably changed.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
INTERFERON (IFN) is secreted by
activated T lymphocytes and natural killer cells. It induces the
activation and differentiation of mononuclear phagocytes producing
profound antiviral and immunomodulatory activities.1 Many
investigators have reported that IFN also inhibits hematopoiesis in
vitro, including both granulocytic and erythroid
progenitors.1-9 Experiments in our laboratory have shown
that IFN inhibited erythroid colony formation, cell proliferation,
and differentiation of highly purified human day-3 to day-6
burst-forming units-erythroid (BFU-E) in a dose-dependent manner and
also produced profound erythroblast apoptosis that was demonstrated by
nuclear condensation and fragmentation plus flow cytometry after in
situ end-labeling.9
Apoptosis, or programmed cell death, has a major role in normal
development, tissue homeostasis, defense against viral invasion, immune
modulation, and, when dysregulated, modulation of autoimmune and clonal
or neoplastic diseases.10,11 It is characterized by cell
shrinkage, chromatin condensation, internucleasomal DNA cleavage,
membrane blebbing, and the formation of apoptotic bodies that are
phagocytosed by other cells.12,13 Many factors, such as the
ligation of the Fas (APO-1; CD95) or tumor necrosis factor (TNF)
receptors, growth factor withdrawal, toxic or chemotherapeutic chemicals, viruses, radiation, and heat shock, trigger
apoptosis.10 The Fas/Fas ligand (Fas L) system constitutes
a very important cellular pathway responsible for the initiation of
apoptosis. Fas is a 45-kD membrane glycoprotein belonging to the TNF
receptor family and is widely distributed among diverse tissues and
cells.8,10,11,14 Fas contains a 70 amino acid cytoplasmic
domain, designated as a death domain, that is necessary for
transduction of the apoptotic signal,15,16 whereas FasL is
a 40-kD type II protein member of the TNF family. In contrast to Fas,
FasL was initially thought to be relatively restricted in its cell and
tissue distribution,17,18 but recent investigations have
shown FasL to be present in thyroid and erythroid
cells.11,14
Ligation of Fas results in aggregation of the intracellular death
domains, leading to the recruitment of a set of signaling proteins and
the formation of the death-inducing signaling complex (DISC), which
includes Fas, FasL, an adopter molecule, Fas-associated protein with
death domain (FADD), and FLICE (FADD-like ICE).19-21 FADD
is recruited to Fas upon its ligation and binds to Fas via interactions
between the death domains. The N-terminal region, termed the death
effector domain (DED), is responsible for downstream signal
transduction. FLICE (caspase-8) contains two N-terminal DED domains
through which it binds FADD19-21 and belongs to a family of
cysteine proteases (caspases). After binding to FADD, FLICE can produce
an activation of proteolytic activity and trigger the interleukin-1
converting enzyme (ICE)-like protease cascade.22,23 At
least 10 caspases have been identified, including caspase-1 (ICE),
caspase-2 (Nedd2, ICH-1), caspase-3 (CPP32, Yama, apopain), caspase-4
(ICH-2, TX/ICErel II), caspase-5 (ICErel III,
TY), caspase-6 (Mch2), caspase-7 (Mch3, ICE-LAP3, CMH-1), caspase-8
(FLICE, MACH, Mch5), caspase-9 (Mch6, ICE-LAP6), and caspase-10 (Mch4),
and this family of proteases plays a critical role in the biochemical events governing apoptosis.24,25 The activated ICE-like
members cleave a variety of substrates, such as poly(ADP)-ribose
polymerase (PARP), lamins, fodrin, and protein kinase C, as well as
producing morphologic alterations of the cells and
nuclei.24
FasL is constitutively present in human erythroid progenitors as they
mature from BFU-E to colony-forming unit-erythroid
(CFU-E).14 This has been demonstrated by
reverse transcriptase-polymerase chain reaction (RT-PCR) and flow
cytometric analysis. Whereas only a small percentage of normal human
erythroid progenitors express Fas, and this is at very low level, the
addition of IFN markedly increases the percentage of erythroid
colony-forming cells (ECFC) expressing Fas on the surface of erythroid
progenitors as well as the intensity of Fas expression and this
increase is associated with the inhibitory effect of IFN on cell
proliferation and the production of ECFC apoptosis.14 In
addition, IFN downregulates the erythropoietin (EPO) and stem cell
factor (SCF) receptors,26 whose ligands and activities
protect human ECFC from apoptosis.27 In this study, the
effect of IFN on the expression and activation of several caspases
and the cleavage of PARP has been studied in highly purified human
erythroid progenitor cells. Additionally, the effects of caspase
inhibitors on the activation of these caspases and protection of the
cells from apoptosis have also been examined.
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MATERIALS AND METHODS |
Purification and expansion of human blood ECFC.
Four hundred milliliters of blood was obtained from normal donors after
receiving informed consent approved by the Vanderbilt University and
Department of Veterans Affairs Medical Center Institutional Review
Boards. BFU-E were first isolated by sequential density gradient
centrifugation, depletion of lymphocytes and adherent cells, and
further negative selection of contaminant cells with CD2, CD11b, CD16,
and CD45 monoclonal antibodies (MoAbs), as previously described.9,28 The BFU-E were suspended in 20 mL
Iscove's modified Dulbecco's medium (IMDM) containing 20% fetal calf
serum (FCS), 5% pooled human AB serum, 1% bovine serum albumin
(BSA), 5 × 10 5 mol/L
2-mercaptoethanol (2-ME), 10 µg/mL insulin, 2 U/mL EPO (Amgen, Inc,
Thousand Oaks, CA), 50 U/mL interleukin-3 (IL-3), 50 ng/mL SCF (Amgen
Inc), penicillin at 500 U/mL, and streptomycin at 40 µg/mL in
polystyrene flasks to generate ECFC. After incubation at 37°C in
5% CO2/95% humidified air for 4 days (day-5 cells) or 5 days (day-6 cells), the cells were collected and further enriched by
centrifugation through 10% BSA and over Ficoll-Hypaque, as necessary.
The cells were then incubated in liquid culture containing 20% FCS,
5% pooled AB serum, 1% BSA, 10 µg/mL insulin, 2 U/mL EPO, and
penicillin/streptomycin. IFN (4.75 × 107 U/mg;
Genzyme Corp, Cambridge, MA) and/or SCF were added, as further
delineated, in some cultures. The cells were collected after incubation
for the indicated times to perform protein or RNA extractions, and
plasma clot cultures were made to measure the number of ECFC.
Plasma clot culture of ECFC.
Cells were plated at a concentration of 103/mL in an IMDM
mixture containing 20% FCS, 5% pooled human AB serum, 1% deionized BSA, 10 µg/mL insulin, 2 U/mL EPO, penicillin, streptomycin, 2 mg/mL
fibrinogen (Sigma, St Louis, MO), and 0.2 U/mL bovine thrombin (Parke-Davis, Morris Plains, NJ). In some experiments, IFN was added
at the indicated concentrations. The clots were fixed on day 15 and
stained with 3,3' demethoxybenzidine and hematoxylin. Colonies of
two or more hemoglobinized cells were scored as ECFC. ECFC purity was
determined by the plating efficiency and data were expressed as the
means ± SD with significance calculated using the t-test.
The average purity of the ECFC in our experiments was 61% ± 8%
(day 6), 68% ± 9% (day 7), 86% ± 5% (day 8), 90% ± 4%
(day 9), and 89% ± 3% (day 10). Because the cell number and colony-forming capacity decreased after incubation of the cells with
IFN for 72 hours or longer,9 we used an equal amount of
RNA or protein from control and IFN-treated cells for RNA protection
assays and Western blot analyses.
RNase protection assay.
Day-5 cells were incubated with or without 500 U/mL of IFN for 72 hours and total RNA was isolated using ULTRASPEC (Bioticx Laboratories,
Inc, Houston, TX). Twenty-microgram RNA samples were analyzed for the
presence of transcripts of mRNAs related to apoptosis. An hApo-1
Multi-Probe Template Set including probes for caspases-1, -2, -5, -6, -7, -8, and -9 and granzyme B was purchased from PharMingen (San Diego,
CA). L32 and GAPDH were included as internal controls. RNA protection
assays were performed with the MAXIscript and RPA II Ribonuclease
Protection Assay Kit (Ambion, Austin, TX), according to the
manufacturer's recommendations. Protected transcripts were separated
by denaturing polyacrylamide gels and quantified by autoradiography.
Western blot analysis.
Cell extracts were prepared by lysing 107 cells in 100 µL
lysis buffer containing 1% Triton X-100, 20 mmol/L Tris-HCl, pH 7.5, 10% glycerol, 140 mmol/L NaCl, 100 mmol/L sodium fluoride, 10 mmol/L
EDTA, 2 mmol/L vanadate, 0.2 mmol/L phenylmethylsulfonyl fluoride, and
0.15 U/mL aprotinin at 4°C. Insoluble materials were removed by
centrifugation for 20 minutes at 14,000g and
4°C.29 The samples were quantitated using a Bio-Rad
protein assay kit II (Bio-Rad, Hercules, CA) and were boiled for 5 minutes in a sodium dodecyl sulfate (SDS) sample buffer before 8% or
13% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The
concentration of protein per cell was nearly identical between normal
and IFN-treated cells. After SDS-PAGE, the proteins were transferred
to nitrocellulose. The membranes were blocked by incubation in
Tris-buffered saline with 0.05% Tween-20 (TBST) and 5% nonfat milk
powder at 4°C overnight. After a brief rinse, the blots were
incubated for 2 hours at room temperature in TBST-5% milk powder with
one of the following antibodies.
Primary antibodies were diluted as follows: MoAb for FasL (Transduction
Laboratories, Lexington, KY) had a dilution of 1:1,000; MoAb for FADD
(Transduction Laboratories) had a dilution of 1:250; MoAb for caspase-3
(Transduction Laboratories) had a dilution of 1:1,500; polyclonal
rabbit antibody for caspase-3 (a gift from Dr Donald Nicholson, Merck
Frosst Canada Inc, Quebec, Canada), which can recognize both the
proenzyme and its fragments, had a dilution of 1:10,000; polyclonal
goat antibodies for caspase-8, p10, and p20 (Santa Cruz Inc, Santa
Cruz, CA) had dilutions of 1:200; polyclonal rabbit antibody for
caspase-1 and p10 (Santa Cruz) had a dilution of 1:200; and MoAb for
PARP (Enzyme Systems Products, Livermore, CA) had a dilution of
1:10,000.
Immunoblotting was followed by washing in TBST and 2 hours of
incubation at 26°C with antimouse (Amersham Corp, Arlington Heights, IL), antirabbit, (Amersham), or antigoat (Santa Cruz) IgG-peroxidase conjugate (1:2,000) in TBST-5% milk powder. After washing 5 times for 5 minutes each time, the immunoblots were developed
using the ECL method (Amersham). Some blots were stripped by incubation
in a buffer containing 2% SDS, 62.5 mmol/L Tris-HCl for 30 minutes at
50°C (and were then reblocked and reprobed). Density scanning was
performed using an LKB 2222-010 UltraScan XL Laser Densitometer (LKB
Produkter AB, Bromma, Sweden). Caspase-3 inhibitor DEVD-fmk
and caspase-8 inhibitor IETD-fmk were purchased from Enzyme Systems
Products, whereas YVAD-cho and DEVD-cho were purchased from BIOMOL
(Plymouth Meeting, PA).
| |
RESULTS |
Caspases-1, -2, -6, -8, and -9 mRNAs are upregulated by IFN.
Day-5 cells were treated with or without 500 U/mL of IFN for 72 hours and the total RNAs were isolated. RNase protection assays were
performed using a multiprobe set including 7 members of the caspase
family and granzyme B, as well as two housekeeping genes, L32 and
GAPDH, which delineate RNA loading on the gels. Two separate
experiments demonstrated that the expressions of caspase-1, -2, -6, -8, and -9 mRNAs were significantly increased in the cells incubated with
IFN. Caspase-5 and -7 mRNAs were not changed by incubation with
IFN (Fig 1).
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| Fig 1.
Effects of IFN on caspase mRNAs. Day-5 cells were
incubated for 72 hours with or without 500 U/mL of IFN, and RNase
protection assays were performed for members of the caspase family. Two
separate experiments were performed (lanes 1/2 and lanes 3/4).
Twenty-six micrograms of total RNA from each cell group was analyzed
with the hApo-1 Multi-Probe Template Set (Pharmingen) to detect
caspases-1, -2, -5, -6, -7, -8, and -9 and granzyme B mRNAs.
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IFN upregulates and activates caspase-8/FLICE but not FADD.
Because FADD and caspase-8, the most receptor-proximal ICE-like
protease, are associated with the CD95 DISC and the DISC is essential
for induction of apoptosis by Fas,22 we studied the effect
of IFN on FADD and caspase-8. Human day-6 ECFC were treated with
IFN (500 U/mL) for 24 to 96 hours and immunoblots were made of the
cell protein extracts. This concentration of IFN has previously been
shown to be optimum in this system.9,14 These studies showed that FADD is constitutively expressed in human erythroid progenitors from day 6 to day 10 and that the amount of FADD is not
altered by incubation with IFN (Fig 2).
FasL expression in the ECFC was similarly examined and also
demonstrated constitutive expression without any quantitative change
after incubation with IFN, similar to the pattern previously
demonstrated by flow cytometric analysis.14 In contrast,
procaspase-8 was upregulated 30% to 150% by IFN after as little as
24 hours of incubation and remained upregulated through 96 hours
(Fig 3). Proteolytically cleaved forms of
caspase-8 (p31, p20) were clearly observed in our cells after IFN
treatment, and this cleavage occurred also at and after 24 hours of
incubation.
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| Fig 2.
FADD and FasL protein expression in human ECFC is
constituitive and not affected by IFN. Day-6 cells were cultured in
medium with or without 500 U/mL IFN for 24 to 96 hours and then
whole cell protein lysates were prepared at each time for immunoblot
analysis
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| Fig 3.
Caspase-8/FLICE is upregulated and activated by IFN.
In two separate experiments, day-6 cells were cultured in medium with
or without 500 U/mL of IFN for 24 to 96 hours. Cell protein lysates
were prepared and immunoblot analysis was performed with
anti-caspase-8 antibodies. Each lane contained 150 µg of total
protein. The top panel, using antibody to Mch5 p10 and a 3-minute
exposure, shows that pro-caspase-8 increased after incubation with
IFN for 24 hours. The bottom panel shows the result with antibody to
Mch5 p20 and 15 minutes of exposure. Activation products of caspase-8,
p31, and p20 are clearly observed in the IFN-treated cells by 24 hours.
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IFN upregulates and activates caspase-1/ICE.
Two groups have reported that STAT1 is required for efficient
constitutive expression of caspase-1 in some human cell lines and that
caspase-1 is essential for IFN-induced apoptosis.30,31 Previous experiments in this laboratory have shown that IFN induces a strong STAT1 activation (C.H. Dai, unpublished data).
To observe the expression and activation of caspase-1 in ECFC, the
cells were cultured in medium with or without IFN for 24 to 96 hours and the total cell lysates from each time point were prepared for
immunoblot analysis using anti-caspase-1 antibody. The results are
shown in Fig 4 and demonstrate that
procaspase-1 expression significantly increased after incubation with
IFN for 24 hours. Two additional experiments, with lesser exposure,
clearly confirmed this (data not shown). In addition, the cleavage of
caspase-1 was also increased by 24 hours of incubation with IFN and
persisted for 96 hours. A slight decrease in control caspase-1 with
time of incubation also was evident.
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| Fig 4.
ICE/caspase-1 is upregulated and activated by IFN in
human ECFC. Day-6 cells were incubated in liquid medium or medium
containing 500 U/mL of IFN for 24 to 96 hours and immunoblot
analysis was performed using whole cell lysates and antibody to
caspase-1. An increase in caspase-1 and its activation fragments was
observed in IFN-treated cells by 24 hours.
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Caspase-3/CPP32/ apopain is upregulated and activated by IFN.
Because the activation of caspase-3 to either of its catalytically
active p17 or p12 subunits has been demonstrated in cells undergoing
apoptosis,32-34 we analyzed caspase-3 expression in human
erythroid progenitors at various times after IFN treatment. These
experiments indicate that caspase-3 is very highly expressed in human
erythroid progenitors and increases from day 7 to day 10 of cell
incubation with IFN (Fig 5). An increase
of the proenzyme level of caspase-3 was observed after 24 hours of
IFN treatment using two different anti-CPP32 antibodies. The mean
increase in concentration of procaspase-3 at 24, 48, 72, and 96 hours
of incubation with IFN, compared with the controls, in four
experiments, as detected by scanning, was 15%, 31%, 145%, and 134%,
respectively. Activation of caspase-3, identified by the appearance of
the 17-kD peptide fragment, occurred at and after 48 hours of IFN
stimulation (Fig 5).
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| Fig 5.
Upregulation and delayed activation of
caspase-3/CPP32/apopain in human ECFC after incubation with IFN.
Day-6 cells were cultured in liquid medium or medium containing 500 U/mL of IFN for 24 to 96 hours and the cell proteins were analyzed
by immunoblotting in three separate experiments. The top panel shows
intact caspase-3 expression using MoAb that only reacts with the
proenzyme. A polyclonal antibody reacting to both the intact enzyme and
its fragments was used in the bottom panel. The proenzyme is
significantly increased and a clear cleavage of caspase-3 occurs by 48 hours after incubation with IFN.
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PARP is cleaved in the presence of IFN.
PARP, one of the proteolytic substrates of the caspases during
apoptosis, has been reported to be cleaved to 24- and 89-kD fragments,
which represent the N-terminal DNA binding domain and the C-terminal
catalytic domain of the enzyme, respectively.35 Whereas
most of the caspases can cleave PARP at high concentrations in vitro,
it appears that caspase-3 and -7 are primarily responsible for PARP
cleavage during apoptosis.36 Because caspase-3 was activated in the presence of IFN, we further investigated whether this activation, as well as the activation of other caspases, would
lead to the cleavage of PARP. As shown in
Fig 6, an increase of the 89-kD PARP
cleavage product was observed at 48 hours of IFN incubation,
approximately the same time as caspase-3 activation. A comparison of
the intact PARP band in control and IFN-treated samples showed that
a decrease of the inactive form of PARP of up to 60% accompanied the
formation of the activation fragment after 48 to 96 hours of incubation
with IFN. In the control cultures not treated with IFN, a smaller
cleavage of PARP also was noted.
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| Fig 6.
Effect of IFN on PARP cleavage. Day-6 cells were
incubated with or without 500 U/mL of IFN for the indicated times
and whole cell protein lysates were prepared for immunoblotting. The
IFN-treated cells showed an enhanced cleavage of PARP at 48 hours,
denoted by the appearance of the p89 activation fragment.
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Effects of caspase inhibitors on caspase activation and apoptosis.
To obtain further evidence for the involvement of the caspase system in
IFN-induced apoptosis of human ECFC, we tested the capacities of
peptide inhibitors of the caspase family to inhibit caspase activities
in vitro and reduce apoptosis. DEVD is a relatively potent inhibitor of
caspases-1, -3, and -8, whereas YVAD is a potent and selective
inhibitor of caspase-1 and IETD is known to inhibit
caspase-8.37,38 Day-6 cells were treated with or without
the inhibitor(s), in the absence or presence of IFN, for 96 hours
and then cell lysates were prepared for immunoblot analysis. As shown
in Fig 7, either IETD-fmk or DEVD-fmk
effectively inhibited the cleavage of caspase-3 and -8 as well as the
PARP substrate.
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| Fig 7.
Inhibition of caspase-8 and -3 and PARP cleavage by DEVD
and IETD. Day-6 cells were cultured in medium with or without 500 U/mL
of IFN for 96 hours in the presence or absence of DEVD-fmk and
IETD-fmk at 40 µmol/L. The cell protein lysates were electrphoresed
using a 13% acrylamide gel to separate caspase-8 and -3 and an 8% gel
for PARP.
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To observe the effects of the inhibitor on the reduction of cell growth
induced by IFN, two classes of caspase inhibitors, -fmk and -cho,
were used. These experiments indicated that the addition of either DEVD
or IETD to IFN-treated ECFC for 96 hours partially blocked the
reduction in cell growth induced by IFN (Table 1). The addition of similar
concentrations of IETD-fmk or DEVD-fmk to IFN-treated plasma clot
cultures of day-6 ECFC reduced the number of erythroid colonies with
cellular nuclear fragmentation, indicative of apoptosis, by 33% and
38%, respectively (data not shown).
| |
DISCUSSION |
Previous experiments have demonstrated that IFN produces apoptosis
of erythroblasts and reduces the number and size of erythroid colonies
as well as the degree of differentiation.9 IFN
downregulates the EPO and SCF receptors of ECFC and upregulates Fas
mRNA and protein, whereas FasL remains constitutively produced without apparent change as the cells mature.14 To further
investigate the manner in which the Fas/FasL pathway is involved in
human erythroid progenitor cell apoptosis induced by IFN, we
examined the expression and activation of several members of the
caspase family that have been reported to mediate apoptosis in many
cells of different origin.
Although several models for apoptosis-induced by IFN through the
Fas/FasL system have been proposed,39 the data presented here seem most consistent with the model presented by Fraser and Evan.40 First, ligation of Fas recruits other proteins,
such as FADD, and forms the CD95 DISC.19-21 Caspase-8/FLICE
is a third component of the DISC and is the first caspase in a cascade
of ICE-like proteases activated by CD95. Upon binding to FADD, through the DED, caspase-8 is converted to its active subunits, which are
released to the cytosol and can activate a cascade of ICE-like preteases.22 Hence, caspase-8 is called an initiator
protease. Overexpression of caspase-8 resulting in apoptosis has been
reported.24 When we measured FADD levels, no major change
was evident in IFN-treated human ECFC. In contrast, procaspase-8 was
upregulated and cleavage of caspase-8 was detected in the cells by 24 hours of incubation with IFN. The kinetics of caspase-8 expression
were similar to the kinetics of Fas upregulation that occurs after
incubation of ECFC with IFN.14
Although the precise role of caspase-1/ICE in apoptosis is still
uncertain, ectopic expression of ICE in some cells has resulted in
apoptosis41 and a reduction of apoptosis in
ICE/ cells provides evidence for a
requirement of ICE in IFN-induced cell death. Because ICE appears to
be activated near the apex of an ICE-related protease cascade, ICE has
been called an amplifier protease, which amplifies the initial
caspase-8 activation signal.21,40 Both increases in the
expression of ICE and the cleavage of ICE, demonstrated by the
appearance of activation fragments, were detected at and after 24 hours
of incubation with IFN. These observations indicate that ICE is
activated in IFN-treated cells and that activation of ICE may
precede activation of other caspase, such as caspase-3.
Caspase-3/CPP32/apopain has been called an executioner protease, and it
appears to have a critical role during apoptosis. It has been shown to
be responsible either partially or totally for the proteolytic cleavage
of many key proteins, such as PARP, DNA-PK, and U1-70 kD.24
Activation of caspase-3 has been reported in human hematopoietic cells
during apoptosis induced by EPO deprivation as well as IL-3
deprivation.42,43 In this study, an increased expression of
intact caspase-3 was observed in the human ECFC at and after 24 hours
of treatment with IFN. A distinct activation of caspase-3 and its
substrate, PARP, demonstrated by the appearance of proteolytically
fragments, was detected 24 hours later, as compared with caspase-8 and
caspase-1. This correlates with the appearance of apoptotic changes
induced by IFN and occurs later than the increase in FAS, caspase-8,
and caspase-1.
It has been reported that IFN enhanced the activity of several
apoptosis-related genes in HT-29 cells, a human colon adenocarcinoma cell line, including Fas, and caspases-1, -3, -4, -7, -8, and -10, but
not caspases-2 and -6.44 Some gene mRNAs were induced or
upregulated by 1 hour of IFN treatment, such as those for Fas and
caspases-7, -8, and -10. Caspase-3 and -4 mRNAs were weakly induced,
whereas others, such as those for Fas and caspases-1, -8, and -10, were
strongly induced by IFN.42 However, no studies were
reported on the presence, activation, or changes in concentration of
the caspase proteins.
To further determine whether caspases had an intermediary role in the
induction of apoptosis in human ECFC by IFN, two classes of caspase
inhibitors were tested. Both prevented at least 90% of the IFN
induced cleavage of caspases-8 and -3, as well as PARP. However,
addition of the inhibitors to the IFN-treated cells only partially
prevented apoptosis. This suggests that other, alternative pathways for
apoptosis also may be activated by IFN, or that the inhibitors may
be active for a limited time during the 96-hour cell incubations.
In conclusion, FADD and FasL were constitutively present in human ECFC,
whereas Fas expression was strongly upregulated by IFN. Caspase-1,
-3, and -8 were not only upregulated but also activated when these
cells were induced to undergo apoptosis by IFN. Cleavage of PARP
appeared at nearly the same time as caspase-3 activation and both were
delayed compared with the activation times for caspase-8 and -1. The
caspase inhibitors, IETD and DEVD, very efficiently blocked the
activation of caspase-8 plus -3 and the cleavage of PARP, but only
reversed the inhibition of cell growth induced by IFN by
approximately 30%. Therefore, the Fas/FasL pathway may be only
partially responsible for production of ECFC apoptosis by IFN.
| |
ACKNOWLEDGMENT |
The authors are very grateful to Dr Donald Nicholson and Merck Frosst
Canada, Inc for their gift of polyclonal antibody to caspase-3.
| |
FOOTNOTES |
Submitted November 2, 1998; accepted January 7, 1999.
Supported by a Veterans Health Administration Merit Review Grant and
Grants No. DK-15555 and 5 T32-DK07186 from the National Institutes of Health.
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.
Address reprint requests to Sanford B. Krantz, MD, Department of
Medicine-Hematology/Oncology, Vanderbilt University Medical School, 547 Medical Research Bldg II, Nashville, TN 37232-6305.
| |
REFERENCES |
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ABSTRACT
INTRODUCTION