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NEOPLASIA
From the Clinical Research Division, Fred Hutchinson
Cancer Research Center and the Department of Medicine, University of
Washington; Immunex; and Hematologics, Seattle, WA.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a
member of the tumor necrosis factor (TNF) family, binds to several
cell-surface receptors with distinct functions (agonistic receptors 1 and 2 [TRAIL-R1, TRAIL-R2]; decoy receptors 3 and 4 [TRAIL-R3,
TRAIL-R4]). Expression and function was characterized in patients with
myelodysplastic syndromes (MDSs). While normal marrow showed negligible
expression of TRAIL and receptors (except TRAIL-R3), TRAIL and all
receptors were constitutively expressed in MDS marrow. Following TRAIL
exposure, MDS marrow showed significant increases in apoptosis, whereas
normal marrow, except for a subset of CD34+ precursors, did
not (P = .012). Marrow from 21 patients with MDS was then
propagated in long-term cultures in the presence or absence of TRAIL.
While in advanced MDS (refractory anemia with excess blasts in
transformation [RAEB-T] and tAML [MDS transformed into
AML]), colony numbers decreased in the presence of TRAIL (63.0% ± 10.4% of untreated group [100%]), numbers
increased in patients with RA or RAEB (160.2% ± 90.5% of untreated
group). TRAIL eliminated preferentially clonally abnormal cells
as identified by chromosomal markers. Thus, TRAIL and receptor
expression differed significantly between normal and MDS marrow, and
TRAIL modulated in vitro hemopoiesis in MDS dependent upon disease
stage but not, to a detectable extent, in normal marrow.
(Blood. 2001;98:3058-3065) The myelodysplastic syndromes (MDSs) comprise a
heterogeneous group of hemopoietic stem cell disorders usually
characterized by a cellular marrow with dysplastic features, peripheral
blood cytopenias, and a variable incidence of transformation into acute myeloid leukemia (AML).1 MDS generally occurs in elderly
patients, the median age at diagnosis being 65 to 70 years.2 Allogeneic hemopoietic stem cell transplantation
is currently considered the only curative treatment modality for MDS;
however, age and performance status of patients and lack of
histocompatible donors limit the applicability of this treatment. Also,
regimen-related toxicity is a major problem in patients with MDS
undergoing transplantation.3,4 It would be desirable,
therefore, to develop new therapeutic strategies to improve or restore
normal hemopoietic function.
One mechanism invoked to explain the apparent discrepancy between
cellular marrow and peripheral blood cytopenias in patients with MDS is
programmed cell death (apoptosis), which occurs with increased
frequency in MDS marrow.5,6 Several cytokines or ligands
known to have proapoptotic properties, such as interleukin-1 The objective was to exploit the potential selective killing of
clonal and, presumably, malignant cells by human TRAIL. Thus, we
determined the effect of TRAIL on apoptosis and hemopoiesis in
long-term marrow cultures (LTMC) of normal and MDS marrow, and
characterized the expression of TRAIL and its receptors. We also
characterized the expression of FLIP in marrow cells from patients with
MDS and healthy donors.
Marrow cells
Cell lines
Antibodies and reagents The following monoclonal antibodies (mAbs) were obtained from commercial sources: fluorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated mouse anti-human CD3 (IgG2a), CD13 (IgG1), CD14 (IgG2a), CD34 (HPLC-2, IgG1), and CD45 (IgG1) (Becton Dickinson, San Jose, CA, or Caltag, San Francisco, CA); FITC or PE-conjugated mouse F(ab')2 IgG1 or IgG2a or IgG2b (Becton Dickinson) or mouse IgM (Catalag) as isotype controls; FITC or PE-conjugated goat F(ab')2 anti-mouse IgG (H+L) or IgM (Caltag) as secondary antibodies; rabbit anti-mouse IgG to carboxy terminus human FLIP long form for Western blot (Calbiochem, La Jolla, CA). The mouse anti-human IgM STRO-1 mAb, which recognizes a stromal cell surface antigen,21 was obtained from Developmental Studies Hybridoma Bank (Iowa City, IA). The monoclonal mouse anti-human TRAIL (IgG1, M180, blocking; M184, nonblocking), TRAIL-R1 (IgG2a, M271), TRAIL-R2 (IgG1, M413), TRAIL-R3 (IgG1, M430), TRAIL-R4 (IgG1, M444), as well as stem cell factor (SCF), and leucine zipper (LZ)-tagged soluble trimeric human TRAIL were supplied by Immunex (Seattle, WA). IL-1, IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were purchased from R & D Systems (Minneapolis, MN).Phenotyping of marrow cells Phenotypic analysis of marrow cells was carried out by flow cytometry as described.5 For determination of the expression of TRAIL and its receptors, 105 cells were incubated with 10 µg/mL mAb against human TRAIL or TRAIL-R1 to -R4 for 30 minutes on ice. After washes in phosphate buffered saline (PBS), cells were incubated for 30 minutes on ice with PE-conjugated (secondary) goat F(ab')2 anti-mouse IgG. For 2-color immunofluorescence, FITC-conjugated mAbs for CD3, CD19 (for the lymphocyte gate), CD13 (for the granulocyte gate), CD14 (for the monocyte gate), and CD34 (for the hemopoietic progenitor cell gate) were incubated after staining with mAbs to human TRAIL and TRAIL-R1 to -R4, PE-conjugated (secondary) goat F(ab')2 anti-mouse IgG. Blast cells were isolated on the basis of orthogonal light scatter and CD45 labeling as described.22,23 Cells labeled with irrelevant isotype-matched mAb and incubated with the secondary detection antibody served as negative controls. Data on at least 10 000 events were collected using a FACScan (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson).Reverse transcription-polymerase chain reaction amplification RNA preparation and reverse transcription (RT) were carried out as described.5,18 Total RNA was prepared from cell pellets using the RNeasy Total RNA kit (Qiagen, Chatsworth, CA). Cells were lysed in buffer RLT (4 M guanidinium isothiocyanate, 0.5% sarcosyl, and 0.1 M 2 -mercaptoethanol) and homogenized using a QIAshredder
spin column. After centrifugation, an equal volume of 70% ethanol was
added to the lysate, and the mixture was applied onto RNeasy spin
columns. Total RNA, which binds to the silica gel membrane under high
salt-buffer conditions, was washed with buffers RW1 and RPE (supplied
with the kit), and then eluted in DEPC-treated water. Finally,
any remaining DNA was removed by digestion with RNase-free DNase. The
concentration and purity of the RNA was determined by measuring the
absorbance at A260/A280 and by gel electropheresis.
To synthesize cDNA, 1 µg the total RNA (generally corresponding to
The cDNA was amplified by PCR in a reaction mixture consisting of 50 mM
KCl, 20 mM Tris-HCl (pH 8.4), 0.1% Triton X-100, 1 mM DTT, 2.0 mM
MgCl2, all 4 dNTPs (each at 0.2 mM), 10 pmol of each
oligonucleotide primer (see below), and 2.5 units
Taq DNA Polymerase (Boehringer Mannheim). Amplification was
performed in 0.5-mL Gene Amp tubes in a final volume of 50 µL. The
PCR mixes were overlaid with mineral oil and amplified for 30 to 40 cycles (see below) denaturation (at 94°C for 1 minute),
annealing (at 55°C to 65°C [see below] for 1 minute, and
extension at 72°C for 1 minute). PCR products were size-fractionated
and analyzed by 1.5% agarose gel electropheresis and normalized
according to the amount of PCR reactions were performed using the following primers:
Western blot analysis Western blots were performed as previously described.18 Briefly, marrow mononuclear cells (MMNCs) from patients with MDS or from healthy donors were lysed in PBS containing 1% Nonidet P-40, 0.35 mg/mL PMSF, 9.5 µg/mL leupeptin, and 13.7 µg/mL pepstatin A. The lysed cells were washed, and protein concentrations of the extracts were determined by colorimetric assay (Bio-Rad, Richmond, CA). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidine difluoride (PVDF; Bio-Rad), and blocked with 5% nonfat dry milk in 1 × Tris buffered saline for 1 hour. The membranes were then incubated with the FLIP antiserum (diluted 1:1000) overnight, washed and incubated with an anti-rabbit horseradish peroxidase antibody (diluted 1:10 000; Amersham, Arlington Heights, IL) for 1 hour. Following several washes, the blots were developed by chemiluminescence reagent (Pierce, Rockford, IL).Apoptosis assay Marrow cells from healthy donors and from patients with MDS were incubated in 48-well plates (5 × 105 cells/well) with progressively increasing concentrations of purified LZ-human TRAIL (0, 10, 30, 100, 300, 1000, and 3000 ng/mL) for 3, 6, 12, and 24 hours.12,13,18 Apoptotic changes were identified by staining with FITC-conjugated Annexin V (Immunotech, Westbrook, ME)24,25 and by means of DNA histograms to identify hypodiploid nuclei (sub-G1 peak) using flow cytometric analysis as described.26 To determine a possible effect of LZ-TRAIL on the marrow "microenvironment" and on stroma cells, adherent cell cultures were established.27 Adherent cell layers from marrow cells and STRO-1-positive/glycophorin-negative sorted cells were established in T25 tissue culture flasks (Corning, NY) using Iscoves modified Dulbecco medium (Gibco, Grand Island, NY) supplemented with 12.5% each of heat-inactivated horse and fetal calf serum (FCS), 0.4 mg/mL L-glutamine, 1 mM sodium pyruvate, 10-6 M hydrocortisone, 10-4 M 2-mercaptoethanol, 100 U/mL penicillin, and 100 µg/mL streptomycin.28 Flasks were demidepleted of cells at weekly intervals. After 4 weeks, nonadherent cells and monocytes were removed by 3 trypsinization passes, and negative selection by PE-conjugated anti-CD14 mAb. Apoptosis was determined as described.Cytogenetic and fluorescence in situ hybridization analysis To determine whether TRAIL-mediated apoptosis occurred preferentially in clonal cells (identifiable by a chromosomal marker), we performed cytogenetic and fluorescence in situ hybridization (FISH) analysis on marrow cells from 9 patients with MDS before and after exposure to LZ-TRAIL (300 ng/mL).29 We also determined the proportion of cytogenetically abnormal cells among apoptotic (Annexin V-stained) and nonapoptotic (Annexin V-negative) marrow cells from 3 patients with MDS following sorting by flow cytometry after exposure to LZ-TRAIL (300 ng/mL).In vitro hemopoiesis Marrow cultures were carried out using the Dexter method.30,31 Stromal layers were established in T25 tissue culture flasks (Corning) using Iscoves modified Dulbecco medium. Flasks were demidepleted of cells at weekly intervals. At 2 to 3 weeks, the stromal cells were irradiated with 1800 cGy, removed from the flask by a brief exposure to 0.05% trypsin, and 1.25 × 105 viable cells/mL were plated as a supportive layer in flat-bottomed 48-well plates (Costar, Cambridge, MA). After 1 to 5 days, MMNCs (0.5 × 106 cells/well) were layered onto the stromal layers in replicates of 3. LZ-TRAIL (300 ng/mL) and M180 as a blocking Ab to TRAIL (300 ng/mL) were added to culture wells as indicated. Cultures were maintained at 33°C in a humidified air of 5% CO2 atmosphere. After 1 week, nonadherent cells were collected, plates were washed twice with Ca++- and Mg2+-free Hanks balanced salt solution, and 100 µL 0.05% trypsin was added to each well. Adherent cells were removed and combined with nonadherent cells and washed once. The cell mixture was then resuspended in 3.5 mL 2 × Dulbecco medium containing 40% fetal calf serum, mixed with an equal volume of 0.06% agar (Difco, Detroit, MI) and aliquoted into 35 × 10 mm culture dishes (2 mL/dish). Ten µL of a cytokine mixture (including IL-1, IL-3, IL-6, G-CSF, GM-CSF, and SCF at concentrations of 10 µg/mL each) was added to each dish.27 Untreated cells served as controls. After 2 weeks, the plates were scored for colony numbers. Groups of at least 40 cells were counted as colonies.Statistical analysis The Mann-Whitney U test was used to compare the levels of expression of TRAIL and TRAIL receptors between normal and MDS marrow. A Wilcoxon signed-rank test was used to compare apoptosis percentages and colony numbers between untreated and LZ-TRAIL-treated normal and MDS marrow. Significant differences between data from specific groups were defined as those with P .05.
TRAIL-induced apoptosis in normal and MDS marrow In ancillary dose/response experiments, apoptosis was determined in the TRAIL-sensitive cell lines Colo 205, Jurkat, and KG1. The extent of apoptosis was assessed by Annexin V-staining and DNA histograms (data not shown). Based on those results, marrow from healthy donors and patients with MDS was exposed to TRAIL at concentrations as high as 1000 ng/mL for up to 24 hours. Results are summarized in Figures 1 and 2. In unfractionated mononuclear cells from normal marrow, even at the highest TRAIL concentrations and exposure for 24 hours, no increase in the proportion of apoptotic cells was detectable. In MDS marrow, in contrast, apoptosis increased above baseline already at TRAIL concentrations of 10 ng/mL and reached a maximum/plateau with a 100% increase at TRAIL concentrations of 300 ng/mL and exposure durations of 6 to 12 hours. An increase in apoptosis was observed in all subpopulations of cells studied (Figure 2): total marrow, 24.8% ± 14.0% increased to 36.3% ± 18.0%, P = .011; CD3+, 34.3% ± 20.0% to 44.4% ± 24.9%, P = .002; CD14+, 19.2% ± 17.2% to 29.7% ± 24.2%, P = .006; CD13+, 27.9% ± 24.5% to 37.1% ± 27.1%, P = .004; CD34+, 33.3% ± 35.1% to 43.2% ± 30.7%, P = .043). There was no difference between CD34+ /CD33+ and CD34+/CD33
precursors. Two examples, MDS blasts and CD34+ precursors,
are shown in Figure 3. The most prominent
increase in apoptosis was seen in MDS blasts (22.9% ± 12.4% to
39.6% ± 16.1%). The increase in TRAIL-mediated apoptosis was
prevented in the presence of the blocking anti-TRAIL mAb, M180 (data
not shown). In normal marrow, no change in the proportion of apoptotic cells following LZ-TRAIL exposure was detected in the CD3+,
CD14+, or CD13+ gates. However, in the gate of
CD34+ precursors, the proportion of apoptotic cells was
4.3% ± 2.8% before and 7.3% ± 4.3% after LZ-TRAIL exposure
(n = 7; P = .06). This finding suggests a physiologic
role for TRAIL and its receptors in the regulation of normal
hemopoiesis at a CD34+ precursor stage as recently
suggested by others.32
Differences in regard to susceptibility to apoptosis were also observed
among adherent cell layers, whereas TRAIL exposure failed to have a
significant effect on adherent layers derived from normal marrow
(10.2% apoptosis before TRAIL exposure, 14.4% after TRAIL
exposure), the proportion of apoptotic cells in adherent layers derived
from MDS marrow increased significantly after TRAIL exposure from
23.6% ± 7.1% to 47.4% ± 9.4% (P = .04).
Apoptosis was limited to CD14+ and other mononuclear cells;
no apoptosis was observed in STRO Surface expression of TRAIL and its receptors on normal and MDS marrow cells The higher TRAIL susceptibility of malignant cells may be due to differential expression of the agonistic (TRAIL-R1 and -R2) and decoy (TRAIL-R3 and -R4) receptors.15,16 We determined surface expression of TRAIL and its receptors on marrow cells from 21 patients with MDS and from 10 healthy donors. Results are summarized in Figure 4A. In normal marrow, the expression of TRAIL and its receptors was negligible except for TRAIL-R3. On MDS marrow cells, in contrast, TRAIL and receptors R1, R2, R3, and R4 were constitutively expressed: TRAIL, 1.4% ± 0.6% (mean ± SD) of cells in normal vs 7.9% ± 10.0% in MDS marrow (P = .011); TRAIL-R1, 1.4% ± 0.4% vs 16.3% ± 23.1% (P = .017); TRAIL-R2, 1.4% ± 0.6% vs 14.9% ± 21.2% (P = .001); TRAIL-R3, 28.3% ± 20.7% vs 23.6% ± 17.2% (P = .495); TRAIL-R4, 1.6% ± 0.6% vs 7.0% ± 11.3% (P = .043). TRAIL-R3 expression on normal cells was restricted to the granulocyte gate (CD13+) (65.0% ± 25.8%). TRAIL-R2 expression in MDS marrow was significantly higher than in normal marrow in the lymphocyte gates (CD3+ or CD19+), in the monocyte gate (CD14+), and in the granulocyte gate. In purified CD34+ cells or blasts, a distinct pattern of expression was noted especially for TRAIL-R2. Among CD34+ cells from MDS marrow, 17.9% ± 34.3% expressed TRAIL-R2 vs 0.2% ± 0.1% in normal marrow. Among MDS blasts, 50.5% ± 48.1% were TRAIL-R2-positive vs 1.4% ± 1.2% in normal marrow.
To estimate TRAIL sensitivity on the basis of expression of TRAIL receptors, we calculated the ratio of agonistic (TRAIL-R1 and -R2) to antagonistic (TRAIL-R3 and -R4) receptors. As shown in Figure 4B, in MDS marrow this ratio was significantly higher than in normal marrow (18.4% ± 20.3 vs 5.1% ± 6.3, P = .015). These data are compatible with the higher susceptibility to TRAIL-induced death observed in MDS marrow. Expression of mRNA for TRAIL and its receptors in marrow cells Estimation of mRNA expression was based on band intensities of electropheresed RT-PCR products. Although mRNA for TRAIL and all TRAIL receptors was detected in cells from both normal and MDS marrow, in agreement with the data on cell surface expression, the ratio of agonistic TRAIL-R1 and -R2 to decoy TRAIL-R3 and -R4 receptors was higher in MDS than in normal marrow (Figure 5). When only CD34+ cells were considered, the ratio was again higher in CD34+ cells from MDS marrow. Further, within MDS marrow, there was a higher ratio in blast cells than in the non-blast cell population. This suggests that blast cells might be particularly vulnerable to TRAIL-induced apoptosis, a fact that might explain the phenomenon that a clonal (blast) population can exist in MDS marrow for extended periods of time without expanding and developing into frank leukemia.
Expression of apoptosis inhibitory protein FLIP In addition to the expression of agonistic and decoy receptors, differences in intracellular modification of TRAIL signals could explain differential effects in normal and malignant cells. FLIP, a cytoplasmatic inhibitor of apoptosis, can interfere with TRAIL-induced cell death.33 Griffith et al described a correlation between the level of expression of FLIP and sensitivity to TRAIL in a melanoma cell line.18 Thus, we compared the level of FLIP expression in normal and MDS marrow at the mRNA and protein levels. FLIP mRNA was present in both normal and MDS marrow, though quantitative differences were observed in MDS marrow. Differences in RNA levels for FLIP were reflected in differences in the protein levels as determined by Western blots (Figure 6). The ratio of mRNA expression of agonistic (R1 and R2) receptors to the cytoplasmatic inhibitor FLIP was higher in MDS marrow than in normal marrow, and within MDS marrow this ratio was higher in blasts than in the remainder of cells. This observation was consistent with the notion that apoptosis is facilitated in blasts, thereby controlling the size of the clonal (blast) pool.
Cytogenetics and FISH analysis To determine whether TRAIL-mediated apoptosis eliminated selectively or preferentially clonal (malignant) cells, we performed FISH analysis on the marrow from patients with MDS with a cytogenetic marker before and after TRAIL exposure. Based on results of ancillary time course studies, cells were exposed to TRAIL for 6 hours. Results in 8 individuals are summarized in Table 1. After TRAIL exposure, the proportion of cytogenetically abnormal cells decreased in 6 of 9 MDS marrows studied, especially in marrow from patients with RA. While the results suggest that LZ-TRAIL eliminated predominantly abnormal cells in certain MDS marrows, results indicate considerable heterogeneity.
In vitro hemopoiesis To determine whether the effects of human LZ-TRAIL on marrow cells were of functional relevance, we characterized in vitro hemopoiesis of marrow mononuclear cells from 21 patients with MDS (RA n = 8, RARS n = 3, RAEB n = 3, CMML n = 1, RAEBtn = 4, and tAML from MDS n = 2) and 6 healthy donors were propagated in LTMC in the presence or absence of human TRAIL (Figure 7). As postulated, numbers of colonies from normal marrow did not change in the presence of LZ-TRAIL (104% ± 5% of untreated group [100%]). In MDS marrow, colony numbers were slightly increased in the presence of LZ-TRAIL when results of all MDS marrows were combined (114% ± 14% of untreated group). However, results were strikingly dependent upon disease stage. In advanced MDS (RAEB-T and tAML) colony and cluster numbers decreased significantly in the presence of TRAIL (65% ± 7% of untreated marrow; P = .004 compared to normal marrow), whereas in patients with less advanced MDS (RA, RARS, and RAEB), colony numbers increased in the presence of TRAIL (157% ± 21% of untreated marrow, P = .037 compared to normal). These results suggest complex regulatory mechanisms and reflect the great heterogeneity of MDS. Nevertheless, the data are consistent with a model that proposes that hemopoiesis in less advanced MDS is suppressed by negative signals, presumably generated by clonal precursors,5 and elimination of those cells should result in improved hemopoiesis. Conversely, in advanced MDS, the abnormal precursor pool has expanded and is a major contributor to overall hemopoiesis.34 As a consequence, elimination of those cells should result in a decline in colony formation. An alternative consideration might be that cellular responses to TRAIL signaling differed for different disease stages.
Increased cellular proliferation, along with exaggerated programmed cell death (apoptosis), is one paradigm that has been used to explain the presence of a cellular or even hypercellular marrow concurrently with peripheral blood cytopenias in patients with MDS.35 Proliferation is likely to be due to dysregulated cytokine expression, while apoptosis could be mediated by either a lack of positive (survival) signals or an overproduction of negative (death) signals.5,8,27,36 Such a pattern could arise with an excess of bifunctional cytokines showing a positive impact on early precursors and mediating negative signals for cells at later stages of differentiation.27 What is striking, however, in patients with MDS is the fact that even though clonally abnormal precursors (with malignant potential) are detectable early in the disease course, they often expand only slowly, and patients may have life expectancies of years without requiring cytotoxic therapy.1 Such a course may be related to slow proliferation or to continuous elimination of clonal precursors that would keep the proportion of clonal cells at low levels. We have previously shown that one cytokine, TNF Another mechanism to explain selective killing of tumor cells by TRAIL is the differential expression (or function) of intracellular inhibitors of apoptosis such as FLIP.18,33 Our results show that FLIP mRNA was consistently expressed in marrow from healthy donors. FLIP was also detected in marrow cells from MDS patients but protein levels varied considerably. Although the coexistence of normal and clonal cells in MDS marrow rendered the interpretation difficult, these results suggest the possibility that variations in FLIP expression modify apoptotic responses in addition to the effects related to cell-surface-receptor expression. Apoptosis in MDS marrow was most prominent in the blast cell population, and a preferential effect on clonal cells was, in principle, supported by the results of cytogenetic and FISH analysis. TRAIL-exposed cells from MDS marrow showed decreased proportions of clonal cells (identified by FISH) in 7 of 9 marrows tested. Such a pattern is also consistent with other reports that describe a selective killing effect of TRAIL on transformed cells.11-13 The fact that a small population of CD34+ precursors from normal marrow was also sensitive to TRAIL-mediated apoptosis does not invalidate such a concept. In fact, those findings are in good agreement with a recent study by Zamai et al who showed TRAIL sensitivity in a population of glycophorin Adim precursors in normal marrow.32 Taken together, the available data suggest a physiologic role for TRAIL at a certain stage of hemopoietic cell differentiation. Furthermore, they may suggest that one factor in MDS marrow leading to increased apoptosis in normal cells is the up-regulation of TRAIL and the increase in apoptotic signals for these susceptible precursors. Further studies are needed. In agreement with other reports, we also showed an increase in
apoptotic cells in the microenvironment of MDS marrow.6,37 To define the effect of TRAIL on stroma proper in MDS marrow, we
examined cells sorted by STRO-1 expression (STRO+
glycophorin Of note was the observation that following TRAIL exposure, hemopoietic colony formation increased in cases of less advanced MDS, most likely due to the elimination of clonal cells (as shown above) that provide inhibitory signals for normal hemopoiesis.5,27 Conversely, in patients with advanced MDS, which is associated with an expansion of CD34+ precursors, exposure to TRAIL reduced colony numbers, presumably because TRAIL sensitive clonal precursors accounted for a progressively increasing proportion of marrow cells. In summary, there were distinct differences in TRAIL and TRAIL receptor expression between normal and MDS marrow. TRAIL induced significant apoptosis only in MDS, but not in normal marrow, and tended to eliminate cytogenetically indentifiable clonal cells from MDS marrow. These data suggest that TRAIL might play a role in the regulation of hemopoiesis in MDS marrow.
We thank Cassandra Beckham for technical support; Andrew Berger, Michele Black, and Cherie Green for their help with flow cytometric and Kate Rupert for cytogenetic analysis; Beverly Torok-Storb, Shelly Heimfeld, Deborah Banker, Vladimir Lesnikov, and Marina Lesnikova for helpful discussions; Bruce Clurman, Matthew Smitherman, and Jherek Swanger for their help with Western blots; and Bonnie Larson and Helen Crawford for typing the manuscript.
Supported by PHS grants HL36444, CA18029 and CA87948. H.J.D. is also supported by a grant from the Gabrielle Rich Leukemia Fund.
Submitted March 14, 2001; accepted July 6, 2001.
Ray G. Goodwin owns stock in Immunex, which is codeveloping TRAIL/Apo2 ligand with Genentech.
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: H. Joachim Deeg, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, D1-100, PO Box 19024, Seattle, WA 98109-1024; e-mail: jdeeg{at}fhcrc.org.
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R. C. Briggs, K. E. Shults, L. A. Flye, S. A. McClintock-Treep, M. H. Jagasia, S. A. Goodman, F. I. Boulos, J. W. Jacobberger, G. T. Stelzer, and D. R. Head Dysregulated Human Myeloid Nuclear Differentiation Antigen Expression in Myelodysplastic Syndromes: Evidence for a Role in Apoptosis. Cancer Res., May 1, 2006; 66(9): 4645 - 4651. [Abstract] [Full Text] [PDF]
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T. Braun, G. Carvalho, A. Coquelle, M.-C. Vozenin, P. Lepelley, F. Hirsch, J.-J. Kiladjian, V. Ribrag, P. Fenaux, and G. Kroemer NF-{kappa}B constitutes a potential therapeutic target in high-risk myelodysplastic syndrome Blood, February 1, 2006; 107(3): 1156 - 1165. [Abstract] [Full Text] [PDF]
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R. T. Means Jr Closing the TRAIL to the DISC Blood, January 15, 2006; 107(2): 418 - 419. [Full Text] [PDF]
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D. M. B. Kerbauy, V. Lesnikov, N. Abbasi, S. Seal, B. Scott, and H. J. Deeg NF-{kappa}B and FLIP in arsenic trioxide (ATO)-induced apoptosis in myelodysplastic syndromes (MDSs) Blood, December 1, 2005; 106(12): 3917 - 3925. [Abstract] [Full Text] [PDF]
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Y.-E. Claessens, S. Park, A. Dubart-Kupperschmitt, V. Mariot, C. Garrido, S. Chretien, F. Dreyfus, C. Lacombe, P. Mayeux, and M. Fontenay Rescue of early-stage myelodysplastic syndrome-deriving erythroid precursors by the ectopic expression of a dominant-negative form of FADD Blood, May 15, 2005; 105(10): 4035 - 4042. [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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R. Tehranchi, B. Fadeel, A.-M. Forsblom, B. Christensson, J. Samuelsson, B. Zhivotovsky, and E. Hellstrom-Lindberg Granulocyte colony-stimulating factor inhibits spontaneous cytochrome c release and mitochondria-dependent apoptosis of myelodysplastic syndrome hematopoietic progenitors Blood, February 1, 2003; 101(3): 1080 - 1086. [Abstract] [Full Text] [PDF]
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P. Secchiero, A. Gonelli, G. Ciabattoni, E. Melloni, V. Grill, B. Rocca, G. Delbello, and G. Zauli TNF-related apoptosis-inducing ligand (TRAIL) up-regulates cyclooxygenase (COX)-1 activity and PGE2 production in cells of the myeloid lineage J. Leukoc. Biol., November 1, 2002; 72(5): 986 - 994. [Abstract] [Full Text] [PDF]
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P. Secchiero, A. Gonelli, P. Mirandola, E. Melloni, L. Zamai, C. Celeghini, D. Milani, and G. Zauli Tumor necrosis factor-related apoptosis-inducing ligand induces monocytic maturation of leukemic and normal myeloid precursors through a caspase-dependent pathway Blood, September 18, 2002; 100(7): 2421 - 2429. [Abstract] [Full Text] [PDF]
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 P. L. Greenberg, N. S. Young, and N. Gattermann Myelodysplastic Syndromes Hematology, January 1, 2002; 2002(1): 136 - 161. [Abstract] [Full Text]
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Top
Abstract
Introduction
(TNF
0.5 × 106 cells) was combined with 1 µL Oligo
(dT)12-18 (500 µg/mL; Gibco/BRL, Gaithersburg, MD) and
heated for 5 minutes to 70°C; the resulting heteroduplex was
resuspended to 20 µL in RT buffer and incubated for 30 minutes at
37°C with 200 U Moloney murine leukemia virus (M-MLV) reverse
transcriptase (Gibco/BRL). After adding a second 100 U aliquot M-MLV,
the reaction was continued for an additional 15 minutes prior to
heat-inactivating the enzyme for 2 minutes at 80°C. cDNA was stored
at 
/+
glycophorin
