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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 440-446
RAPID COMMUNICATION
Deletion of a Critical Internalization Domain in the G-CSFR in Acute
Myelogenous Leukemia Preceded by Severe Congenital Neutropenia
By
Melissa G. Hunter and
Belinda R. Avalos
From the Molecular, Cellular, and Developmental Biology Program, Bone
Marrow Transplantation Program, The Ohio State University, Arthur G. James Cancer Hospital and Research Institute, Columbus, OH.
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ABSTRACT |
Acquired mutations in the granulocyte colony-stimulating factor
receptor (G-CSFR) occur in a subset of patients with severe congenital
neutropenia (SCN) who develop acute myelogenous leukemia (AML). These
mutations affect one allele and result in hyperproliferative responses
to G-CSF, presumably through a dominant-negative mechanism. Here we
show that a critical domain in the G-CSFR that mediates ligand
internalization is deleted in mutant G-CSFR forms from patients with
SCN/AML. Deletion of this domain results in impaired ligand
internalization, defective receptor downmodulation, and enhanced growth
signaling. These results explain the molecular basis for G-CSFR
mutations in the pathogenesis of the dominant-negative phenotype and
hypersensitivity to G-CSF in SCN/AML.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE GRANULOCYTE colony-stimulating factor
receptor (G-CSFR) is a major regulator of in vivo
granulopoiesis.1 Mice deficient in G-CSFR expression due to
a targeted null mutation in the G-CSFR gene have chronic neutropenia
and exhibit reduced numbers of marrow progenitors and impaired terminal
granulocytic differentiation.2 These observations have led
to speculation that defects in the G-CSFR may contribute to disorders
of granulopoiesis.
The G-CSFR is a member of the Type-I cytokine receptor family and
exists as a single-chain molecule that dimerizes on ligand binding to
form high-affinity receptor complexes.3-5 Distinct functional domains have been identified in the cytoplasmic portion of
the human G-CSFR.6 The membrane-proximal cytoplasmic region of 53 amino acids generates mitogenic signals, whereas the distal carboxy-terminal 98 amino acids are required for transduction of
myeloid maturation signals.7-9
Acquired mutations in the G-CSFR gene have recently been reported in a
subset of patients with severe congenital neutropenia (SCN) progressing
to acute myelogenous leukemia (AML).10-14 A direct relationship between the occurrence of these mutations and leukemic progression in patients with SCN has been suggested. These mutations localize to a critical region in the cytoplasmic domain spanning nts
2384-2429 and all have been nonsense mutations that introduce a
premature stop codon leading to truncation of the distal cytoplasmic tail of the G-CSFR that is critical for maturation and growth arrest
signaling. In the cases studied, the mutations have been found to
affect cells of the myeloid lineage only. Both mutated and normal
alleles of the G-CSFR are expressed in these patients. Interestingly,
coexpression of wild-type (WT) and mutant G-CSFR forms in transfected
myeloid cell lines to mimic the in vivo situation in patients with
SCN/AML, interferes with terminal maturation by the WT receptor and
leads to hyperproliferative responses to G-CSF through a presumed
dominant-negative mechanism.8,10,11
The mechanisms promoting the dominant-negative phenotype in SCN/AML
have remained elusive. Ligand binding in cells coexpressing both mutant
and WT G-CSFR forms should induce mutant receptor homodimerization and
mutant/WT receptor heterodimerization, as well as the formation of
functionally active WT receptor homodimers. However, a defect in
receptor processing or degradation leading to prolonged expression of
mutant G-CSFR forms could lead to preferential formation of dimeric
receptor complexes containing mutant receptor forms and produce a
dominant-negative phenotype. To evaluate this possibility, we examined
ligand internalization, receptor processing, and receptor degradation
in cells expressing WT and mutant G-CSFR forms.
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MATERIALS AND METHODS |
Reagents.
Reagents for maintenance of cell lines were purchased from GIBCO-BRL
(Grand Island, NY). [I125] G-CSF (>800 ci/mmol) and
Pro-mix [35S] in vivo cell labeling mix (>1000 ci/mmol)
were obtained from Amersham (Arlington Heights, IL).
[Methyl-3H] thymidine (82 ci/mmol) was from DuPont NEN
(Wilmington, DE). All other reagents were obtained from Sigma (St
Louis, MO) unless otherwise indicated.
DNA constructs.
For the construction of pCDM8-WT, the wild-type human Class-I G-CSFR
cDNA (generously provided by Dr A. Larsen, Seattle, WA) was excised
from pBluescript SK+, ligated to Bst XI linkers and inserted
into the Bst XI site of pCDM8. The p309 plasmid containing the
neomycin resistance gene was cotransfected with the G-CSFR cDNA plasmid
to establish stable clones as previously described.15 The
pCDM8 vector was a gift from Dr B. Seed (Cambridge, MA) and the p309
vector was generously provided by Dr J. Lang (Columbus, OH). The G-CSFR
 716 clone was generated by introducing a stop codon with a C to T
point mutation at nt 2384 of the WT cDNA by PCR. The oligonucleotides
used to generate this mutant were: forward primer F1 (5 -CCACC
TAGCCCCAATCCCAGTCTGGC-3), and reverse R4 (5-GATCGCTGGTGCCAGACTGGGATTGGGGCTAGG-3); the
underlined nucleotides indicate the position of the point mutations. In
the first PCR reaction, primer F2 containing a 5 restriction
site for BamHI and corresponding to nts 2257-2273 of the WT
cDNA was used in conjunction with the R4 primer. In the second PCR
reaction, primer F1 was used with primer R2 that was designed to
contain an XhoI restriction site and corresponded to nts
2584-2601 of the WT cDNA. The extension products from both reactions
were ligated and further amplified using primers F2 and R2. The PCR
fragment was subcloned into pBluescript SK+, excised from the plasmid
by Cfr 10 I and Bst E II digestion, and ligated into
the Cfr 10 I and Bst E II sites of pCDM8-WT. DNA
sequencing was done to confirm the introduced point mutation.
Transfections and cell culture.
The pro-B Ba/F3 cell line was maintained in RPMI 1640 with 10% fetal
bovine serum (FBS) and 10% WEHI-3 conditioned media. Ba/F3 cells were
stably transfected with the WT and 716 G-CSFR forms, as previously
described.15 Clones were selected in G418-containing media.
Neomycin-resistant clones were examined for binding of 125I-G-CSF and the binding data analyzed by Scatchard
analysis as previously described.15 Six WT and four 716
G-CSFR clones expressing similar receptor numbers and binding
affinities were selected from a total of 32 and 11 clones,
respectively, for use in all subsequent experiments. G-CSFR expression
was confirmed by RT-PCR and DNA sequencing. COS-7 cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS
and were transiently transfected by calcium phosphate
precipitation of plasmid DNA.16 An equivalent amount of
plasmid DNA was used for single transfections in COS-7 cells. A 1:1
molar ratio of pCDM8-WT and pCDM8-716 was used for cotransfections
in COS-7 cells.
Binding assays and competition studies.
Binding of [I125] G-CSF to transfected Ba/F3 cells was
assayed as previously described.15 Binding of
[I125] G-CSF to transfected COS-7 cells (plated at 5 × 104 cells/well ) was performed 72 hours after
transfection of the cells in 12-well plates. Binding of
[I125] G-CSF was examined in the absence and presence of
varying concentrations of unlabeled G-CSF (0.1nmol/L to 1000nmol/L).
Incubations were for 4 hours at 4°C, after which the cells were
washed twice with ice-cold phosphate-buffered saline (PBS) and then
lysed with 1 mol/L NaOH. Equivalent aliquots (400µL) from each well
were counted in a gamma counter to determine bound [I125]
G-CSF. Receptor numbers and binding affinities were calculated using
the Ligand computer program.17 Percent competition was calculated as described by Dittrich et al.18,19
Proliferation studies.
Stably transfected Ba/F3 cells were serum and cytokine deprived for 2 to 4 hours in RPMI 1640, 0.1% bovine serum albumin (BSA), and 2mmol/L
glutamine. The cells were washed once in PBS and resuspended at 1 × 105/mL in RPMI 1640 media containing 10% FBS and
2mmol/L glutamine. A total of 5 × 103 cells/well were
seeded in 96-well microtiter plates with varying concentrations of
G-CSF (0.002-2000 pmol/L). Duplicate plates were also set up in the
presence of IL-3 without G-CSF. The plates were incubated for a total
of 72 hours at 37°C in 5% CO2, and pulsed with 0.5 µCi/well of [methyl-3H] thymidine for the last 8 hours
of incubation. Samples were harvested onto glass fiber filters and
counted in scintillation fluid.
Internalization studies.
Cells were incubated with 500pmol/L [I125] G-CSF for 2 hours at 4°C. Internalization was examined by temperature shifting
to 37°C for varying times from 30 to 360 minutes. After incubation at 37°C, the cells were incubated for an additional 2 hours at 4°C. Unbound ligand was removed from the cells with cold PBS
containing 1mmol/L MgCl2, 0.1mmol/L CaCl2, and
0.2% BSA. Surface-bound [I125] G-CSF was determined
after the addition of 0.5 mol/L NaCl (pH1.0) for 3 minutes. The acid
strip solution and wash were both collected to determine bound ligand.
Internalized ligand was quantified by lysis of the cells with 1 mol/L
NaOH for 1 minute. The data were expressed as percent receptor
internalization over time.18,19
Receptor degradation.
Confluent monolayers of transfected COS-7 cells grown in T-75 flasks
were incubated with short-term labeling media (RPMI 1640 media
containing 10% dialyzed FBS without methionine and cysteine) for 15 minutes at 37°C to deplete intracellular pools of methionine and
cysteine. The cells were then metabolically labeled with
[35S] Cysteine/Methionine Pro-mix at 0.15mCi/mL in
short-term labeling media for 1 hour at 37°C as previously
described.18-20 The cells were washed with incubation media
(RPMI 1640 containing 10% FBS, 1 nmol/L G-CSF, and unlabeled
methionine and cysteine) and incubated for varying times, then washed
once in PBS, scraped, and pelleted. The cell pellets were lysed in
buffer containing 1% NP-40 (Boehringer Mannheim Biochemical,
Indianapolis, IN), 1 mmol/L EDTA (pH 8.0), 20 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 0.15 U/mL aprotinin, 10 µg/mL leupeptin, 10 µg/mL
pepstatin A, and 1 mmol/L sodium vanadate, then cleared of insoluble
material. Lysates were precleared with protein A sepharose (Pharmacia
Biotech, Piscataway, NJ), immunoprecipitated with 8 µg anti-G-CSFR
antibody recognizing the N-terminal portion of the G-CSFR (Pharmingen,
San Diego, CA), and analyzed under reducing conditions by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gels
were treated with Entensify (DuPont-NEN) according to the
manufacturer's instructions and dried. The labeled proteins were
visualized by autoradiography.
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RESULTS |
Similar binding affinities of WT and mutant G-CSFR forms.
To study the role of the distal portion of the G-CSFR deleted in
patients with AML preceded by SCN in the process of internalization, we
constructed a deletion mutant designated 716 by introducing a
premature stop codon through a C-to-T substitution at nt 2384 of the WT
human (Class I) G-CSFR cDNA. This mutation deletes the most distal 98 carboxy-terminal amino acids of the G-CSFR and is the most frequent
mutation that has been identified in patients with
SCN/AML.10-14 Both the WT and 716 G-CSFR forms
retain the conserved box 1 and box 2 regions that are essential for
proliferation.
We initially examined the binding properties of the WT and 716
receptor forms stably transfected in Ba/F3 cells or transiently expressed in COS-7 cells. For COS-7 transfectants, studies were performed 3 days after the cells were transfected when receptor expression was maximal (data not shown). Cells were incubated with
500pmol/L [I125] G-CSF for 4 hours at 4°C in the
presence of increasing amounts of unlabeled ligand. Scatchard analysis
of the equilibrium-binding data showed expression of a single class of
high-affinity receptors on the surface of both Ba/F3 and COS-7
transfected cells (data not shown). 716 receptors displayed similar
binding affinities as the WT receptor. Dissociation constants for both
G-CSFR forms were in the range of 4.2 to 61.0 × 10-11
mol/L (Table 1). A 100-fold higher level of
receptor expression was consistently observed in COS-7 cells making the
COS-7 transfectants an ideal system for further studying receptor
expression and processing. The reasons for the higher level of receptor
expression on COS-7 cells are not clear, although other investigators
have made similar observations with the G-CSFR and other cytokine
receptors using COS-7 cells.4,18,21
Hyperproliferative responses in cells expressing 716 receptors.
The mitogenic capacities of the WT and 716 G-CSFR forms in response
to G-CSF were examined in stably transfected Ba/F3 cells (Fig 1). Ba/F3 cells transfected with
either the WT or the 716 mutant responded to G-CSF in a
dose-dependent fashion. Notably, the 716 transfectants responded to
a 100-fold lower concentration of G-CSF than WT transfectants. The
shift to the left in the dose-response curve for 716 transfectants
indicates that cells expressing this receptor form are hypersensitive
to G-CSF, an observation also reported by other
investigators.8,10
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| Fig 1.
Proliferative responses of G-CSFR transfectants. Ba/F3
cells stably transfected with the WT or 716 G-CSFR were serum and
cytokine deprived for 4 hours before cytokine stimulation. Cell
proliferation was assayed in the presence of G-CSF or IL-3. The data
are expressed as the percent [methyl-3H] thymidine uptake
in cells grown in G-CSF versus maximal growth in IL-3 containing media.
Error bars represent the standard deviation from three independent
experiments.
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An internalization domain in the distal region of the G-CSFR is
deleted in the 716 receptor.
We next examined ligand internalization over time in COS-7 and Ba/F3
cells transfected with the WT receptor alone or the 716 receptor
alone. Because G-CSFR mutations in patients with SCN/AML affect only
one allele, we also examined ligand internalization in
cells expressing both the WT and 716 receptors
(WT/716 ) to mimic the in vivo situation. Cells were incubated with
500 pmol/L [I125] G-CSF for 2 hours at 4°C. The
temperature was shifted to 37°C for different times after
which the cells were returned to ice for another 2 hours.
Surface-bound G-CSF was eluted by high salt/low pH incubation for
3 minutes, and internalized G-CSF was determined after solubilization
of the cells in 1mol/L NaOH.
As shown in Fig 2, internalization of G-CSF
in COS-7 cells was apparent within 30 minutes in WT transfectants, and
by 2 hours surface binding was nearly undetectable indicating rapid
downregulation of WT receptors (left panel). However, ligand
internalization was delayed in 716 transfectants and significant
amounts of surface binding of G-CSF could still be detected at 6 hours
(middle panel). The detection of persistent surface binding beyond 2 hours suggests a defect in receptor downregulation in 716
transfectants. Nearly identical results as observed with 716
transfectants were obtained with COS-7 cells coexpressing both the WT
and 716 receptors (WT/716), as would be expected if the 716
mutant conferred a dominant-negative phenotype. Ligand internalization
was also examined in Ba/F3 transfectants and similar results were
obtained as observed with COS-7 transfectants (data not shown).
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| Fig 2.
Internalization of surface-bound [125I]
G-CSF by WT and mutant  716 G-CSFR complexes. Three days after
transfection COS-7 cells were washed, incubated with 500 pmol/L
[125I]-G-CSF for 2 hours at 4°C, then shifted to
37°C for the indicated times. Cells were returned to ice again for
2 hours, then washed to eliminate unbound ligand. Surface-bound ligand
( ) was determined after acid stripping in 0.5 mol/L NaCl/HCl (pH1.0)
for 3 minutes. Internalized ligand ( ) was measured after lysis of
the cells in 1 mol/L NaOH. Data are expressed as a percentage of
initial binding of G-CSF at 4°C. Values represent the mean of
duplicate points. The data shown are from three independent
experiments. Standard deviations are indicated by error bars.
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Delayed degradation of the 716 receptor.
To determine whether altered receptor degradation could account for the
defects observed in receptor modulation in 716 transfectants, we
examined receptor processing and degradation in pulse-chase experiments
with COS-7 transfectants. Three days after transfection, COS-7 cells
expressing either the WT or 716 receptors alone, or coexpressing
both the WT and 716 receptor forms (WT/716) were metabolically
labeled with [35S] methionine and [35S]
cysteine for 1 hour. The cells were then chased with 1 nmol/L G-CSF in
DMEM containing unlabeled methionine and cysteine for 0 to 8 hours. The
cells were lysed, immunoprecipitated with an antibody recognizing the
amino-terminus of the G-CSFR, and analyzed by SDS-PAGE. In all of the
transfectants an upward shift in molecular weight of the receptor was
observed by 1 hour. This suggests that the receptor is glycosylated to
a more mature form of 150 kD for the WT receptor and 127 kD for the
truncated 716 receptor (Fig 3A). In the
presence of G-CSF, the mature WT receptor (left panel, arrow) was
largely degraded by 4 hours. In contrast, even at 8 hours there was
little evidence of degradation of the mature 716 receptor (middle
panel), consistent with delayed receptor degradation in cells
expressing the mutant 716 G-CSFR form. In COS-7 cells coexpressing
both receptor forms (WT/716), degradation of each receptor form was
similar to that observed in corresponding single transfectants (right
panel). To account for the differences in intensity of labeled bands
between blots, the blots were individually subjected to densitometry
with standardization at time 0 and the fold-change in intensity
calculated (Fig 3B). Densitometric analysis further confirmed delayed
receptor degradation in 716 transfectants.
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| Fig 3.
G-CSF stimulated degradation of the wild-type (WT) and
716 mutant G-CSFR. (A) Confluent monolayers of COS-7 cells,
transfected with the wild-type (WT) G-CSFR alone, mutant 716
receptor alone, or cotransfected with both the WT and 716 receptors
(WT/716), were incubated for 1 hour in DMEM containing
[35S] methionine and [35S] cysteine.
Thereafter, the cells were incubated in DMEM containing 1nmol/L G-CSF
and unlabeled methionine and cysteine for 0 to 8 hours. The cells were
lysed, immunoprecipitated with antibody to the aminoterminus of the
G-CSFR, and analyzed by SDS-PAGE followed by autoradiography. Both
receptor forms are glycosylated to more mature forms (arrows). (B) The
autoradiographs from (A) were analyzed by densitometry. Data are
expressed as the fold-change compared to time 0.
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DISCUSSION |
SCN or Kostmann's syndrome is a disorder of granulopoiesis
characterized by an absolute neutrophil count of 200 to 500/µL, a
maturation arrest of neutrophil precursors in the bone marrow at the
promyelocyte/myelocyte stage of differentiation, and frequent severe
bacterial infections.22,23 The disease may be inherited in
an autosomal recessive manner or occur sporadically. In the majority of
patients with SCN, treatment with pharmacologic doses of G-CSF improves
peripheral neutrophil counts and reduces infection-related events.24,25 However, approximately 10% to 15% of
patients with SCN develop AML.13,14
The molecular mechanisms underlying SCN remain unknown. Defects in
G-CSF production or G-CSFR expression do not appear to play a role in
the pathogenesis of SCN. Serum G-CSF levels are usually high in
patients with SCN and G-CSFR numbers are normal to increased with
normal binding affinities.26-29 Despite elevated serum
G-CSF levels in most patients with SCN, pharmacologic doses of G-CSF
generally restore in vivo granulopoiesis.24,28 GM-CSF treatment, however, is usually ineffective.30 These
observations have led to the suggestion that a defect in G-CSFR signal
transduction may be involved in the pathogenesis of SCN.
In the subset of patients with SCN progressing to AML, acquired
mutations in the G-CSFR have been detected in the patients examined.
These mutations affect one allele and delete the maturation signaling
domain of the G-CSFR.11-14 Enforced expression of these mutations in myeloid cell lines in vitro7,8 and in cells from knock-in mice in vivo results in increased sensitivity to G-CSF
through a dominant-negative mechanism. Both the number and size of
colonies grown in the presence of G-CSF have been found to be increased
in knock-in mice in colony-forming assays.31,32 These
observations have led to the hypothesis that G-CSFR mutations are
involved in the pathogenesis of AML arising from SCN.
We considered the possibility that cells from patients with SCN/AML may
have a defect in processing or degradation of the G-CSFR leading to the
dominant-negative phenotype. Aberrant receptor processing has been
shown to play a role in the pathophysiology of several human diseases
including Laron dwarfism, type-A insulin resistance, and familial
hypercholesterolemia.33-35 Mutations in the erythropoietin
receptor, another member of the cytokine receptor superfamily, have
recently been identified in patients with familial polycythemia, and
these mutations confer a dominant-negative phenotype.36
We show that ligand internalization and downmodulation of receptor
expression are defective in cells coexpressing WT and mutant G-CSFR
forms as observed in patients with SCN/AML. Delayed receptor degradation results in prolonged expression of mutant G-CSFR forms and
the formation of dimeric receptor complexes containing mutant receptors. Enhanced expression of mutant receptor complexes leads to
the dominant-negative phenotype.
A role for phosphatidylinositol 3-kinase (PI3-K) in ligand
internalization and receptor downmodulation was recently reported for
receptors for CSF-1 and PDGF. Attenuated rates of internalization and
degradation were observed with CSF-1 and PDGF receptor mutants that
failed to bind PI3-K.37,38 In the case of the G-CSFR, we
have previously shown that activation of PI3-K requires the region
spanning residues 682 to 716. We showed that 716 cells could
activate PI3-K in response to G-CSF stimulation.39 Thus, similar to the Flt3/Flk2 receptor, PI3-K does not appear to be required
for internalization and downmodulation of the G-CSFR.40
A dileucine motif corresponding to the amino acid sequence STQPLL was
previously identified in the box 3 region of gp130 and was shown to
mediate interleukin-6 (IL-6) internalization and downregulation of the
IL-6 receptor (IL-6R).18,19,41 An identical dileucine motif
is present in the distal tail of the WT G-CSFR but is deleted in G-CSFR
forms from patients with SCN/AML such as the 716 mutant
(Fig 4). Because the G-CSFR shows
significant homology to gp130 and contains the identical dileucine
motif in its conserved box 3 region, we speculate that this motif also mediates internalization of G-CSF and modulates receptor expression. Additional studies are in progress to directly test the role of the
dileucine motif in ligand internalization and receptor modulation using
G-CSFR forms with targeted mutations in the dileucine motif.
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| Fig 4.
Schematic diagram of dileucine internalization motif. The
extracellular (EX), transmembrane (TM) and intracellular (ID) domains
are indicated for gp130 and the wild-type (WT) and mutant 716 G-CSFR
forms. A dileucine motif (underlined) that modulates ligand
internalization and receptor expression localizes to the distal region
of box 3 in gp130. An identical motif is present in the WT G-CSFR
(underlined). Mutations in the critical region in patients with SCN/AML
as in the 716 mutation result in the introduction of a premature
stop codon (STOP) and deletion of the carboxy-terminal tail of the
G-CSFR (diagonal lines) within which lies the dileucine motif
(underlined).
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We are investigating the specific signaling molecules affected to
explain the hyperproliferative response observed with 716 cells. We
recently showed that the distal tail of the G-CSFR that is deleted in
716 mutants is required for recruitment of the negative growth
regulator SH2-containing inositol phosphatase (SHIP).39 Recruitment of SHIP was found to
correlate with decreased proliferative responses. Thus, loss of the
domain required for SHIP recruitment and downregulation of
proliferative responses seems a likely explanation for the enhanced
growth of 716 cells.
Mutant receptor forms from patients with SCN/AML also delete the
maturation signaling domain. These receptors fail to generate differentiation signals but transduce proliferation signals that are
enhanced. Expression of mutant G-CSFR forms in myeloid
progenitors could increase their self-renewal capacity and
predispose to leukemia. Additionally, high-dose G-CSF therapy as used
in patients with SCN could further promote expansion of abnormal clones
harboring G-CSFR mutations.
Our results provide an explanation for the dominant-negative phenotype
observed in SCN/AML and suggest a mechanism by which G-CSFR mutations
result in hypersensitivity to G-CSF. Because the majority of patients
with SCN are treated with G-CSF, continued close monitoring including
molecular studies for G-CSFR mutations will be necessary to determine
whether chronic administration of G-CSF alters the latency or incidence
of AML in these patients.
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FOOTNOTES |
Supported by NCI Grant No. CA75226.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Belinda R. Avalos, MD, The Ohio State
University, Bone Marrow Transplantation Program, A437A Starling-Loving
Hall, 320 W Tenth Ave, Columbus, OH 43210; e-mail:
avalos-1{at}medctr.osu.edu.
| |
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Abstract
Introduction