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Prepublished online as a Blood First Edition Paper on May 31, 2002; DOI 10.1182/blood-2002-01-0047.
HEMATOPOIESIS
From the Department of Morphology and Embryology, Human
Anatomy Section, University of Ferrara; Institute of Morphological
Sciences, University of Urbino; and the Department of Human Normal
Morphology, University of Trieste, Italy.
Treatment of the human HL-60 cell line with tumor necrosis factor
(TNF)-related apoptosis-inducing ligand (TRAIL) resulted in rapid
(6-24 hours) cytotoxicity associated with progressive maturation of the
surviving cells along the monocytic lineage. The occurrence of
monocytic maturation was demonstrated by a significant increase of both
CD14 and CD11b surface expression, the acquisition of morphologic
features typical of mature monocytes, and phagocytic capacity in
TRAIL-treated cultures. By using selective pharmacologic inhibitors, it
was possible to demonstrate that activation of the caspase cascade
played a crucial role in mediating TRAIL cytotoxicity and monocytic
maturation of HL-60 cells. Moreover, experiments performed using
agonistic polyclonal antibodies, which mimic the interactions between
TRAIL and each TRAIL receptor, indicated that TRAIL-R1 was responsible
for mediating the TRAIL-induced maturation. Importantly, the
maturational effects of TRAIL were observed also in primary normal
CD34+ cells, seeded in serum-free liquid cultures for 4 to
8 days in the presence of SCF + GM Tumor necrosis factor (TNF)-related
apoptosis-inducing ligand (TRAIL),1 also known as Apo-2
ligand,2 is a member of the structurally related TNF family
of cytokines. A subset of these cytokines, including TNF, CD95L, DR3
ligand (TWEAK), and TRAIL, is known to activate the cell death program
(reviewed in Gruss and Dower3 and Baker and
Reddy4). These cytokines, also called death ligands, induce
apoptosis by activating their cognate surface receptors. These death
receptors are members of the TNF receptor superfamily and include
TNFR1, CD95 (Fas/Apo-1), death receptor (DR)-3, DR-4, DR-5, and DR-6.
In particular, TRAIL, which exists as either a type 2 membrane protein
or as a soluble protein,5 specifically binds and induces
apoptosis by way of DR-4 (also known as TRAIL-R1) and DR-5 (also known
as TRAIL-R2).1,2,4 The unique feature of TRAIL, with
respect to CD95L and TNF- TRAIL induces cell death through recruitment of the adapter molecules
FADD15,16 and a hierarchical cascade of cysteine preoteases, termed caspases, that are synthesized as proenzymes and
activated by cleavage through upstream caspases or by intermolecular auto-proteolysis.17 They can be grouped into 3 major
families: (1) the ICE subgroup (caspases 1, 4, and 5) corresponds to
proteases involved in apoptosis and cytokine maturation; (2) the CPP32
subgroup (caspases 3, 6, and 7) includes caspases that mediate
downstream steps of proteolysis with preferential cleavage of the DEVD
sequence; (3) caspases 2, 8, 9, and 10, which are characterized by the
presence of a long prodomain and are related to events of
receptor-dependent initiation of apoptosis.17 Initial
studies15,16 have outlined the central role of caspase 8 in
mediating the apoptotic signal of TRAIL, whereas more recent work has
demonstrated that caspases 8 and 10 are recruited by the interaction of
TRAIL with either DR-4/TRAIL-R1 or DR-5/TRAIL-R2.18 By
inducing proximity between pro-caspase-8 and -10 molecules, which have
weak protease activity, death receptor clustering facilitates
trans-proteolysis and consequent activation of the cell
death machinery.17,18
It should be emphasized that, although TRAIL has potential as an
anticancer therapeutic, its physiologic role is presumably more complex
than merely activating caspase-dependent cell death of cancer cells. In
this respect, it has been shown by us and other groups of investigators
that TRAIL induces a stage of development-specific inhibitory effects
on normal immature erythroblasts.19,20 Moreover, it has
been shown that caspase activation plays an essential role in the
terminal differentiation of human normal erythroblasts,21 disclosing a novel role for caspases in the control of hemopoiesis in
addition to that of mediating apoptotic cell death. Therefore, because
little is still known about possible nonapoptotic functions induced by
TRAIL, these data suggest that TRAIL might play different roles in the
regulation of normal human hemopoiesis.
The experiments illustrated in this study were designed to investigate
the biologic activity of TRAIL on myeloid cells in terms of
cytotoxicity or modulation of maturation along the monocytic lineage.
As an experimental system, we have chosen the HL-60 myeloblastic leukemia cell line, which can be induced to undergo terminal
differentiation along the monocytic and granulocytic lineages by a
variety of chemical and biological agents.22-25 Parallel
experiments were carried out using primary normal myeloid cells,
derived from CD34+ hemopoietic progenitors, to ascertain
whether TRAIL also exhibited a maturation-inducing activity on normal
myeloid cells.
Reagents
Lipopolysaccharide (LPS; Sigma Chemical, St Louis, MO) and polymyxin B
(Calbiochem, La Jolla, CA) were used at the final concentration of 10 to 100 ng/mL and 10 µg/mL, respectively. LY294002, a pharmacologic inhibitor of the PI 3-kinase/Akt signal transduction pathway, was
purchased from Calbiochem and was used at the final concentration of 10 µM.
For neutralization experiments, either recombinant TRAIL or the
supernatant derived from apoptotic TRAIL-treated HL-60 cultures was
preincubated with TRAIL-R1-Fc or TRAIL-R2-Fc chimeras, according to the
supplier's instructions (R&D, Minneapolis, MN).
The broad caspase inhibitor Cbz-Val-Ala-Asp (Ome)-fluoromethyl ketone
(z-VAD-fmk), the peptide control Cbz-Phe-Ala-fluoromethyl ketone
(z-FA-fmk), the selective caspase 8 Cbz-Ile-Glu(Ome)-Thr-Asp(Ome)-CH2F(z-IETD-fmk), and caspase
9 Cbz-Leu-Glu(Ome)-His-Asp(Ome)-CH2F (z-LEHD-fmk) inhibitors were from Calbiochem. The activity of most effector caspases3,4,7 and of caspase 1 is blocked by z-VAD-fmk, whereas z-IETD-fmk and z-LEHD-fmk specifically block the activity of
caspase 8 and 9, respectively. All caspase inhibitors were dissolved in
dimethyl sulfoxide, stocked in aliquots at Cells
In some experiments, 24 hours after TRAIL treatment, dead HL-60 cells were removed from the cultures by using the Dead Cell Removal kit (Miltenyi Biotech, Auburn, CA). In other experiments, TRAIL-treated HL-60 cultures were enriched in CD14+ cells by positive selection using CD14 MicroBeads (Miltenyi Biotech). Cord blood (CB) specimens, collected according to institutional
guidelines, were obtained during normal full-term deliveries. CB
mononuclear cells were isolated by density-gradient centrifugation (Ficoll/Histopaque 1077 g/mL). CB CD34+ cells were
then isolated by using a magnetic cell sorting program, Mini-MACS, and
the CD34 isolation kit (Miltenyi Biotech) in accordance with the
manufacturer's instructions. The purity of CD34+ ranged
between 90% and 98%. Myeloid cultures were obtained by seeding
CD34+ cells in X-vivo (BioWhittaker) medium, supplemented
with nucleosides (10 µg/mL each), 0.5% BSA (fraction V of Chon),
10 Assessment of apoptosis and cell differentiation After TRAIL treatments, cytotoxicity and maturation-inducing activity were assessed. In particular, at different times (1-4 days) after treatment with TRAIL, samples were analyzed by (1) counting the total number of viable cells by trypan blue dye exclusion; (2) evaluating the degree of apoptosis by propidium iodide (PI) staining and flow cytometry analysis; (3) monitoring cell surface antigen expression by using flow cytometry (for this purpose, surface expression of CD33, CD14, CD15, and CD11b antigens was analyzed); (4) examining the morphology of the cells by staining with May-Grünwald-Giemsa solution or with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) followed by either light or fluorescence microscopy examination, respectively, and by transmission electron microscopy; (5) examining the presence of nonspecific monocyte-associated esterase (NSE) by cytochemical procedure with the NSE kit (Sigma); (6) measuring solid-particle phagocytosis activity by flow cytometry after the addition of 5 × 105/mL FITC-latex microbeads (diameter, 2.0 µm; Polysciences, Warrington, PA) to the cells for 2 hours at 37°C, as previously described.27Flow cytometry analyses Apoptosis and surface cellular antigens were analyzed by flow cytometry. For apoptosis detection and quantification, samples containing 2-5 × 105 cells were harvested by centrifugation at 200g for 10 minutes at 4°C, fixed with cold 70% ethanol for at least 1 hour at 4°C, and treated as previously detailed.28 After PI staining, analysis of PI fluorescence was performed by FACScan flow cytometer with the FL2 detector in a linear mode using Lysis II software (Becton Dickinson, San Jose, CA). For quantitative evaluation of apoptosis, the subdiploid (less than 2n) DNA content was calculated as described28 and expressed as a percentage of apoptotic versus nonapoptotic cells, regardless of the specific cell cycle phase.To examine the presence of surface antigens, aliquots of 0.5 × 106 cells per experimental point were subjected to single- or multiple-label staining, as described previously.29 In particular, CD33, CD15, CD11b, and CD14 expression was analyzed by direct staining with either phycoerythrin (PE)- or fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (Becton Dickinson). Nonspecific fluorescence was assessed by using isotype-matched controls. The expression of TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 was analyzed by indirect staining using goat anti-human TRAIL-R1, TRAIL-R2, TRAIL-R3, and TRAIL-R4 polyclonal antibodies (all from R&D System, Minneapolis, MN) followed by PE-conjugated rabbit anti-goat IgG (Sigma). Nonspecific fluorescence was assessed by using normal goat IgG followed by a second layer, as described above. In TRAIL-treated cultures, surface antigens and endocytic activity were analyzed in the gated area detecting viable cells, as established according to the PI staining-negative area, and the forward and side scatter intensities established by multicolor analysis. Data collected from 10 000 cells are reported as either percentage of positive cells or mean fluorescence intensity (MFI) values. All analyses were performed by using a FACScan flow cytometer (Becton Dickinson) and Lysis II software. Assays for caspase activity Caspase activity was measured in HL-60 cell extracts by using the fluorometric CaspACE assay system (Promega, Madison, WI). To determine ICE (Caspase-1) and CPP32 (Caspase-3) protease activities, assays were performed in triplicate in 96-well, flat-bottom plates by incubating 10 µg cell proteins/sample with the specific fluorigenic substrates according to the manufacturer's instructions. After 1 hour of incubation, the products of reaction were measured by using a plate reader (Victor 1420; Wallac, Freiburg, Germany). Increases in fluorescence were linear over time and extract concentration.Caspase activation was also monitored by using the CaspACE FITC-VAD-FMK in situ marker (Promega) according to the manufacturer's instructions. This compound is a FITC-conjugate of the cell-permeable broad caspase inhibitor VAD-fmk, which can be delivered to the cells where it binds to activated caspase. The bound marker was localized by fluorescence detection and quantified by flow cytometry. Analysis of TRAIL receptor transcripts The presence of TRAIL receptor mRNA transcripts was examined by reverse transcription-polymerase chain reaction (RT-PCR). RNA purification from HL60 and primary myeloid cultures was performed using the SV total RNA isolation system (Promega) following the manufacturer's protocol. Synthesis of first-strand cDNA and amplification were performed using the Access RT-PCR system (Promega) and specific primer sets according to the manufacturer's protocol. As a control for DNA contamination, equal amounts of RNA were used for PCR without template retro-transcription. RT-PCR reactions were performed using the following primer sets: TRAIL-R1 (forward: 5'-CTG AGC AAC GCA GAC TCG CTG TCC AC-3'; reverse: 5'-TCC AAG GAC ACG GCA GAG CCT GTG CCA T-3'); TRAIL-R2 (forward: 5'-GCC TCA TGG ACA ATG AGA TAA AGG TGG CT-3'; reverse: 5'-CCA AAT CTC AAA GTA CGC ACA AAC GG-3'); TRAIL-R3 (forward: 5'-GAA GAA TTT GGT GCC AAT GCC ACT G-3'; reverse: 5'-CTC TTG GAC TTG GCT GGG AGA TGT G-3'); TRAIL-R4 (forward: 5'-CTT TTC CGG CGG CGT TCA TGT CCT TC-3'; reverse: 5'-GTT TCT TCC AGG CTG CTT CCC TTT GTA G-3'); -actin (forward: 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; reverse:
5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'). Resultant PCR products from
RT-PCR were resolved on 2% agarose gels and visualized with
ethidium bromide.
Statistical analysis Data were analyzed using the 2-tailed, 2-sample t test (Minitab statistical analysis software, State College, PA). Values of P < .05 were considered significant.
TRAIL induces a rapid cytotoxicity on HL-60 cells coupled to maturation of the surviving cells along the monocytic lineage Following treatment with recombinant Histidine6-tagged TRAIL, containing the TRAIL protein fused to a tag of 6 histidine residues, or with a control Histidine6-peptide (His-Pept),26 the extent of apoptosis was determined by PI staining followed by flow cytometry analysis, and cell viability was determined by trypan blue dye exclusion. Although cultures treated with the control His-Pept were indistinguishable from the untreated ones, addition of TRAIL induced a dose-dependent increase of apoptosis (Figure 1), accompanied by a drastic decline in the total number of trypan blue dye-negative viable cells (data not shown).
Phenotypic analysis of untreated and His-Pept-treated HL60 cells
showed a high expression of CD33 and CD15 early myeloid markers, low
expression of CD11b late myeloid marker, and virtually absent expression (less than 7%) of CD14 (Figure
2A). Although CD33 and CD15 antigens were
unaffected by TRAIL treatment, a significant (P < .01)
subset (39% ± 10%; n = 27 independent experiments) of HL-60
cells surviving to the cytotoxic activity of TRAIL unexpectedly showed
a selective induction of the surface 55-kD glycoprotein CD14 (Figure
2A). It should be noted that CD14 represents an excellent marker of
monocytic maturation because it is undetectable on the surface of
monocyte precursors and increases dramatically during their
differentiation to monocytes.30-32 Surface CD11b, the
We next investigated whether these phenotypic changes were accompanied
by the appearance in culture of cells with monocytic features.
Therefore, HL-60 cultures were depleted of the necrotic cells and
debris 24 hours after TRAIL treatment and were cultured for an
additional 2 to 5 days before morphologic (Figure
3) and cytochemical (NSE) analyses (data
not shown). During light (Figure 3A) and electron (Figure 3B)
microscopy, cells with morphologic features of the monocytic lineage,
such as condensation and cleaving of the nucleus, were hardly
detectable in untreated and His-Pept-treated cultures. On the
contrary, by day 3 of TRAIL treatment, cells with monocytic features
represented a significant percentage of the surviving cell population,
ranging from 20% to 50%.
Specificity of the proapoptotic and maturational effects of TRAIL was
next demonstrated combining different approaches. To completely rule
out the effect of potential contaminating LPS in the TRAIL
preparations, cells were treated in the presence of polymyxin, an
inhibitor of LPS. TRAIL-mediated cytotoxicity (Figure
4A) and up-regulation of surface CD14
(Figure 4B) were unaffected by co-treatment of HL-60 cells with
polymyxin. On the other hand, polymyxin completely abrogated the
ability of LPS (100 ng/mL) to induce a small increase of surface CD14.
The specificity of the effect was confirmed by preincubation of TRAIL
with TRAIL-R1-Fc or TRAIL-R2-Fc chimeric proteins, which abrogated the
TRAIL-mediated maturational effects (Figure 4A) without exhibiting by
themselves any effect on the expression of surface markers in HL-60
cells (Figure 4B). In addition, we considered the possibility that
apoptosis of a large proportion of cells, as a consequence of TRAIL
treatment, could leave a subpopulation undergoing differentiation in
response to signals derived from apoptotic cells, related to the stress of the elimination of large proportions of cells. Therefore, to evaluate whether the differentiation of the surviving cells was truly
dependent on a direct TRAIL signal, in some experiments the
supernatants of His-Pept (control)- and TRAIL-treated cells were
harvested and filtered (0.4 µm) after 24 hours of culture. Incubation
of HL60 cells with control supernatant did not result in any modulation
of maturative markers. On the other hand, a significant increase of
CD14 and CD11b surface expression and the induction of apoptosis was
observed on 24-hour incubation of HL60 cells with the supernatant
derived from TRAIL-treated cultures (Figure 4A-B). However, such
effects were abrogated by preincubating the TRAIL-treated supernatant
with the TRAIL-R1-Fc chimeric protein, supporting a direct and specific
role of residual TRAIL in the differentiation process (Figure 4A-B).
This conclusion was further strengthened by the observation that
treatment of HL-60 cells with LY294002, a pharmacologic inhibitor of
the PI 3-kinase/Akt survival pathway, induced a significant increase in
the percentage of apoptosis without inducing any maturative effect in
the surviving HL-60 cells (Figure 4A-B).
TRAIL-treated HL-60 cells were next analyzed by a functional assay, consisting of the evaluation of the phagocytic activity of solid particles. The phagocytic activity in TRAIL-treated cells was significantly (P < .05) increased, as demonstrated by the higher incorporation of microbeads-FITC with respect to untreated cells or cells treated with His-Pept, as evaluated by flow cytometry (Figure 4C). TRAIL-induced monocytic maturation of HL-60 cells requires the activation of the caspase cascade We have recently demonstrated that TRAIL activates effector caspases and NOS pathways in the K562 erythroleukemic cell line and that both pathways contribute to the cytotoxicity of TRAIL on these leukemic cells.29 Moreover, it has been shown that certain types of AML can be induced to differentiate along the monocytic lineage by NO.35 Therefore, in the next group of experiments, we sought to investigate whether these pathways were involved in the maturational effects induced by TRAIL in the myeloblastic HL-60 cell line.The activity of the effector caspase subgroup (including caspase 3) and
the ICE subgroup (including caspase 1) was assessed at different time
points after treatment with either control His-Pept or with TRAIL.
Although no activation of the ICE subgroup was observed on treatment
with TRAIL, the activity of the caspase 3 subgroup showed a significant
(P < .01) and persistent increase (Figure
5A) with respect to untreated or
His-Pept-treated cells. Activation of the caspase 3 subgroup was
independently demonstrated by using a fluorometric marker revealed by
flow cytometry (Figure 5B).
The role of the caspase pathway in TRAIL-mediated maturation of HL-60
cells was next investigated by using selective pharmacologic inhibitors. In this set of experiments, cells were preincubated with
each inhibitor for 45 minutes, followed by the co-incubation of TRAIL
for an additional 24 to 72 hours. After treatment with TRAIL,
cytotoxicity and degree of maturation were examined. Cell viability and
expression of surface antigens were not affected by any of the
inhibitors, used at indicated concentrations, in control HL-60 cells
(data not shown). Incubation of HL-60 cells with the broad inhibitor of
effector caspases, z-VAD-fmk, completely abrogated the TRAIL-induced
apoptosis (P < .01) (Figure
6A) and almost completely prevented
(P < .01) the TRAIL maturational effect (Figure 6B). On
the other hand, the NOS inhibitor, L-NAME, did not affect
(P > .1) the degree of maturation along the monocytic lineage, thus suggesting that effector caspases play a crucial role in
TRAIL-mediated maturation, whereas the NO pathway is not involved in
this process.
It has been previously shown that members of the TNF superfamily activate 2 main signal transduction pathways. The type 1 pathway is mediated by high levels of caspase 8, which directly activates effector caspases. On the other hand, the type 2 pathway is mediated by mitochondrial dysfunction, which precedes the activation of caspase 9.36 Therefore, the maturation activity of TRAIL was also evaluated in the presence of selective inhibitors for caspase 8 and caspase 9. As shown in Figure 6, z-IETD-fmk, a selective caspase 8 inhibitor, significantly (P < .01) reduced the cytotoxic activity of TRAIL (Figure 6A) and was as efficient as z-VAD-fmk in blocking (P < .01) the TRAIL-induced maturation along the monocytic pathway (Figure 6B). On the other hand, z-LEHD-fmk, a selective caspase 9 inhibitor, was unable to significantly prevent TRAIL-mediated cytotoxicity and maturative effect (Figure 6A-B). Maturational effect of TRAIL is mediated by TRAIL-R1 It should be emphasized that an extreme complexity characterizes the expression and function of TRAIL receptors (reviewed in Baker and Reddy4). In fact, at least 5 TRAIL receptors have been described so far. TRAIL-R1 (DR4) and TRAIL-R2 (DR5) transduce apoptotic signals on the binding of TRAIL, whereas TRAIL-R3 (DcR1), TRAIL-R4 (DcR2), and osteoprotegerin are homologous to DR4 and DR5 in their cysteine-rich extracellular domain but lack intracellular death domain and apoptosis-inducing capability. Although HL-60 cells expressed the mRNA of all transmembrane TRAIL receptors (TRAIL-R1, -R2, -R3, and -R4) (Table 1), flow cytometry analysis showed detectable surface levels of TRAIL-R1 and TRAIL-R2 expression coupled to low or undetectable surface expression of TRAIL-R3 and TRAIL-R4 (Table 1). Therefore, to elucidate whether the maturational effects of TRAIL on HL-60 cells were specifically mediated by one or more TRAIL receptors, HL-60 cells were challenged with agonistic polyclonal anti-TRAIL-R1, -R2, -R3, and -R4 antibodies, which mimic the interaction between TRAIL and each TRAIL-R without cross-reacting among each other.37
Remarkably, treatment of HL-60 with anti-TRAIL-R1, but not with
anti-TRAIL-R2, anti-TRAIL-R3 or anti-TRAIL-R4, induced a significant (P < .05) increase in apoptosis (Figure
7A) and CD14 (Figure 7B) surface
expression. The biologic activity of anti-TRAIL-R1 antibody was
significantly lower (P < .05) than that of recombinant
TRAIL, likely because of their differential ability to induce TRAIL-R1 oligomerization.
TRAIL induces maturation along the monocytic lineage in primary normal CD34-derived myeloid cells In the last group of experiments, we investigated whether TRAIL affected the maturation of primary normal myeloid cells. For this purpose, we set up serum-free liquid cultures of CB CD34+ hemopoietic progenitors supplemented with SCF + GM-CSF. Cells were monitored for phenotypic expression of myeloid maturative surface antigens every 2 days, and TRAIL was typically added in culture between day 4 and day 8, when the cells still showed low expression of CD14. At this time point, primary normal myeloid cells expressed the mRNA for all TRAIL receptors, and, at flow cytometry, dim expression of TRAIL-R, TRAIL-R2, and TRAIL-R3 was clearly observed, whereas TRAIL-R4 was undetectable (Table 1). Thus, with the exception of surface TRAIL-R3, the phenotypic pattern of primary myeloid cells at this culture time (days 4-8) resembled that of HL-60 myeloblastic cell line (Table 1). In agreement with the data obtained on the HL-60 cell line, the addition of TRAIL for 3 days significantly (P < .01) increased the surface expression of CD14 (from 15% ± 7% to 33% ± 6%; n = 6 independent experiments) and CD11b (from 26% ± 8% to 47% ± 9%, n = 6 independent experiments) antigens with respect to untreated or control cells treated with His-Pept (Figure 8A). It is particularly remarkable, however, that TRAIL did not induce any significant cytotoxicity on primary normal myeloid cells, as evaluated in terms of apoptosis (from 15 ± 4 to 20 ± 6; n = 6 independent experiments) and viable cell counts (from 126 × 104 to 132 × 104; n = 6 independent experiments). At light microscopy examination, untreated or His-Pept-treated control cultures were characterized by a heterogenous population of cells comprising monoblasts, granulocyte precursors, monocytes, and rare macrophages (Figure 8B). The frequency of monocytes and macrophages in control cultures varied from 19% to 40% in the different experiments performed. In TRAIL-treated cultures, the cell population was still heterogeneous, but cells with morphologic features of mature monocytes and macrophages were more easily detectable and their frequency ranged from 35% to 58%.
We have here shown for the first time that, besides inducing a marked cytotoxicity on HL-60, TRAIL promotes maturation of the surviving cells along the monocytic lineage, as shown by combining phenotypic, morphologic, and functional analyses. The possibility that monocytic maturation was the consequence of stress factors released by apoptotic HL-60 cells cannot be completely excluded, but it is unlikely on the basis of experiments performed by using the culture supernatant of apoptotic HL-60 cells that was unable to induce monocytic maturation when added to viable cells in the presence of TRAIL-R1-Fc. Moreover, the induction of apoptosis by LY294002, a pharmacologic compound interfering with the PI 3-kinase/Akt survival pathway, did not induce any monocytic maturation in HL-60 cells. It is particularly remarkable that similar maturational effects were observed in TRAIL-treated primary normal myeloid cells in the absence of cytotoxicity. Therefore, the ability of TRAIL to promote maturation along the monocytic pathway is not confined to the HL-60 experimental cell model, but it involves primary normal myeloid cells. Moreover, the fact that the maturational activity of TRAIL in primary myeloid cells took place in the absence of cytotoxicity strongly suggests that a major physiological role of the TRAIL/TRAIL receptor system in hemopoiesis is to promote normal monocytic development. It should be emphasized that this represents the first demonstration of a positive regulative effect of TRAIL on primary normal cells, whereas previous studies of our and other groups have demonstrated the inhibitory effects of TRAIL on primary normal immature erythroblasts19,20 in the absence of cytotoxicity on normal myeloid cells.11 The ability of TRAIL to promote monocytic differentiation of the HL-60 leukemic cell line, besides inducing cytotoxicity, envisions an additional role of TRAIL as an antineoplastic agent, at least for certain types of acute myeloid leukemia. In fact, inducing death by terminal differentiation represents an alternative approach to cytodestruction of cancer cells by conventional antineoplastic agents with important biologic implications. In fact, it indicates that the malignant state is not an irreversible one and suggests that certain cancers may eventually be treated with agents that initiate terminal maturation, presumably with less morbidity than that produced by cytodestructive agents.38 In this respect, retinoid acids are well-known inducers of granulocytic differentiation of primary acute promyelocytic leukemia (APL) blasts and leukemic cell lines, apparently through the transcriptional regulation of genes critical to this process.39 In fact, retinoids and vitamin D3 interact with nuclear receptors, members of the steroid-thyroid hormone receptor superfamily of transcription factors.40 These are ligand-inducible trans regulators that modulate the transcription of genes, which play a critical role in the control of cell growth and differentiation by interacting with retinoic acid or vitamin D3 cis-acting DNA responsive elements.39,40 However, though some retinoids are used in the treatment of APL, the M3-type of AML, agonists able to induce monocytic differentiation in vitro, such as vitamin D3, have not demonstrated efficacy in clinical trials performed in leukemic patients with different types of AML, mainly because of secondary hypercalcemia that limited the dose of this agent that could be administered.41 Interestingly, it has been proposed that one of the mechanisms of action of retinoic acid in APL is the induction of TRAIL expression in cells differentiating along the granulocytic lineage.42 It has been shown that HL-60 cells become progressively more resistant to TRAIL cytotoxicity as all-trans retinoic acid-induced granulocytic differentiation proceeds.43 Our present study suggests that a therapeutic regimen including TRAIL would optimize the anti-leukemic activity against neoplastic cells by a 2-fold mechanism: cytotoxicity and induction of differentiation along the monocytic lineage. In this respect, TRAIL has garnered the most interest therapeutically, as several studies have demonstrated in vivo tumoricidal activity in animal models without inducing significant toxicity in mice44,45 or nonhuman primates.46 Moreover, recombinant TRAIL synergizes with conventional chemotherapy and radiotherapy in inducing cytotoxicity in acute leukemias,37,47-49 which lack functional p53 wild-type genes or overexpress Bcl-2 or MDR genes. We also started to explore the mechanisms underlying the maturational
activity of TRAIL by demonstrating that the activation of effector
caspases plays an essential role in this process. These data are in
agreement with previous studies showing that the caspase activation in
intact cells does not necessarily lead to cell death and that argue for
a checkpoint in the apoptotic pathway downstream of caspases. In
particular, it has been demonstrated that the terminal differentiation
of erythroblasts requires a transient activation of
caspases,21 thus indicating that the caspase family of
proteases mediates a broader range of biologic activities than
originally thought, including the activation and proliferation of
quiescent T lymphocytes.50,51 Interestingly, 2 distinct
caspase-dependent pathways have been described in response to CD95
ligand,36,52 the member of the TNF superfamily showing the
closest amino acid structure to TRAIL. The type 1 pathway triggers the
activation of large amounts of caspase 8 followed by the rapid cleavage
of caspase-3 before the loss of mitochondria transmembrane potential
( We could demonstrate that in HL-60 cells, z-IETD-fmk, a selective pharmacologic inhibitor of caspase 8, almost completely reproduced the ability of z-VAD-fmk, a broad inhibitor of effector caspases, to block TRAIL-mediated cytotoxicity and maturational effects. On the other hand, z-LEHD-fmk, a selective pharmacologic inhibitor of caspase 9, was unable to interfere with TRAIL biologic activities. These findings clearly indicate that the type 2 intrinsic pathway, which has been involved mainly in the induction of apoptosis,36 does not play a role in the maturational effects of TRAIL. Theoretically, because TRAIL preferentially triggers the type 1 pathway in leukemic HL-60 cells, TRAIL with or without chemotherapeutic drugs might substantially enhance the antileukemic activity of conventional chemotherapy in hematologic malignancies.36,52 The experiments performed with agonistic antibodies have indicated that, although TRAIL-R1 and -R2 were expressed on the surfaces of HL-60 cells, only TRAIL-R1 induced maturation along the monocytic lineage. We have not used these agonistic antibodies in primary normal myeloid cultures because of the limited number of cells. However, we have observed that TRAIL-mediated maturation took place only in 4- to 8-day cultures, when primary myeloid cells expressed surface TRAIL-R1, -R2, and -R3. On the other hand, no changes in the surface expression of CD14 and CD15 were observed at later culture times (days 18-22), when TRAIL-R3 was the only TRAIL-R expressed, as also shown by other authors (Zhang et al11 and data not shown). Thus, TRAIL-R1 likely plays an important role in mediating monocytic maturation, possibly by activating effector caspases in primary cells. Moreover, the coexistence of TRAIL-R3 with -R1 and -R2 on the surfaces of primary normal myeloid cells likely protects these cells from apoptosis. In conclusion, the findings derived from this study might have important implications for understanding the biology of the TRAIL/TRAIL receptor system in normal and leukemic hematopoiesis. In fact, our data disclose a novel physiological role for TRAIL as a positive regulator of monocytic maturation. Moreover, the dichotomous effect of TRAIL on malignant cells (early induction of apoptosis and maturation of the surviving cells) might have therapeutic implications for the treatment of acute myeloid leukemia and possibly of other hematologic malignancies.
Submitted January 15, 2002; accepted May 14, 2002.
Prepublished online as Blood First Edition Paper, May 31, 2002; DOI 10.1182/blood-2002-01-0047.
Supported by Consiglio Nazionale delle Ricerche (CNR) and Associazione Italiana per le Ricerca sul Cancro (AIRC) funds. A.G. is supported by a Fondazione Italiana per le Ricerca sul Cancro (FIRC) fellowship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Paola Secchiero, Department of Morphology and Embryology, Human Anatomy Section, University of Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy; e-mail: secchier{at}mail.umbi.umd.edu.
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© 2002 by The American Society of Hematology.
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  J.-W. Hsu, H.-C. Huang, S.-T. Chen, C.-H. Wong, and H.-F. Juan Ganoderma lucidum Polysaccharides Induce Macrophage-like Differentiation in Human Leukemia THP-1 Cells via Caspase and p53 Activation Evid. Based Complement. Altern. Med., August 20, 2009; (2009) nep107v1. [Abstract] [Full Text] [PDF]
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P. Secchiero, F. Corallini, E. Rimondi, C. Chiaruttini, M. G. di Iasio, A. Rustighi, G. Del Sal, and G. Zauli Activation of the p53 pathway down-regulates the osteoprotegerin expression and release by vascular endothelial cells Blood, February 1, 2008; 111(3): 1287 - 1294. [Abstract] [Full Text] [PDF]
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P. Mirandola, C. Ponti, G. Gobbi, I. Sponzilli, M. Vaccarezza, L. Cocco, G. Zauli, P. Secchiero, F. A. Manzoli, and M. Vitale Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity Blood, October 15, 2004; 104(8): 2418 - 2424. [Abstract] [Full Text] [PDF]
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P. Secchiero, E. Melloni, M. Heikinheimo, S. Mannisto, R. Di Pietro, A. Iacone, and G. Zauli TRAIL regulates normal erythroid maturation through an ERK-dependent pathway Blood, January 15, 2004; 103(2): 517 - 522. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
CSF. After treatment with
TRAIL for 3 additional days, a significant increase in CD14 and CD11b
expression, coupled with an increased number of mature monocytes and
macrophages, was noticed in the absence of cytotoxicity. These data
disclose a novel role for TRAIL as a positive regulator of myeloid
differentiation. Moreover, the dichotomous effect of TRAIL on malignant
cells (early induction of apoptosis and monocytic maturation of the
surviving cells) might have important therapeutic implications for the
treatment of acute myeloid leukemia.
(Blood. 2002;100:2421-2429)
, is considered its ability to induce
apoptosis in a variety of malignant cells, including several of
hemopoietic origin,
4 M BSA-adsorbed cholesterol, 10 µg/mL insulin, 200 µg/mL iron-saturated transferrin, 5 × 10
m). In the type 2 pathway, low concentrations of caspase 8, insufficient to allow an effective downstream activation of caspase 3, induce the loss of