The loss of IAP expression during HL-60 cell differentiation is caspase-independent

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1 The loss of IAP expression during HL-60 cell differentiation is caspase-independent B. T. Doyle,*, A. J. O Neill,*, 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 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. J. Leukoc. Biol. 71: ; Key Words: retinoic acid CD11b zvad-fmk CD33 INTRODUCTION 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 antiand 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-tothree 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 19]. The BIR 2 repeat is believed to be directly involved in the inhibition [20], although there is evidence that suggests Correspondence: R. William G. Watson, Ph.D., Department of Surgery, University College Dublin, Mater Misericordiae Hospital, 47 Eccles Street, Dublin 7, Ireland. research@profsurg.iol.ie Received February 21, 2001; revised July 23, 2001; revised October 9, 2001; accepted October 9, Journal of Leukocyte Biology Volume 71, February

2 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 Chemicals and reagents Dulbecco s modified Eagle s medium (DMEM), fetal calf serum (FCS), L- glutamine, penicillin/streptomycin solution, and fungizone were purchased from Gibco-Life Technologies (Paisley, UK). CD11b leu -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 activecaspase 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-cm 2 -vented culture flask and incubated at 37 C in a humidified atmosphere of air and 5% CO 2. HL-60 cells were differentiated into mature neutrophil-like cells by treating with 0.5 M 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 ( ) were centrifuged at 1100 rpm for 5 min, then gently resuspended in 400 l 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 ( )/100 l DMEM were treated with 10 l anti-cd11b/cd33 antibody and left at 4 C for 20 min. The cells were washed three times with 400 l cold PBS at 1100 rpm for 10 min and finally resuspended in 400 l Isoton II solution on ice before they were analyzed using the flow cytometer. Caspase activity assay HL-60 cell lysates were prepared from cells over 5 days differentiation using caspase isolation buffer [25 mm HEPES, ph 7.8, 5 mm MgCl 2, 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 l) were diluted in caspase incubation buffer (40 l) and 20 mm Ac-LEHD-AMC (caspase 9; 5 l) 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/ g protein. Western blot analysis Total protein was isolated from 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 M leupeptin, 1 M aprotinin, and 2 M pepstatin). Isolated protein was measured by the Bradford Assay Protein Detection kit (Bio-Rad, Hercules, CA) and loaded at 50 g 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 ( ) 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 g) was resolved on 1.5% formaldehyde agarose gel to assess RNA integrity, and 20 g 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-IT gel Version 5.1 program. Statistics Statistical analysis was carried out using analysis of variance (ANOVA) oneway analysis of variance with Student Newman correction, and the Student s t-test. Significance was assumed for values of P Journal of Leukocyte Biology Volume 71, February

3 the percentage of cells undergoing spontaneous apoptosis (Fig. 1c). 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 timedependent 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). 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 Fig. 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 M) 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 ( ) were incubated with 10 L 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 ( ) cells with 10 L 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. RESULTS 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. 1a), 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 Fig. 2. The effects of RA differentiation on IAP RNA expression. RA (0.5 M)-treated HL-60 cells ( ) 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 g) 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. Doyle et al. Decreased IAP expression during differentiation 249

4 Fig. 3. The effects of RA differentiation on IAP protein expression. HL-60 cells were incubated with RA (0.5 M) for 5 days at 37 C. Total protein was extracted every 24 h from HL-60 cells. Protein concentration was assayed using the Bio-Rad assay, and equal amounts (50 g) 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. 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. 5A). 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. 7A), and a corresponding increase in its activity peaked at day 4 (Fig. 7B). 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 M) 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. 4. The effects of RA on caspase 3 mrna expression. Total RNA was extracted from RA (0.5 M)-treated HL-60 cells ( ) 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 g) 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. 250 Journal of Leukocyte Biology Volume 71, February

5 Fig. 5. The effects of RA on caspase 3 (A) expression and (B) activity. (A) HL-60 cells were incubated with RA (0.5 M) to induce differentiation. Total protein was extracted every 24 h from the HL-60 cells ( ) up to 5 days. Protein concentration was assayed using the Bio-Rad assay, and equal amounts (50 g) 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 ( ) 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. (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. 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. 9A). 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. 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 phos- Fig. 6. The effects of RA on caspase 9 mrna expression. Total RNA was extracted from HL-60 cells ( ) every 24 h following RA (0.5 M) 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 g) 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. Fig. 7. The effects of RA on caspase 9 (A) expression and (B) activity. (A) Total protein was extracted from RA (0.5 M)-treated HL-60 cells ( ) for 5 days at 37 C every 24 h. The protein concentration was measured using the Bio-Rad protein assay, and equal protein (50 g) 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 ( ) 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. Doyle et al. Decreased IAP expression during differentiation 251

6 Fig. 8. The effects of zvad-fmk on HL-60 cell differentiation and apoptosis. HL-60 cells were assessed before and after RA (0.5 M) with or without the general caspase inhibitor zvad-fmk (100 M). 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. phate (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 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 Fig. 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 M) and treated with the pan caspase inhibitor zvad-fmk (100 M) every 24 h. Total protein was extracted from HL-60 cells ( ) on days 0 and 5. The protein concentration was assessed using the Bio-Rad protein assay, and equal protein (50 g) 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. 252 Journal of Leukocyte Biology Volume 71, February

7 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 caspasemediated 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. ACKNOWLEDGMENT This study was funded by a project grant from the Irish Health Research Board. REFERENCES 1. 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