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Prepublished online as a Blood First Edition Paper on June 14, 2002; DOI 10.1182/blood-2002-03-0852.
NEOPLASIA
From the Departments of Hematology and Oncology,
Virology, and Immunology, Istituto Superiore di Sanità, Rome;
Department of Cellular Biotechnology and Hematology, University "La
Sapienza," Rome, Italy; and Kimmel Cancer Center, Thomas Jefferson
University, Philadelphia, PA.
We have investigated the expression of interleukin-3 receptor Blood cells are derived from a small number of
pluripotent hemopoietic stem cells (HSCs) endowed with the capacity to
self-renew and to differentiate into hemopoietic progenitor cells
(HPCs) progressively committed to proceed along one of the maturation pathways.1 Survival, growth, and differentiation of HPCs
are, at least in part, regulated by a network of hematopoietic growth factors (HGFs) called colony-stimulating factors (CSFs) or
interleukins (ILs).
Acute leukemias are characterized by an arrest of cell maturation and
the accumulation of undifferentiated cells in marrow, blood, and other
tissues.2 As observed in normal hematopoiesis, most
leukemic cells descend from a relatively small pool of progenitor cells
with high proliferative activity. In line with this hypothesis, recent
studies have shown that acute myeloid leukemia (AML) cells with the
membrane phenotype CD34+Thy-1 Acute leukemia cells have usually retained responsiveness to HGF
stimulation in the promotion of cell survival and cell proliferation; however, leukemic cells show little maturation under stimulation with
HGFs.6 More particularly, recombinant IL-3 and granulocyte macrophage-CSF (GM-CSF) induce leukemic colonies and activate DNA
synthesis in more than 80% of AMLs.7-10 No clear
relationship between IL-3 and GM-CSF responses and the
French-American-British (FAB) classification of acute leukemias was
observed.6 Furthermore, leukemic cells may produce one or
more of the principal HGFs, including IL-3 and
GM-CSF.11,12 Thus, the concomitant expression of receptors
for IL-3 and GM-CSF and the production of the respective ligands by
leukemic cells determine the formation of complete autocrine circuits
of HGF stimulation. According to these observations, it was suggested
that autonomous mechanisms of growth contribute to the clinical biology
of leukemia.
IL-3 and GM-CSF exert their biological activities through interaction
with cell surface receptors that consist of 2 subunits, the Studies on AML blasts have shown that receptors for IL-3 and GM-CSF are
often coexpressed on these cells.20-22 Furthermore, specific IL-3 binding was observed in approximately 50% of
B-ALL.23 Finally, a recent study showed that IL-3R Other studies have shown that in a significant proportion of myeloid
and lymphoid acute leukemias, transducers of the signal originated from
IL-3R/GM-CSFR, such as Janus kinase 2 (JAK2) and signal transducer and
activator of transcription (Stat5), are constitutively
activated.25,26 On the other hand, studies carried out on
IL-3-dependent cell lines have shown that the overexpression of
IL-3R According to the ensemble of these observations, it seemed of interest
to investigate the pattern and the level of IL-3R Cells
Cell culture
TF-1 cells were grown in RPMI 1640 medium supplemented with 10% FCS and 10 ng/mL GM-CSF. In some experiments TF1 cells were deprived for 24 hours of GM-CSF and then exposed for short periods of time either to IL-3 (100 U/mL) or to GM-CSF (10 ng/mL). Analysis of cell surface antigens Analysis of cell surface antigens was performed by flow cytometry using a FACScan Flow Cytometer (Becton Dickinson, Bedford, MA). The following antibodies to membrane antigens were used: anti-CD3, -CD7, -CD11a, -CD11b, -CD11c, -CD14, -CD18, -CD19, -CD20, -CD21, -CD22, -CD33, -CD34, -CD38, -CD41, -CD61, -CD71, -CD90, -CD117, glycophorin A, and HLA-DR. Cells were labeled with these antibodies and were analyzed as previously reported.31Hematopoietic growth factor receptor expression Phycoerythrin (PE)-labeled anti-IL-3R chain monoclonal
antibody (mAb) clone 9G532 was purchased from PharMingen
(San Diego, CA); PE-labeled anti-c-kit and anti-M-CSFR
chain mAbs were obtained from Immunotech (Marseilles, France); and
purified anti-c-fms, clone 3-4A433 and
anti-IL-3R/GM-CSFR  c (clone 5-16) were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Cells were incubated with optimal
concentrations of the antibodies and were processed for flow cytometry
analysis as described above.
For G-CSF receptor analysis, cells were incubated for 60 minutes at 4°C with PE-labeled G-CSF, washed, and analyzed by flow cytometry. All controls necessary to verify the specificity of each of these reagents are reported.34 Cell sorting Leukemic blasts stained with PE-labeled anti-IL-3R antibody
were sorted according to fluorescence intensity into
IL-3Rdim and IL-3Rbright fractions using
a FACS Vantage (Becton Dickinson).
IL-3 binding and internalization assays Binding reactions were performed in 25-mm × 75-mm polypropylene tubes in RPMI 1640 containing 0.1% bovine serum albumin (BSA, Fraction V; Sigma, Milan, Italy). Cell concentrations were 10 × 106 cells/mL. Cells were incubated for 60 minutes at 4°C in the presence of 20 ng/mL sodium iodide I 125 IL-3 (125I-IL-3; Amersham Italia, Milan, Italy). Unbound ligand was removed by centrifugation of the cells through a density cushion, as described previously.35 After incubation, 200-µL aliquots of the cell suspension were layered over 150 µL dibutyl phthalate and dinonyl phtalate (Merck, Darmstadt, Germany) mixture, to a final density to 1.0125, in 400-µL plastic microcentrifuge tubes and were centrifuged in an Eppendorf (Milan, Italy) microcentrifuge (10 000g for 2 minutes). At the end of centrifugation, the supernatant and most of the dibutyl phtalate cushion were aspirated. Vial tips containing the cell pellet were then cut off with a scalpel and were transferred to plastic vials, and radioactivity was measured in a counter. Total binding
corresponded to the radioactivity in the cell pellet when cells were
incubated with 125I-IL-3 alone. Nonspecific binding was
represented by the radioactivity bound to the cells in the presence of
radioactive IL-3 (20 ng/mL) and cold IL-3 (20 µg/mL). Specific
binding was the difference between total and nonspecific binding.
IL-3 internalization on intact cells was evaluated as described
previously.36 Cells were incubated for 90 minutes at 4°C in RPMI 1640 medium with 10 ng/mL 125I-IL-3, rinsed twice
at 4°C in phosphate-buffered saline (PBS), and incubated in RPMI 1640 medium at 37°C. At different times, aliquots of cells were removed
and processed as follows: (1) cells were first centrifuged (2 minutes,
3000 rpm, 4°C), and the radioactivity present in the supernatant was
counted; (2) cell pellet was incubated for 2 minutes at 4°C with
saline acid solution (pH 3), a procedure that allows detachment of
surface-bound IL-336; and (3) cells were then centrifuged,
and the radioactivity present in the supernatant and cell pellet was
counted in a Evaluation of apoptosis Apoptosis of leukemic cells was evaluated by double staining with fluorescein isothiocyanate (FITC)-labeled annexin V and propidium iodide (PI).37 Briefly, 2 × 104 cells were washed twice in cold PBS and were resuspended in 0.25 mL binding buffer (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline solution supplemented with 0.25 mM CaCl2). Five microliters FITC-annexin V and 5 µL PI reagents were added to the cells, and the mixtures were gently vortexed and incubated for 15 minutes at room temperature in the dark. Within 1 hour, cells were analyzed at 488 nm in a FACS Sort Cytometer (Becton Dickinson).Cell-cycle analysis Cell-cycle analysis was carried out on nuclei stained with PI, as described.38 Briefly, the cells were first washed in Ca++- and Mg2+-free PBS and were fixed overnight in cold 90% ethanol; after 3 washings, the cells were resuspended in PBS containing 1% BSA, 50 µg/mL PI, and 1 mg/mL boiled ribonuclease A (RNase A; Sigma). Cells were then analyzed by flow cytometry using a FACS scan equipped with software for cell-cycle analysis.Western blotting Cell samples were lysed at a concentration of approximately 1 × 107 cells/mL in cell lysis buffer (20 mM HEPES, pH 7.20, 50 mM NaCl, 10 mM EDTA [ethylenediaminetetraacetic acid], 2 mM EGTA [ethyleneglycoltetraacetic acid], 0.5% nonidet P-40 [NP40], 0.5 mM phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL, 0.5 mM dithiothreitol [DTT], 10 mM Na2MoO4 · H2O, 10 mM Na3VO4, 100 mM NaF), incubated for 20 minutes on ice, and centrifuged at 10 000 rpm for 10 minutes to remove cell debris. The resultant protein lysate was then aliquoted and stored at 80°C until analysis. For immunoblot analysis, lysates were first adjusted to contain equal amounts of proteins (4 µg total proteins), using the Bradford assay and were then boiled for 5 minutes in an equal
volume of sodium dodecyl sulfate (SDS) sample buffer before loading on
a 7.5% SDS polyacrylamide gel. Proteins were then transferred onto a
nitrocellulose membrane (Amersham Life Sciences, Arlington Heights,
IL). Filters were blocked with 5% low-fat milk and incubated overnight
with antibodies against human Stat5 or phosphorylated Stat5 A/B
(Upstate Biotechnology, Lake Placid, NY) diluted 1:1000 (1 µg/mL
final concentration). Western blots were developed using horseradish
peroxidase-conjugated secondary antibody goat antirabbit or
antimouse immunoglobulin (1:3000 dilution; Bio-Rad, Hercules, CA) and
enhanced chemiluminescence (Amersham) according to the
manufacturer's protocol.
DNA electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) experiments were performed on total cell extracts, as previously described.39 Where indicated, 1 µL anti-Stat5a-b (Santa Cruz Biotechnology) was added to 20 µg cell extract in the reaction mixture. Analysis of DNA-protein complexes was carried out on 6% polyacrylamide gels with 0.5× TBE (1× TBE is 50 mM Tris-borate [pH 8.2], 1 M EDTA), as previously described.39 The oligonucleotide probe used was casein Stat binding element
(SBE) 5'-AGATTTCTAGGAATTCAATCC-3'.
RT-PCR analysis of IL-3R chain was evaluated by
RT-PCR using primers and conditions previously reported.34
2-Microglobulin (2-M) mRNA levels were used for the normalization
of RNA.34
Clinical outcome Response to therapy was evaluated in 34 AML patients receiving intensive chemotherapy; patients with acute promyelocytic leukemia (APL) and elderly patients with AML who received only palliative or supportive therapy were not included in this analysis. Intensive therapy consisted of an induction phase with anthracycline and cytarabine with or without etoposide, followed by one cycle of consolidation using the same drugs and, in patients younger than 60 years, by autologous or allogeneic bone marrow transplantation.Statistical analysis Patient characteristics and complete remission rates were compared using the 2 analysis. Overall survival time was
calculated from the rate of diagnosis until death or last follow-up
examinations. Survival curves were estimated using the product-limit
method of Kaplan-Meier and were compared using the log-rank square test.
IL-3R and c chain
expression in leukemic blasts derived from 111 patients with acute leukemia, classified according to FAB and immunophenotypic criteria (79 AML, 25 B-ALL, and 7 T-ALL). Among 79 patients with AML, IL-3R chain
was constantly expressed on more than 60% of leukemic blasts (Figure
1). In acute lymphoid leukemias, a more
heterogeneous situation is foundin all patients with B-ALL, IL-3R
chain was expressed, whereas in most patients with T-ALL, this receptor chain was only scarcely expressed (Figure 1). Analysis of the fluorescence intensity labeling of IL-3R chain provided some interesting findings (Figure 1): (1) normal HPCs exhibited positivity ranging from 50 to 70 (data expressed in arbitrary units of
fluorescence intensity); (2) approximately 46% of patients with AML
exhibited fluorescence intensity values significantly higher than those of normal HPCs, and the remaining patients had values in the range of
healthy controls; (3) approximately 40% of patients with B-ALL had
fluorescence intensity values distinctly higher than those observed for
normal HPCs. Among AML patients, the frequency of occurrence of
different leukemia subtypes (M0 to M7) was similar in the group of
patients with normal IL-3R fluorescence intensity values (up to 100)
compared with those with high IL-3R fluorescence intensity values
(more than 100) (data not shown).
We evaluated also the levels of mRNA encoding IL-3R
In parallel, we have evaluated in the same patients the expression of
GM-CSFR Finally, we evaluated the expression of IL-3/GM-CSFRs
Interestingly, the expression of receptors with a more limited spectrum of biological activity, such as the M-CSFR, was restricted to AML (data not shown). Similarly, c-kit expression was observed in most (71%) patients with AML, but relatively few patients with B-ALL (7%) expressed this membrane receptor (data not shown). In AML patients a possible correlation between these parameters was
evaluated. No significant correlation was observed between IL-3R Leukemic blasts do not down-modulate IL-3R observed in
approximately 45% of AML patients, we evaluated whether incubation of AML blasts with exogenous IL-3 down-modulated the surface IL-3R chain. Control experiments performed on purified HPCs (Figure 4A, B) and in TF-1 erythroleukemic cells
(data not shown) showed that incubation with IL-3 (100 U/mL) leads to a
marked and rapid down-modulation of surface IL-3R chain. In
contrast, leukemic blasts showed only a moderate IL-3R chain
down-modulation; more particularly, in the first hours following the
addition of IL-3, leukemic blasts did not modify IL-3R chain
expression (Figure 4A), but in the days following ligand addition, a
moderate decline in IL-3R chain was observed (Figure 4B). Similarly,
leukemic blasts failed to down-modulate GM-CSFR chain following
incubation with GM-CSF (data not shown).
To determine whether the lack of IL-3R down-modulation observed in
leukemic cells could be attributed to defective internalization, specific experiments were performed in which the cells were first incubated at 4°C with 125I-IL-3 and then were shifted at
37°C to allow the internalization of surface-bound IL-3. These
experiments showed that leukemic blasts and TF-1 cells (used as a
positive control) are able to bind with high affinity and to
internalize IL-3 with comparable efficiency (Figure
5). This observation indicates that the
deficient IL-3R
Stat5 expression and activation in AML To evaluate whether the differences in IL-3R expression in AML
correlate with differences in the activation of Stat5, the key
transcription factor in IL-3 signaling, EMSA and Western blot analysis
were performed. Total cell extracts were prepared from patients with
AML who displayed normal IL-3R chain levels and from patients with
AML who had elevated IL-3R chain levels, treated or not treated with
IL-3. Stat5 activation was evaluated with EMSA using a labeled,
double-stranded oligonucleotide corresponding to the SBE present in the
-casein gene promoter (the sequence is indicated in "Materials and
methods"). Composition of the specific complex observed was
determined by supershift analysis with antibodies recognizing Stat5.
The results of this analysis indicate that the extent of specific Stat5
activation correlates with the level of IL-3R chain expression:
patients displaying low or normal IL-3R expression exhibited after
IL-3 stimulation a level of Stat5 activation distinctly lower than that
observed in patients with high IL-3R levels (Figure
6A). Constitutive Stat5 activation was
observed in only 2 of 15 patients studied; interestingly, the 2 patients with constitutive Stat5 activation were part of the group of
AML patients with elevated IL-3R expression. Western blot analysis
(Figure 6B), performed with anti-Stat5 phosphotyrosine antibody,
confirmed the results obtained using EMSA.
Leukemic blasts with elevated expression of IL-3R chain
observed in a significant proportion of acute leukemias may offer a
growth advantage to leukemic blasts, we have sorted within the leukemic
population, present in each patient, cells displaying strong and low
reactivity with anti-IL-3R chain mAb. We then determined
immediately after sorting the proportion of cycling cells within the
IL-3Rbright and IL-3Rdim populations.
This analysis, performed in 12 AML patients, showed that
IL-3Rbright blasts displayed a significantly higher
proportion of cycling cells (Figure 7A)
than IL-3Rdim blasts (8.6 ± 2.8 vs 2.08 ± 1.6;
P < .001).
Interestingly, Stat5 DNA-binding activity was preferentially observed
in the IL-3R In some patients we evaluated whether IL-3R
We also evaluated the membrane phenotype of IL-3R
IL-3R chain are cycling (see above), it was of interest to evaluate a possible correlation between IL-3R levels and number of leukemic blasts detected at diagnosis. This analysis showed that a good correlation exists between
IL-3R levels and the number of leukemic blasts at diagnosis among AML patients (P < .001), suggesting a role for
IL-3R in leukemic blast proliferation/survival.
This observation prompted us to evaluate whether the level of IL-3R
Leukemogenesis and, more generally, tumorigenesis are considered multistep processes by which a sequence of transformation events progressively modifies the capacity of hemopoietic progenitors to proliferate, survive, and differentiate. Recent studies have shown that several molecular mechanisms are responsible for the autonomous proliferation and increased survival of leukemic cells.40 Acute leukemia cells are known to respond to exogenous HGFs, showing an induction of cell proliferation uncoupled to the induction of cell differentiation.6 Studies have shown that in most patients, however, leukemic cells are endowed with a consistent capacity of autonomous proliferation.41 The level of autonomous proliferation represents a major determinant of prognosis in AML in that a high rate of autonomous proliferation is associated with a highly aggressive phenotype and a poor prognosis.41 Several lines of evidence suggest that the autonomous proliferation of leukemic blasts may be related to autocrine mechanisms of HGF production6 or to constitutive activation of the signal transduction machinery triggered by HGFR.42,43 Furthermore, other studies have shown that AML patients, whose leukemic cells have a positive proliferation response to IL-3 and Kit ligand (KL), have poorer outcomes that result in lower remission rates and shorter survival times.44 In the present study we have investigated the level of IL-3R The mechanisms through which elevated IL-3R The mechanisms underlying the elevated IL-3R Our results also suggest that the elevated IL-3R
We thank M. Blasi, M. Fontana, and A. Zito for editorial assistance.
Submitted March 19, 2002; ac | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
in acute myelogenous leukemia is
associated with enhanced blast proliferation, increased
cellularity, and poor prognosis
Top
Abstract
Introduction
,
