,
* 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
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Key Words: retinoic acid • CD11b • zVAD-fmk • CD33
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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.
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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 manufacturer’s 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 1–2 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 Student—Newman correction,
and the Student’s t-test. Significance was assumed for
values of P < 0.05.
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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.
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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) .
 View larger version (62K): [in a new window] |
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 manufacturer’s
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.
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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)
.
 View larger version (19K): [in a new window] |
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 manufacturer’s 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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 View larger version (10K): [in a new window] |
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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 View larger version (21K): [in a new window] |
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 manufacturer’s 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.
|
 View larger version (29K): [in a new window] |
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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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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 View larger version (51K): [in a new window] |
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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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 cell’s 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.
Received February 21, 2001; revised October 9, 2001; accepted October 9, 2001.
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