(Journal of Leukocyte Biology. 2002;71:247-254.)
© 2002
by Society for Leukocyte Biology
The loss of IAP expression during HL-60 cell differentiation is caspase-independent
B. T. Doyle*,
,
A. J. ONeill*,
,
P. Newsholme
,
,
J. M. Fitzpatrick*,
and
R. W. G. Watson*,
* Department of Surgery, Mater Misericordiae Hospital,
Department of Biochemistry, and
Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland
Correspondence: R. William G. Watson, Ph.D., Department of Surgery, University College Dublin, Mater Misericordiae Hospital, 47 Eccles Street, Dublin 7, Ireland. E-mail: research{at}profsurg.iol.ie
 |
ABSTRACT
|
|---|
Human promyelocytic leukaemia cells (HL-60) differentiate into
neutrophil-like cells that die spontaneously by apoptosis when treated
with retinoic acid (RA). Inhibitors of apoptosis proteins (IAP) bind to
and inhibit caspases 3, 7, and 9 activity and the induction of
apoptosis. In this study, we demonstrate that undifferentiated HL-60
cells express IAP. During their differentiation, IAP expression is
decreased at the mRNA and protein levels. In addition, we show that
there is a corresponding increase in the expression and functional
activity of active caspases 3 and 9. This activity was associated with
the cleavage of XIAP, NAIP, and cIAP-2. Most importantly, we
demonstrate that blocking caspase activity does not alter the decrease
in IAP protein expression during differentiation but prevents caspase
activation, IAP cleavage, and the induction of apoptosis. This result
shows that the loss of IAP expression is independent of the induction
of apoptosis and is solely related to the differentiation process.
However, IAP cleavage is caspase-dependent. Terminal differentiation
results in an altered apoptotic phenotype that is associated with the
induction of HL-60 cell apoptosis.
Key Words: retinoic acid CD11b zVAD-fmk CD33
 |
INTRODUCTION
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Retinoic acid (RA) differentiation of the human promyelocytic
leukaemia (HL-60) cell line is a well-characterized in vitro model for
the generation of neutrophil-like cells [1
]. These
differentiating cells acquire neutrophil-like morphological
characteristics and biochemical functions. When terminally
differentiated, HL-60 cells subsequently die by apoptosis
[1
, 2
]. Differentiation is associated with
a decrease in Bcl-2 expression and a corresponding increase in the
expression of proapoptotic caspase family members [3
,
4
]. This altered apoptotic phenotype contributes to cell
susceptibility to spontaneous and chemically induced apoptosis [5,
6].
The caspase family of proteases is the main biochemical machinery
involved in the execution of apoptosis. Activation of the caspases
occurs through their cleavage by autoactivation or other activated
caspases [7
]. These activated caspases stimulate a
cascade of cell death pathways [8
]. Upon activation, the
caspases cleave specific proteins of the cell. This degradation process
is essential for the apoptotic process and leads to its characteristic
morphology. The caspases have a wide range of specific substrates that
are cleaved during apoptosis. Recently XIAP, itself an inhibitor of the
caspases, has been identified as a substrate of caspases 3 and 7
activity [9
]. Inhibiting caspase activity has been shown
to block the apoptotic cascade, indicating their importance in the cell
death pathway. Inhibition of apoptosis can occur at a number of sites
[10
]. One site is at the level of the receptor with
decoy receptors such as DcR4 or proteins that inhibit the activation of
the death domain; i.e., FLICE inhibitory protein
[11
] is expressed to block the apoptotic signal.
Inhibition can also occur at the level of the mitochondria where the
antiapoptotic members of the Bcl-2 family of proteins stabilize the
mitochondrial membrane and prevent the release of cytochrome c, thus
blocking apoptosis.
HL-60 cell differentiation and induction of apoptosis are associated
with altered expression of the Bcl-2 family of anti- and proapoptotic
proteins. These differentiated HL-60 cells are similar to mature human
neutrophils, which do not express Bcl-2 [12
,
13
]. However, neutrophils do express the proapoptotic bax
and bak at the mRNA level but do not express Bcl-2, which may explain
their short half-life. Studies have shown that HL-60 cells used as in
vitro models of precursor neutrophil cells express bak, bik, bax, and
Bcl-2 at mRNA and protein levels. Then, some of these expressions are
lost during differentiation into neutrophil-like cells
[3
, 4
].
Another family of antiapoptotic proteins is the inhibitors of apoptosis
proteins (IAP). Initially discovered in the baculovirus, there are now
seven mammalian homologues: NAIP, XIAP, cIAP-1, cIAP-2, survivin,
livin, and apollon [14
, 15
]. The IAP family
of proteins is distinguished by the presence of one-to-three
baculovirus-inhibition repeat (BIR) domains that are positioned at the
N-terminal domain. At the C-terminal end of XIAP, cIAP-1, cIAP-2, and
survivin, there is a ring zinc-finger domain [14
,
16
]. The primary function of the IAP is to bind to and
inhibit the caspases, in particular caspases 3, 7, and 9
[17
18
19
]. The BIR 2 repeat is believed to be directly
involved in the inhibition [20
], although there is
evidence that suggests both the BIR 1, BIR 3, and zinc-finger domains
are also involved. Different domains may be specific for different
caspases [21
]. Previously, it was understood that the
IAP did not inhibit the cascade upstream of the cytochrome-c release,
however it has been demonstrated that livin blocks Bax-induced
cytochrome-c release [22
]. XIAP, cIAP-1, and cIAP-2 have
the ability to block a wide range of apoptotic stimuli such as UV
light, tumor necrosis factor (TNF), Fas ligand, caspase family,
radiation, cytochrome c, and chemotoxic drugs [23
,
24
]. IAP are highly expressed in tumor cells such as
glioma, breast, colon, and gastric cancer [15
,
25
]. Genetic defects of neural IAP are associated with
the neurodegenerative disorder spinal muscular atrophy
[26
]. The expression and role of IAP in HL-60 cell
differentiation toward an apoptotic cell are unknown. However, a recent
study has shown alterations in survivin during the differentiation
process [27
, 28
].
We hypothesize that during HL-60 cell differentiation, the loss of IAP
expression represents an important mechanism that alters cell
susceptibility to apoptosis. This study aims to demonstrate that during
differentiation, there is a decrease in IAP protein expression that is
independent of caspase activation and the induction of apoptosis,
however later loss of the IAP is a result of their cleavage by
activated caspases. It may be concluded that the loss of the IAP
expression prepares for enhanced caspase activation and apoptosis.
 |
MATERIALS AND METHODS
|
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Chemicals and reagents
Dulbeccos modified Eagles medium (DMEM), fetal calf serum
(FCS), L-glutamine, penicillin/streptomycin solution, and fungizone
were purchased from Gibco-Life Technologies (Paisley, UK). CD11b
leuTM-15 antibody, CD33 antibody, and pro- and active-caspase 3
antibody (rabbit anti-human) were purchased from Becton Dickinson
(Cambridge, UK). IAP antibodies, XIAP, cIAP-1, cIAP-2, and NAIP (rabbit
anti-human/-mouse), pro- and active-caspase 9 antibody (rabbit
anti-human/-mouse), and
N-benzyloxy-carbonyl-Val-Ala-Asp-fluoromethyl ketone
(zVAD-fmk) pan caspase inhibitor were purchased from R&D Systems
(Oxford, UK). Ac-LEHD-AMC (caspase 9) and Ac-DEVD-AMC (caspase 3)
fluorogenic substrates were purchased from Bio Mol, Affinity Research
Products (Exeter, UK). RNA probes hAPO-5c and hAPO-1b were purchased
from Pharmingen (San Diego, CA). All remaining chemicals were purchased
from Sigma-Aldrich Co. (Dorset, UK), unless otherwise stated.
Cell and culture conditions
The HL-60 cells were grown in DMEM culture medium supplemented
with 20% FCS, 1% glutamine, 1% penicillin/streptomycin solution, and
fungizone. Cells were cultured in a 75-cm2-vented culture
flask and incubated at 37°C in a humidified atmosphere of air and 5%
CO2. HL-60 cells were differentiated into mature
neutrophil-like cells by treating with 0.5 µM RA for 5 days.
Quantification of apoptosis
Spontaneous apoptosis of differentiated HL-60 cells was
quantified by flow cytometry as the percent of cells with hypodiploid
DNA. Cells (1x106) were centrifuged at 1100 rpm for 5 min,
then gently resuspended in 400 µl hypotonic fluorochrome solution
[200 ml phosphate-buffered saline (PBS), 10 mg propidium iodide, 3.4
mM sodium citrate, 1 mM Tris, 0.1 mM ethylenediaminetetraacetate
(EDTA), and 0.1% Triton X-100], and placed on ice for 10 min before
they were analyzed using the Coulter Epics XL-mcl cytofluorometer
[29
]. A minimum of 5000 events were collected and
analyzed. Apoptotic nuclei were distinguished from normal nuclei by
their hypodiploid DNA. All measurements were performed under the same
instrument settings.
Quantification of cell-surface antigen expression
The expression of CD11b and CD33 antigens on the surface of
differentiating HL-60 cells was measured by flow cytometry. Cells
(1x105)/100 µl DMEM were treated with 10 µl
anti-CD11b/CD33 antibody and left at 4°C for 20 min. The cells were
washed three times with 400 µl cold PBS at 1100 rpm for 10 min and
finally resuspended in 400 µl Isoton II solution on ice before they
were analyzed using the flow cytometer.
Caspase activity assay
HL-60 cell lysates were prepared from 10 x 106
cells over 5 days differentiation using caspase isolation buffer [25
mM HEPES, pH 7.8, 5 mM MgCl2, 1 mM EDTA, 10 mM leupeptin, 5
mM pepstatin, 100 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM
dithiothreitol (DTT)] and caspase incubation buffer (100 mM HEPES, pH
7.5, 10% sucrose, 0.1% CHAPS, 10 mM leupeptin, 5 mM
pepstatin, 100 mM PMSF, and 10 mM DTT). Aliquots of the lysates (40
µl) were diluted in caspase incubation buffer (40 µl) and 20 mM
Ac-LEHD-AMC (caspase 9; 5 µl) or 20 mM Ac-DEVD-AMC (caspase 3) and
incubated for 1 h at 37°C. The release of AMC fluorescent tag
was measured using a cytofluorometer II (Perkin Elmer Biosciences,
Birchwood Science, Park North, Warrington, UK) at 380 nm excitation and
460 nm emission. Specific activity was measured as activity/µg
protein.
Western blot analysis
Total protein was isolated from 10 x 106 cells
using Nonidet P-40 (NP-40) isolation solution (0.5% NP-40, Tris 10 mM,
pH 8.0, 60 mM KCL, 1 mM EDTA, pH 8.0, 1 mM DTT, 10 mM PMSF, 1 µM
leupeptin, 1 µM aprotinin, and 2 µM pepstatin). Isolated protein
was measured by the Bradford Assay Protein Detection kit (Bio-Rad,
Hercules, CA) and loaded at 50 µg per well. Samples were then run on
12% sodium dodecyl sulfate (SDS) polyacrylamide gradient gel (140V for
60 min) and electrophoretically transferred to Immobilon P (Millipore,
Bedford, MA; 100V, 80 min). The remaining gel was stained after
transfer with Coomassie blue solution to confirm equal loading. Blots
were incubated with rabbit anti-human antipro- and active-caspase 3
antibody (1:1000), rabbit anti-human antipro- and active-caspase 9
antibody (1:1000), rabbit anti-human/-mouse XIAP, cIAP-1, and cIAP-2
antibodies (1:1000, 1:1000, and 1:1500, respectively), and rabbit
anti-human NAIP antibody (1:1000) in 1% bovine serum albumin (BSA),
Tris-buffered saline (TBS), and 0.1% Tween 20 for 1 h at room
temperature. The blots were then incubated with horseradish
peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) at 1:5000
dilution for 1 h. Blots were developed using the enhanced
chemiluminescence system (ECL; Amersham Pharmacia Biotech,
Buckinghamshire, UK).
Ribonuclease protection assay (RPA)
HL-60 cells (10x106) were harvested and washed in
cold PBS, and total RNA was isolated using Trizol reagent according to
the manufacturers protocol (Gibco-Life Technologies). RNA (5 µg)
was resolved on 1.5% formaldehyde agarose gel to assess RNA integrity,
and 20 µg RNA was used for the RPA. The probes for the RPA were the
hAPO-5c probe set [XIAP, survivin, NAIP, cIAP-1, cIAP-2, TRMP-2, L32,
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] and hAPO-1b
probe set (caspase 8, GranzymeB caspases 3, 6, 5, 2, 7, 1, and 9, L32,
and GAPDH). Probe synthesis, hybridization, proteinase K, and RNase
digestion were carried out according to the protocol supplied by
PharMingen, Becton Dickenson. Samples were resolved on a 5% acrylamide
gel that was dried at 80°C under vacuum. The gel was then placed on
autoradiographic film and incubated at -80°C for 12 days prior to
development. The density of each band was measured using the
UN-SCAN-ITTM gel Version 5.1 program.
Statistics
Statistical analysis was carried out using analysis of variance
(ANOVA)one-way analysis of variance with StudentNewman correction,
and the Students t-test. Significance was assumed for
values of P < 0.05.
 |
RESULTS
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Differentiation characteristics of RA-treated HL-60 cells
HL-60 cell differentiation by RA is a well-characterized model. To
confirm differentiation, three recognized markers were assessed, CD11b,
CD33, and the induction of apoptosis. RA treatment resulted in an
increase in the percentage of cells expressing CD11b (Fig. 1 a
), which is expressed on the surface of mature neutrophils and a
time-dependent decrease in the percent of CD33 expression (Fig. 1b)
. RA
treatment also resulted in the well-characterized, time-dependent
increase in the percentage of cells undergoing spontaneous apoptosis
(Fig. 1c)
.

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Figure 1. Effects of RA on HL-60 cell (a) CD11b, (b) CD33 expression, and (c)
spontaneous apoptosis. HL-60 cells were differentiated into
neutrophil-like cell by RA (0.5 µM) over 5 days. HL-60 cell
differentiation was determined every 24 h by monitoring CD11b and
CD33 expression on the cell surface as well as percent apoptosis. (a)
HL-60 cells (1x105) were incubated with 10 µL CD11b
antibody at 4°C in darkness for 20 min, and the percentage of cells
with positive expression was analyzed using flow cytometry, as
described in Materials and Methods. (b) The percentage CD33 expression
was measured by incubating (1x105) cells with 10 µL CD33
antibody at 4°C in darkness for 20 min and assessed by flow
cytometry. (c) Apoptosis was assessed by propidium iodide DNA binding
using flow cytometry. *, P < 0.05 versus day 0.
|
|
Previous studies have demonstrated that RA-induced differentiation
results in the loss of Bcl-2 family members and gain of caspase
expression resulting in an overall change in the apoptotic phenotype
and induction of apoptosis.
The effects of RA on IAP expression
We analyzed IAP expression at the level of mRNA and protein.
Treatment of the HL-60 cells with RA resulted in a time-dependent
decrease in XIAP, NAIP, cIAP-1, cIAP-2, and survivin mRNA expression
(Fig. 2
). This result was confirmed by corresponding decreases in XIAP,
NAIP, and cIAP-2 protein at day 3 (Fig. 3
). There was no decrease in the protein level of cIAP-1 until day 5
(Fig. 3) , which also had the highest percentage of apoptotic cells
(Fig. 1c) .

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Figure 2. The effects of RA differentiation on IAP RNA expression. RA (0.5
µM)-treated HL-60 cells (10x106) were harvested every
24 h, and total RNA was extracted using the manufacturers
protocol. mRNA was hybridized with a specific radioactive probe
h-APO-5c for IAP detection of XIAP, survivin, NAIP, cIAP-1, and cIAP-2
using the RPA. mRNA samples (20 µg) were loaded equally as indicated
by L32 on a 5% acrylamide gel, run for 1.5 h, and then dried for
1 h. Gels were exposed on an autoradiograghic film overnight at
-80°C and developed. Band intensities were measured against the
intensities of the L32 bands. Gel represents one of three separate
experiments.
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Figure 3. The effects of RA differentiation on IAP protein expression. HL-60
cells were incubated with RA (0.5 µM) for 5 days at 37°C. Total
protein was extracted every 24 h from 10 x 106
HL-60 cells. Protein concentration was assayed using the Bio-Rad assay,
and equal amounts (50 µg) were loaded and separated by molecular
weight on a 12% SDS-polyacrylamide gel electrophoresis (PAGE) gel. The
protein was then transferred to an Immobilin P membrane and stained
with primary antibodies for 1 h specific for XIAP, NAIP, cIAP-2,
and cIAP-1 and rabbit secondary antibody for 1 h. Blots were
developed using ECL. Data shown represent one of three separate
experiments.
|
|
Analysis of IAP protein expression during differentiation by Western
blotting demonstrated that as XIAP, NAIP, and cIAP-2 expression
decreased, there was also a corresponding increase in a smaller subunit
(Fig. 3)
, which corresponds to IAP-cleaved products that are believed
to be caused by the activation of the caspases. The loss of IAP
expression through decreased mRNA or through their cleavage by the
caspases results in a more proapoptotic phenotype. This would indicate
that these cells are more susceptible to apoptotic stimuli or the
induction of spontaneous apoptosis as a result of their loss of
survival proteins.
The effects of RA on caspase expression and function
With the loss in expression of the antiapoptotic IAP protein
during differentiation, we next wanted to determine if there was a
change in the proapoptotic proteins such as the caspases. Previous
studies have shown that RA treatment of HL-60 cells alters caspase 3
expression [3
, 4
]. This study demonstrates
an initial increase in caspase 3 mRNA expression at day 1, which
decreased in a time-dependent manner (Fig. 4
). However, there was no significant alteration in the expression
of procaspase 3 protein (32 kDa) that remained stable over 3 days but
decreased at day 4. The biggest change in caspase 3 was at day 3, which
showed an increased expression of the 17-kDa active protein (Fig. 5 A
). This was confirmed by a corresponding increase in its activity
at day 4 (Fig. 5B)
. Caspase 9 plays an important role in the cleavage
and activation of caspase 3. Caspase 9 mRNA levels were increased at
days 1, 2, and 3 (Fig. 6
). This correlated with a time-dependent increase in procaspase 9.
Similarly to caspase 3, there was an increase in the active 10-kDa
protein by day 3 (Fig. 7 A
), and a corresponding increase in its activity peaked at day 4
(Fig. 7B)
.

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Figure 4. The effects of RA on caspase 3 mRNA expression. Total RNA was extracted
from RA (0.5 µM)-treated HL-60 cells (10x106) every
24 h using the manufacturers protocol. The isolated mRNA was
then hybridized with a specific radioactive probe hAPO-1b, which
detects caspase 3 mRNA. mRNA samples (20 µg) were equally loaded as
indicated by L32 on a 5% acrylamide gel, run for 1.5 h, and then
dried for 1 h. Gels were exposed on an autoradiographic film at
-80°C overnight and developed. The band intensity was measured
against the intensity of the L32 band. The experiment represents one of
three separate experiments.
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Figure 5. The effects of RA on caspase 3 (A) expression and (B) activity. (A)
HL-60 cells were incubated with RA (0.5 µM) to induce
differentiation. Total protein was extracted every 24 h from the
HL-60 cells (10x106) up to 5 days. Protein concentration
was assayed using the Bio-Rad assay, and equal amounts (50 µg) were
loaded and separated by molecular weight on a 12% SDS-PAGE. The gel
was transferred to an Immobilin P membrane and stained with a caspase 3
primary antibody, which detects pro and active proteins, and then a
rabbit secondary antibody for 1 h. Blots were developed using ECL.
The blot represents one of three separate experiments. (B) Cell lysates
were also collected every 24 h from differentiating HL-60 cells
(10x106) over 5 days as described in Materials and
Methods. Caspase 3 activity was assessed by the increased fluorescence
intensity of free, fluoroescent AMC tag cleaved from the Ac-DEVD-AMC.
Results represent one of three separate experiments.
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Figure 6. The effects of RA on caspase 9 mRNA expression. Total RNA was extracted
from HL-60 cells (10x106) every 24 h following RA
(0.5 µM) treatment using the manufacturers protocol. The isolated
mRNA was hybridized with a specific radioactive probe hAPO-1b, which
detects caspase 9 mRNA. The mRNA (20 µg) was loaded equally as
indicated by L32 on a 5% acrylamide gel, run for 1.5 h, and dried
for 1 h. The gels were exposed on an autoradiographic film at
-80°C overnight and developed. The band intensity was measured
against the intensity of the L32 band. The blot represents one of three
separate experiments.
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Figure 7. The effects of RA on caspase 9 (A) expression and (B) activity. (A)
Total protein was extracted from RA (0.5 µM)-treated HL-60 cells
(10x106) for 5 days at 37°C every 24 h. The protein
concentration was measured using the Bio-Rad protein assay, and equal
protein (50 µg) was loaded and separated by molecular weight on a
12% SDS-PAGE. The gel was transferred onto Immobilin P membranes and
stained with a caspase 9 primary antibody, which detects pro and active
proteins, and then a rabbit secondary antibody for 1 h. Blots were
developed using ECL. The blot represents one of three separate
experiments. (B) Cell lysates were also collected every 24 h from
differentiating HL-60 cells (10x106) over 5 days as in
Materials and Methods. Caspase 9 activity was assessed by the increased
fluorescence intensity of free, fluorescent AMC tag cleaved from the
Ac-LEHD-AMC. The results represent one of three separate experiments.
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The effects of zVAD-fmk on CD11b expression and percent apoptosis
To determine if the loss of the IAP is a result of caspase
activation or differentiation, we inhibited caspase activity with a
general pan caspase inhibitor. HL-60 cells were treated with and
without zVAD-fmk (100 µM) before and after RA. After 4 days, the
percent differentiation and apoptosis were assessed. zVAD-fmk inhibited
differentiation-induced apoptosis significantly, indicating that this
is a caspase-dependent process (Fig. 8
). The percent of cells positive for CD11b was increased (Fig. 8)
,
indicating a larger number of cells were differentiating, or less cells
were dying by apoptosis. Overall, zVAD-fmk did not inhibit the
differentiation process.

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Figure 8. The effects of zVAD-fmk on HL-60 cell differentiation and apoptosis.
HL-60 cells were assessed before and after RA (0.5 µM) with or
without the general caspase inhibitor zVAD-fmk (100 µM). Cells were
collected on day 4 and assessed for CD11b expression and percent
apoptosis using flow cytometry (n=3). *, P < 0.05 versus vehicle, and , P < 0.05 versus
vehicle - RA.
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The effects of zVAD-fmk on caspase-protein expression, IAP-protein
expression, and IAP cleavage
As the pan caspase inhibitor showed delayed apoptosis but enhanced
the percentage of cells with a differentiated phenotype, it was then
decided to assess the effects of zVAD-fmk on caspase and IAP
expression. There was no alteration in the expression of procaspase 3,
however it did inhibit the presence of an active caspase 3 band
(Fig. 9 A
). Treatment with the caspase inhibitor did not prevent the
decrease in the IAP expression (Fig. 9B)
, indicating that this
alteration in IAP occurs independently of caspase activation. However,
cleavage of the IAP was inhibited by the zVAD-fmk at 5 days (Fig. 9C)
, indicating that this process of IAP cleavage was dependent on the
caspase.

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Figure 9. The effects of zVAD-fmk on (A) pro and active caspase expression, (B)
IAP protein expression, and (C) IAP cleaved products. HL-60 cells were
differentiated with RA (0.5 µM) and treated with the pan caspase
inhibitor zVAD-fmk (100 µM) every 24 h. Total protein was
extracted from HL-60 cells (10x106) on days 0 and 5. The
protein concentration was assessed using the Bio-Rad protein assay, and
equal protein (50 µg) was loaded and separated on a 12% SDS-PAGE.
The gel was then transferred onto an Immobilin P membrane and stained
with specific caspase 3 and IAP primary antibodies for 1 h and
rabbit secondary antibody for 1 h. The blots were developed using
ECL. Data shown represent one of three separate experiments.
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 |
DISCUSSION
|
|---|
HL-60 cells terminally differentiate into neutrophil-like cells
when treated with RA or dimethyl sulfoxide (DMSO) and die spontaneously
by apoptosis [1
, 2
, 30
].
Differentiation is associated with functional changes
[31
], including increased expression of reduced
nicotinamide adenine dinucleotide phosphate (NADPH) [32
,
33
] and CD11b [4
, 34
]. In
this study, we confirmed this model and demonstrated that RA increased
CD11b expression and decreased CD33 expression [35
] in a
time-dependent manner. One hundred percent differentiation was not
reached because all cells do not differentiate at the same time, and
those that differentiate early, will undergo apoptosis and are removed.
This study indicates that once the cells become positive for CD11b or
negative for CD33, they then initiate the cell death pathway, which
leads to spontaneous apoptosis. Significant apoptosis was detected
after day 4. This work verifies other studies [1
,
31
] demonstrating that HL-60 cells differentiate and die
by spontaneous apoptosis. HL-60 cell transformation, from a highly
proliferating, undifferentiated cell type into a differentiated,
nonproliferating phenotype, is associated with a number of cellular
changes. Previous studies have shown decreases in the antiapoptotic
Bcl-2 family members [3
, 4
, 12
,
36
]. Alterations in the pro- and antiapoptotic proteins
during the differentiation process may be an essential change leading
to apoptosis. For this reason, we wanted to examine the role of the IAP
family of antiapoptotic proteins in HL-60 cell differentiation and
determine if they were associated with an altered apoptotic phenotype
that may lead to the induction of apoptosis.
HL-60 cells are demonstrated in this study to express the IAP family of
proteins. Their expression is decreased during differentiation at the
mRNA and protein levels. Specifically, XIAP, NAIP, and cIAP-2 decreased
in a time-dependent manner, but cIAP-1 only decreased at day 5.
Decreased expression of these survival factors contributes to the
altered apoptotic phenotype of the differentiating HL-60 cells and may
make the cell susceptible to the induction of spontaneous apoptosis.
Recent studies confirmed a decrease in survivin during differentiation
with RA that leads to the induction of apoptosis [27
,
28
].
In addition to alterations in antiapoptotic proteins, previous studies
have demonstrated a correlation between differentiation and increases
in proapoptotic protein expression [3
, 35
,
37
]. In this study, we focused on the activation of
caspases 3 and 9, because these caspases are activated downstream in
the apoptotic pathway. mRNA and protein were assessed to determine if
their activation was a result of the initiation of apoptosis or
differentiation process. It was demonstrated that there was an increase
in caspase 9 mRNA expression up to day 3 and an increase in procaspase
9 expression up to day 4. Expression of procaspase 3 remained stable
with a decrease at day 3. Associated with these changes in mRNA and
protein, there were increases in the expression of the cleaved, active
protein for caspases 3 and 9 and their corresponding functional
activity. These results show that as part of the cells preparation to
undergo apoptosis, there is an increased expression of the main
executioners of apoptosis. Previous studies have shown increases in
caspases 1, 3 [3
], and 8 [37
] expression
and activity during differentiation, whereas other studies have
demonstrated no change in caspases 1 and 9 expression
[37
] and a decrease in procaspases 2, 3, and 10
expression [4
]. Our results would support an increase in
caspases 3 and 9 activity coinciding with the decrease in IAP
expression.
It was also observed that during increased caspase activity and the
induction of apoptosis, three members of the IAP family (XIAP, NAIP,
and cIAP-2) were cleaved to a smaller fragment detected by Western
blotting. These cleaved products occurred at the same time as the
detection of active caspases 3 and 9 proteins. It is interesting that
caspase activity increased when the IAP were cleaved, indicating that
the IAP may have been inhibiting their activity. Previous studies have
demonstrated that XIAP [21
] and cIAP-1
[38
] undergo caspase-mediated cleavage. XIAP cleavage
has been shown to occur during T-lymphocyte-induced apoptosis
[9
], but as yet, the function of this cleaved product is
unknown. The cleavage of IAP may represent an additional mechanism for
the removal of antiapoptotic proteins leading to caspase activity and
the induction of apoptosis. Treatment of the HL-60 cells with the
caspase inhibitor decreased apoptosis and also substantially increased
the percentage of cells undergoing differentiation. This can be
explained by the fact that fewer cells are apoptotic, so more
differentiated cells are available for detection. We demonstrated that
the pan caspase inhibitor repressed the cleavage of procaspase 3 to its
active subunit [21
, 38
]. Despite caspase
inhibition, there was still a time-dependent decrease in IAP
expression. This indicates that decreased IAP expression is a result of
differentiation and not apoptosis. However, the results did demonstrate
that the caspase proteins are involved directly in the cleavage of the
IAP that may contribute to their overall loss and enhance the mechanism
of increased apoptosis. This suggests overall that caspases are
involved only at the cleavage stage of IAP and do not play a role in
the regulation of the mRNA or protein level. The ability of IAP to bind
to and inhibit caspase activity may be involved in preventing the
activation of the caspase cascade. Once the IAP were consumed, because
the mRNA signal is switched off, and protein expression is lost, there
is a decrease in caspase inhibition, and apoptosis can proceed.
This study demonstrates that differentiation leads to a decrease in the
IAP antiapoptotic proteins combined with the loss of other
antiapoptotic proteins such as Bcl-2 family members and the increase in
the proapoptotic proteins, which leads to the induction of apoptosis.
It is also important to recognize that the differentiation process and
the apoptotic process are separate mechanisms regulated by different
proteins, where one process may lead to the induction of the other
depending on the circumstances of the cell. An alteration in the
apoptotic phenotype at the pro- and antiapoptotic level is required for
the induction of HL-60 cell apoptosis during differentiation. Further
identifying the sequence of events leading to this change in phenotype
will lead to a better understanding of the mechanisms involved in the
apoptotic process occurring during the differentiation process.
 |
ACKNOWLEDGEMENTS
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|---|
This study was funded by a project grant from the Irish Health
Research Board.
Received February 21, 2001;
revised October 9, 2001;
accepted October 9, 2001.
 |
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