Journal of Leukocyte Biology BioLegend: Treg, Th17, Stem Cell
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0904488 on February 22, 2005

Published online before print February 22, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0904488v1
77/5/811    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shikama, Y.
Right arrow Articles by Maruyama, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shikama, Y.
Right arrow Articles by Maruyama, Y.
(Journal of Leukocyte Biology. 2005;77:811-819.)
© 2005 by Society for Leukocyte Biology

Accumulation of an intron-retained mRNA for granulocyte macrophage-colony stimulating factor receptor common ß chain in neutrophils of myelodysplastic syndromes

Yayoi Shikama*,{dagger},1, Tsutomu Shichishima{dagger}, Isao Matsuoka*, Paul T. Jubinsky{ddagger}, Colin A. Sieff§ and Yukio Maruyama{dagger}

* Department of Pharmacology and
{dagger} First Department of Internal Medicine, Fukushima Medical University, Japan;
{ddagger} Section of Pediatric Hematology/Oncology, Albert Einstein College of Medicine, Bronx, New York; and
§ Department of Pediatric Oncology and Hematology, Dana-Farber Cancer Institute, Boston, Massachusetts

1 Correspondence: Department of Pharmacology and First Department of Internal Medicine, Fukushima Medical University, Fukushima, 960-1295, Japan. E-mail: yayois{at}fmu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We recently identified a reduction in the neutrophil surface expression of common ß chain (ßc) of the receptor for granulocyte macrophage-colony stimulating factor (GM-CSF) in the patients with myelodysplastic syndromes (MDS). To determine the etiology of the impaired ßc expression, ßc mRNA from neutrophilic granulocytes of MDS patients and healthy controls was analyzed by a combination of direct reverse transcriptiase-polymerase chain reaction-based single-strand conformational polymorphism and sequencing. Nine different ßc transcripts were detected, but none was specific for MDS. However, one of the transcripts (ßc79) containing a 79-base intron insertion between exons V and VI was significantly increased in MDS. This 27-kd isoform consisted of the ßc N-terminal 182 amino acids followed by a new 84-amino-acid sequence. ßc79 was overexpressed in all MDS subtypes. No genomic mutations were detected within the intron or at the intron/exon boundaries. The isoform is predominantly located in the cytoplasm by Western blot analysis and was unable to generate high-affinity binding sites or transduce a signal for proliferation when coexpressed with the receptor for human GM-CSF {alpha} chain. Our study suggests that the accumulation of the abnormal ßc transcripts with intron V retention results in the reduction in cell-surface expression of ßc observed in MDS.

Key Words: MDS • GM-CSF receptor • ßc • splice variant • intron retention


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Granulocyte macrophage-colony stimulating factor (GM-CSF) is a multipotential hematopoietic growth factor that stimulates the proliferation, differentiation, and survival of pluripotent stem cells and myeloid, erythroid, and megakaryocytic progenitors and their progeny [1 2 3 4 ]. It also acts as a potent stimulator of neutrophil phagocytosis [5 ], chemotaxis [6 ], and bactericidal activities [7 , 8 ]. These biological activities of GM-CSF are exerted by its binding to specific receptors on the surfaces of target cells. The receptor for human GM-CSF (GMR), a member of the hematopoietin receptor superfamily [9 ], is composed of an {alpha} chain that binds GM-CSF by itself with low affinity [10 ] and a common ß chain (ßc) that is shared by the interleukin (IL)-3 and IL-5 receptor {alpha} chains [11 ]. Coexpression of the ßc with the {alpha} chains converts the binding affinity generated by the {alpha} chain from low to high, which initiates subsequent signal transduction [12 , 13 ]. The cytoplasmic domain of ßc is required for downstream signal transduction [14 15 16 17 ], while the extracellular domain of ßc is essential for interaction with the {alpha} chain-bound ligand and receptor activation [18 , 19 ]. A number of studies about artificially created ßc mutants have shown that a point mutation in the extracellular or transmembrane domain and a duplication or a deletion of part of the extracellular domain affect receptor functions by altering receptor stoichiometry [20 21 22 ]. Similarly, various types of truncation in the cytoplasmic domain result in incomplete signaling or inactivation of the receptors, regardless of the composition of the receptor complex [14 15 16 17 , 23 , 24 ].

Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by refractory cytopenia, ineffective hematopoiesis, and impaired cellular differentiation [25 26 27 ]. As a consequence of impaired differentiation, mature blood cells have various morphological and functional defects. Although nearly half of the cases with poor prognostic subtypes transform to acute leukemia, the most common causes of death in overall MDS are bacterial infections [28 ] that have been ascribed to impaired functions and decreased numbers of neutrophilic granulocytes [29 ]. Patients with MDS, show impaired responses to GM-CSF, including the chemiluminescence response and production of reactive oxygen species in response to f-Met-Leu-Phe in vitro [30 31 32 33 ].

Administration of high doses of GM-CSF to the patients induces a rapid rise in peripheral blood neutrophil counts resulting from demargination of mature neutrophils, followed by an increase in the cellularity and maturation index of bone marrow as a result of GM-CSF activities on stem cells and progenitors [34 35 36 ]. In contrast, lower doses of GM-CSF sometimes lack a rapid neutrophil increase as a result of demargination, even in the limited number of responders [37 38 39 ], suggesting that mature cells may be more affected in some cases. However, the molecular basis of the poor response of neutrophils to GM-CSF is unknown.

We recently found a neutrophil-specific reduction in the expression of ßc in MDS [40 ]. The levels of decreased expression were correlated with the degree of hypogranular changes in neutrophils, one of the typical morphology alterations in MDS, and were associated with increased susceptibility to bacterial infections, suggesting that impaired expression of ßc may be involved in neutrophil dysplasia in MDS.

To determine if structural abnormalities in ßc cause its impaired cell-surface expression in MDS, sequences of entire ßc-coding mRNAs from neutrophils of MDS patients and healthy controls were analyzed. We observed that mRNA levels of a ßc variant, which retained intron V (ßc79), was significantly increased in MDS compared with controls and that its protein was not stably expressed on the cell surface.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Thirteen patients with MDS were studied after obtaining informed consent and approval from the institutional Human Research Committee. According to the classification by the French-American-British (FAB) group [41 ], five had refractory anemia (RA), two had RA with ringed sideroblasts (RARS), one had chronic myelomonocytic leukemia (CMMoL), three had RA with excess of blasts (RAEB), and two had RAEB in transformation (RAEBt). Patient characteristics and clinical findings are presented in Table 1 . Cases 7 and 9 had cells with defective chromosome 22, where ßc is encoded [42 ]. Two separate blood samples were needed for the studies. Unfortunately, three of the nine patients who provided blood samples (see Fig. 2 ) died (#3 and #9 overt leukemia; #7 sepsis) before the second sample was collected, and Patient #5 was not available. To compensate for this loss, samples from four additional patients (#10–13) were analyzed (see Fig. 3 ). Thirteen age-matched, healthy controls were also studied as controls.


View this table:
[in this window]
[in a new window]
  		Table 1. Patient Characteristics

 


View larger version (74K):
[in this window]
[in a new window]
  		Figure 2. Aberrant ßc transcripts detected by direct RT-PCR-SSCP. cDNA was synthesized from RNA extracted from neutrophils of seven controls and nine MDS patients using oligo-dT primers and was followed by 32 PCR amplification cycles with 12 kinds of IRD 800-labeled primer pairs, 1SA–12SA listed in Table 2 . As controls with correct sequences, intact ßc cDNA was amplified by 20 PCR cycles with the same primers. The PCR products were resolved on the MDE gels. The patient numbers correspond to those in Table 1 . The arrows on the right indicate nine abnormally mobilized bands that were subsequently sequenced.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 3. Comparison of ratios of ßc79 to total ßc transcripts between MDS patients and healthy controls. (a) The positions of the primers used for PCR1 and PCR2. The black bar represents full-length ßc cDNA, and the open bar indicates the 79-base insertion. PCR1 with a sense primer 13S, which extended over the junction of exon V and the insertion, specifically amplified the ßc79. PCR2 amplified total ßc with or without the insertion. (b) Expression of ßc79 and total ßc mRNAs. The upper and lower panels show the 222-bp fragments amplified by 32 PCR1 cycles and the 266-bp fragments by 30 PCR2 cycles, respectively. The patient numbers correspond to those in Table 1 . The ßc79 and intact ßc cDNAs subcloned into vectors were subjected to the same analysis as substitutes for samples in which 100% and 0% of ßc had the insertion, respectively. (c) Ratios of the band intensities of ßc79 to total ßc. The NIH Image program measured the PCR band intensities of each patient, and the ratios of PCR1 to PCR2 were compared between MDS patients and controls. When 100% of ßc was ßc79, the ratio was 1.808 because of higher efficiency of PCR1 than PCR2, as shown by the result with ßc79 cDNA. *, Significant difference; P < 0.001.

 
Cell preparation
Heparinized blood was obtained by venopuncture. Following the removal of erythrocytes by 0.6% dextran (T500, Pharmacia Biotech, Uppsala, Sewden) sedimentation, polymorphonuclear leukocytes were isolated from the 70%/81% Percoll interface by centrifugation at 1000 g for 20 min [43 ]. The morphology of the cell populations was confirmed by examination of cytospins (Shandon Elliott, Runcorn, Cheshire, UK) stained with May-Grünwald and Giemsa solution. Differential cell counts revealed that more than 95% of the cells in the fraction were neutrophilic granulocytes.

Reverse transcriptase (RT)
Total cellular RNA was extracted from polymorphonuclear leukocyte fractions using ISOGEN (Nippongene, Toyama, Japan). Using oligo-dT primers, cDNA was synthesized from the RNA by Superscript RT (Gibco-BRL, Rockville, MD). The cDNA was amplified by TaKaRa Ex Taq DNA polymerase (Takara Shuzo, Otsu, Japan) in a thermal cycler (TaKaRa Shuzo).

Polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP)
Twelve kinds of PCR primer pairs, listed as 1SA–12SA in Table 2 , were labeled with IRD 800 (Aloka Co., Tokyo, Japan) and were used for PCR of reverse-transcribed RNA and intact ßc cDNA, a control with no mutations. Figure 1 indicates the regions amplified with each primer pair in ßc cDNA. The PCR products were denatured at 95°C for 2 min and resolved by electrophoresis in MDE gel (Hydrolink, AT Biochem, Malvern, PA) for 16 h at 650 V in a Li-Cor 4000 automated DNA sequencing system (Aloka Co.). Compared with the positions of the bands derived from intact ßc control, differently mobilized bands were defined as aberrant fragments.


View this table:
[in this window]
[in a new window]
  		Table 2. Primers for Direct PCR-SSCP and Sequencing

 


View larger version (11K):
[in this window]
[in a new window]
  		Figure 1. RT-PCR-SSCP strategy. The boxes with Roman numerals represent exons of ßc. Bars show the positions of the fragments amplified using 12 primer pairs in ßc cDNA. TM, Transmembrane domain.

 
Cloning of full-length ßc-coding region
The full-length ßc-coding cDNA was obtained by PCR with primers 1S and 12A (Table 2) from the reverse-transcribed RNA of patients and controls. The products were subcloned directly into a plasmid vector, pCR4-TOPO (Invitrogen, Carlsbad, CA), using a TA cloning kit (Invitrogen) and transformed into DH5a-T1 cells.

Sequencing of atypical ßc clones and genomic DNA
Five full-length, ßc-containing plasmid clones that generated the same aberrant fragments as detected by the SSCP analysis of reverse-transcribed RNA samples were selected for sequencing. Genomic DNA was extracted using the Generation Capture Column kit (Gentra Systems Inc., Minneapolis, MN) following the manufacturer’s instruction, and the 314-base fragments that include the intron V and flanking exons V and VI were amplified by 28-cycle PCR with primers 14S and 3A for subsequent sequencing. The sequences were determined by the dideoxy chain-termination method using a Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH) and IRD 800-labeled primers (1S–12S for mutated clones and 3A for genomic DNA in Table 2 ) in an automated DNA sequencing system (Aloka Co.).

Semiquantitative PCR
The concentrations of reverse-transcribed mRNA templates were measuered by the band intensities of ß-actin obtained from serially diluted templates by 25-cycle PCR [44 ]. The fragment 10SA within exon XIV, in which no abnormal sequence was detected by SSCP, was amplified by 30-cycle PCR as total ßc transcripts. To obtain similar amounts of amplified 10SA fragments, patients 6, 8, and 13 needed two, four, and 2.5 times more concentrated cDNA than controls, and the same concentration of cDNA as controls was sufficient in the other patients. From the 10SA fragment-adjusted concentrations of template cDNAs, the intron V-retained transcripts were amplified using 3A and an intron-specific primer 13S that extended over the junction of the 3'-end of exon V and the 5'-end of the retained intron. The PCR products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light. The intensities of the bands of PCR products on the gels were measured with the National Institutes of Health (NIH; Bethesda, MD) Image program. The plasmid DNA was isolated using Mo Bio UltraClean 6 Minute Plasmid Prep Kit (Mo Bio Laboratories, Solana Beach, CA).

Cells and cell culture
The IL-3-dependent mouse hematopoietic cell line Ba/F3 was maintained in RPMI 1640 (Invitrogen) supplemented with 5% fetal calf serum (FCS) and 500 pmol/L murine IL-3 (Sigma Chemical Co., St. Louis, MO). It is noted that neither human GM-CSF nor human ßc interacts with the murine IL-3 receptor complex. Cells expressing the GMR {alpha} chain that was transfected along with the neomycin-resistance gene were cultured in the presence of 1 mg/mL G418 (Gibco-BRL). The human embryonic kidney (HEK) cell line was maintained in Dulbecco’s modified Eagle’s medium (Sigma Chemical Co.) supplemented with 5% FCS.

Expression constructs and transfection
The cDNAs encoding intact ßc (a generous gift from Dr. Sumiko Watanabe, University of Tokyo, Japan) or variant were subcloned into pIREShyg2, an expression vector containing the hygromycin-resistance gene (Clontech, Palo Alto, CA). For stable transfection, 20 µg of each subcloned cDNA was introduced into 4 x 106 Ba/F3 cells by electoroporation at 270 V and 975 µF by the Bio-Rad Gene Pulser (Richmond, CA). The electroporated cells were allowed to recover for 2 days in the presense of 500 pmol/L murine IL-3 and were seeded in 96-well tissue-culture plates for selection in 0.3 mg/mL hygromycin. Three independent clones, in which expression of mRNA of each introduced ßc was confirmed by RT-PCR, were selected for a subsequent functional assay. For transient transfection, 4 µg subcloned cDNA of intact or variant ßc was incubated with 25 µL Polyfect transfection reagent (Qiagen, Valenica, CA) at room temperature for 5 min, which was then added to subconfluent HEK cells in 100-mm plates. After 12–16 h, the cells were harvested for Western blot analysis.

In situ hybridization
In situ hybridization was performed as described previously [45 ] with a minor modification. The Ba/F3 transfectants on poly-L-lysine-coated glass slides were fixed in 4% (w/v) paraformaldehyde in PBS. The slides were dehydrated sequentially in 70%, 90%, and 100% ethanol at room temperature for 5 min each and defatted in 100% chloroform for 10 min, followed by dehydration with a series of 100%, 90%, and 70% ethanol. After treatment with 1 µg/mL proteinase K at 37°C for 30 min, the cells were again fixed in 1% paraformaldehyde at room temperature for 30 min. The fixed samples were then prehybridized at 37°C for 30 min in a humid chamber with hybridization buffer containing 50% deionized formamide, 4x saline-sodium citrate (SSC), 10 mM Tris (pH 7.0), 1 mM EDTA, 10 mM dithiothreitol (DTT), 1x Denhardt’s solution (0.02% Ficoll, polyvinylpyrrolidione, 0.02% bovine serum albumin), 0.1% sodium laurylsarkosine, and 100 mg/mL tRNA, followed by incubation for 16 h at 37°C with hybridization buffer containing 5 ng/mL digoxigenin-labeled antisense or sense probes. After washing at 42°C for 20 min with 50% formamide/2x SSC and with 1x SSC three times each, the cells were stained using the DIG nucleic acid detection kit (Boehringer Mannheim Biochemica, Manheim, Germany) according to the manufacturer’s instructions.

Cell staining and flow cytometry analysis
Ba/F3 transfectants were incubated with rat monoclonal antibody (mAb) against human ßc, a generous gift from Dr. Atsushi Miyajima (University of Tokyo) [46 ], mouse mAb against the human GMR {alpha} chain [47 ], or isotytpe-matched controls for 30 min on ice. After washing, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated F(ab')2 goat anti-mouse immunoglobulin G (IgG; Tago, Camarillo, CA) or FITC-conjugated F(ab')2 anti-rat IgG (Organon Teknika Corp., Miami, FL) for 30 min on ice. Five thousand cells were analyzed for binding of mAb or isotype controls by flow cytometry (FACScan, Becton Dickinson, Mountain View, CA).

Proliferation assay
Wild-type Ba/F3 cells, Ba/F3 cells expressing only human GMR {alpha} chains, and Ba/F3 clones transfected with the GMR {alpha} chain and either intact or variant ßc were washed three times. After starvation in growth factor-free RPMI containing 5% FCS for more than 4 h, the cells were cultured in the presence of either 1 ng/mL murine IL-3 or 5 ng/mL human GM-CSF. The numbers of viable cells assessed by trypan blue exclusion were assessed every 12 h.

Binding assay
Radioiodinated human GM-CSF was purchased from Perkin Elmer Life Sciences (Boston, MA). It had a specific activity of 1.44 x 1018 cpm/mol. The binding affinities of GM-CSF to transfectants were analyzed as described previously [48 ]. In brief, washed cells were cultured in the absence of growth factors for 4 h, and then various concentrations of 125I-GM-CSF were incubated with 3 x 106 cells for 16 h at 4°C with or without a 100-fold excess of unlabeled human GM-CSF (Sigma Chemical Co.). The cells were centrifuged through a 3:2 mixture of dibutyryl phthalate (Sigma Chemical Co.):bis-phthalate oil (Sigma Chemical Co.), and radioactivities of the pellets were measured in a {gamma} counter. The binding data were subjected to Scatchard analysis.

Western blot analysis
The Ba/F3 and HEK transfectants were resuspended in lysis buffer containing 1% sodium dodecyl sulfate, 10 mmol/L Tris (pH 7.6), 2 mmol/L disodium ethylenediaminetetraacetic acid, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL leupeptin, and 1 µg/mL pepstatin. The cell lysates were mixed with an equal volume of twofold concentrated Laemmeli sample buffer containing 5% 2-mercaptoethanol, boiled for 5 min, and electrophoresed on a 12.7% polyacrylamide gel. The separated proteins were transferred onto a nitrocellulose membrane using a semi-dry Western transfer apparatus (ATTO, Amherst, NY). After blocking with 5% skim milk dissolved in Tris-buffered saline buffer (10 mmol/L Tris, pH 8.0, 150 mmol/L NaCl), the membrane was incubated for 1 h at room temperature with rabbit polyclonal antibody against human ßc N-terminal peptide (N-20, Santa Cruz Biotechnology, CA) followed by incubation for 1 h at room temperature with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham, Arlington Heights, IL). Signals were detected by enhanced chemiluminescence (Amersham).

Statistical analysis
The Mann-Whitney test was used to compare MDS patients and controls with respect to the ratios of the PCR band intensities derived from variant ßc to those from total ßc.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ßc transcripts detected by direct RT-PCR-SSCP
To detect atypical transcripts of ßc, PCR-SSCP analysis was carried out on reverse-transcribed mRNA prepared from nine MDS patients (Nos. 1–9 in Table 1 ) and seven healthy controls using 12 primer pairs shown in Figure 1 . Compared with the electrophoretic patterns derived from intact ßc cDNA control, at least nine abnormally mobilized bands were detected in 1SA, 2SA, 6SA, 7SA, and 9SA fragments as indicated by A–I in Figure 2 . These abnormal bands always coexisted with predominant, intact fragments in each patient. None of them was specific to MDS, as they were found in patients and controls. The only difference between patients and controls was the intensities of band "B," amplified with a sense primer 2S in exon IV and an antisense primer 2A in exon VI. Band "B" was obviously stronger in MDS than in controls, suggesting that the corresponding aberration was increased in MDS patients compared with healthy individuals. No abnormal transcripts were found in the regions amplified by 3SA, 4SA, 5SA, 8SA, 10SA, 11SA, and 12SA primer pairs (data not shown).

Sequences of the abnormal transcripts
The ßc cDNA clones that generated the same SSCP aberrations as A–I in Figure 2 were sequenced. As presented in Table 3 , the aberration that generated band "B" was an insertion of 79 bases at nucleotide position 578, the junction of exons V and VI. The analyses of three independent clones confirmed that the inserted sequence was completely identical to that of the intron between exons V and VI registered in the database. Sequencing of genomic DNA also revealed that the insertion was intron V in ßc and that neither patients nor controls had mutations in the intron and exon/intron boundaries (data not shown). The insertion caused a frameshift and generated a premature stop codon in the extracellular domain, predicting a truncated ßc isoform composed of conserved N-terminal 182 amino acids followed by a new sequence of 84 amino acids. The other eight alterations detected by SSCP analysis were two silent point mutations (A and D), four missense mutations (E; T1601->C, F; A1525->T, G; T1612->C, I; C2141->A) that altered amino acids in the cytoplasmic domain (Phe534->Ser, Met509->Leu, Phe538->Leu, and Pro714->His, respectively), and two deletions [a deletion of five nucleotides from positions 419 to 423 (C) and a deletion of 104 base pairs from positions 1492 to 1595 corresponding to the entire exon XIII (H)], both of which generated premature stop codons in the extracellular and cytoplasmic domains, respectively.


View this table:
[in this window]
[in a new window]
  		Table 3. Sequences of the Abnormal mRNAs Detected by Direct PCR-SSCP

 
Comparison of the incidence of the intron V-retained ßc variants (ßc79) between MDS and controls by semiquantitative RT-PCR
The ratios of the intron V-retained variants (ßc79) to total ßc transcripts were compared between MDS patients and controls by RT-PCR. The PCR1 specifically amplified the ßc79 as the 222-bp fragments, and 266-bp fragments were obtained by PCR2 regardless of intron V retention (Fig. 3a ). Figure 3b shows that more PCR1 fragment was obtained in each MDS patient compared with healthy controls, whereas similar amounts of PCR2 fragments were amplified with no significant difference in the band intensities between MDS patients and controls (P=0.3329). The measurement of band intensities derived from the ßc79 cDNA indicated that difference in PCR efficiency between PCR1 and PCR2 resulted in a ratio of 1.808 when 100% of the expressed ßc transcripts retained the intron (Fig. 3c) . Statistically, MDS patients showed a significantly higher ratio (0.795±0.188) than healthy individuals (0.112±0.076; P<0.001; Fig. 3c ). The ratios in the patients with RA or RARS (0.863±0.187) were higher than those in RAEB/RAEBt (0.659±0.113; P<0.05), which were still significantly higher than controls (P<0.01). This experiment was performed twice, and similar results were obtained.

ßc expression in transfected Ba/F3
To examine the effects of intron retention on receptor functions, the cDNA of intact ßc or ßc79 was introduced into a murine IL-3-dependent cell line, Ba/F3, along with the human GMR {alpha} chain, and the expression of introduced ßc mRNA in the isolated clones was confirmed by RT-PCR (Fig. 4a ). The intron-containing 222-bp fragments were detected in Ba/F3 clones transfected with ßc79 but not with intact ßc [Fig. 4a , (1)]. The PCR with primer 2S and 3A, which sandwiched the retained intron, generated 387-bp and 466-bp fragments from the intact ßc- and ßc79-transfected clones, respectively [Fig. 4a (2)]. These data confirmed that the clones tested expressed the introduced ßc, at least at the mRNA level. In situ hybridization using an antisense probe complementary to the sequence within the intron V showed that 100% of the cells in each ßc79-transfected clone expressed intron V-containing RNA, and the cells did not react with a sense probe (Fig. 4b) . The clones with intact ßc did not react with either probe. Western blot analysis indicates that the transfected ßc and ßc79 were detected as the expected molecular weights in Ba/F3 (130 kd and 27 kd, respectively; Fig. 4c ).



View larger version (22K):
[in this window]
[in a new window]
  		Figure 4. Expression of transfected ßc in Ba/F3. (a) RT-PCR. cDNA was prepared from total cellular RNA extracted from wild-type Ba/F3, human GMR {alpha} chain-expressing Ba/F3, each of three Ba/F3 clones transfected with GMR {alpha} chain + intact ßc (CE3, EE4, CF10), and GMR {alpha} chain + ßc79 (mG7, mE10, and mF19) using oligo-dT primers for subsequent PCR. ßc79 and intact ßc cDNAs in expression vectors, which were used for the transfections, were also subjected PCR as controls. (1) ßc79 mRNA expression. Twenty-eight cycle PCR amplification with insertion-specific primers 13S and 3A detected 222-bp fragments in the {alpha} + ßc79 clones but not in the {alpha} + intact ßc clones. (2) Expression of intact ßc and ßc79 mRNAs. The primer pair 2S and 3A, which sandwiched the insertion, amplified 387-bp (solid arrow) and 466-bp fragments (open arrows) from the {alpha} + intact ßc and the {alpha} + ßc79 clones, respectively, by 28 PCR cycles. (3) ß-actin. As an internal control, ß-actin expression was confirmed by 25 PCR cycles. (b) In situ hybridization. An antisense probe complementary to 40 bases within the insertion and a sense probe were labeled with digoxigenin. RNA expression in each transfected clone was tested. (c) Western blotting. Cellular proteins derived from Ba/F3 transfected with the GMR {alpha} chain alone (lane 1), GMR {alpha} chain + ßc79 (lane 2), and GMR {alpha} chain + intact ßc (lane 3) were separated on a 12.7% acrylamide gel, transferred onto nitrocellulose membranes, and blotted with an antibody against a ßc N-terminal peptide. A solid arrow indicates intact ßc, and a white arrow indicates ßc79.

 
Effects of the intron V retention on receptor function
To assess the function of the isoform as a signal transducer, proliferation of transfectants in response to human GM-CSF was examined. All transfectants proliferated in the presence of murine IL-3, as did the wild-type and the GMR {alpha} chain-expressing Ba/F3 (Fig. 5a ). Although the cells expressing intact ßc with the {alpha} chain proliferated in response to human GM-CSF, all three clones with ßc79 and GMR {alpha} chain (mG7, mE10, mF10) failed to grow and decreased in numbers, as did wild-type Ba/F3 and Ba/F3 expressing only the GMR {alpha} chain (Fig. 5b) .



View larger version (20K):
[in this window]
[in a new window]
  		Figure 5. Proliferative response of transfectants to murine IL-3 and human GM-CSF. Wild-type murine IL-3-dependent Ba/F3 ({blacktriangleup}), Ba/F3-transfected with GMR {alpha} chain alone (x), Ba/F3 with {alpha} chain + intact ßc, CF10 ({blacksquare}), and Ba/F3 with {alpha} chain + ßc79, mF10 ({square}) were cultured in the presence of (a) 1 ng/mL murine IL-3 or (b) 5 ng/mL human GM-CSF, and the numbers of viable cells were determined every 12 h.

 
As signal transduction via GMR requires association of {alpha} chain with ßc, resulting in high-affinity binding of GM-CSF, it was assessed if the isoform can form a high-affinity complex with the {alpha} chain. Although coexpression of intact ßc with the GMR {alpha} chain converted the low-affinity binding by the GMR {alpha} chain (3.3 nM) to high-affinity binding (39.8 pM), the isoform neither generated high-affinity sites nor interfered with the binding of the ligand to the {alpha} chain (Table 4 ).


View this table:
[in this window]
[in a new window]
  		Table 4. Binding Characteristics of Expressed Receptors

 
Localization of the translated proteins
To determine if ßc79 is expressed at the cell surface, ßc79-transfected Ba/F3 cells were incubated with antibodies against the extracellular domains of human ßc or GMR {alpha} chain and analyzed by flow cytometry. As shown in Figure 6a , ßc79 expression was not detected, and intact ßc was recognized. Similar amounts of the GMR {alpha} chain were present in both transfectants. The localization of transfected ßc proteins was further examined by Western blot analysis of HEK cells transiently transfected with ßc and ßc79. The blot, using whole cell lysates, confirmed that the ßc79 was present as a 27-kd protein (Fig. 6b) . The 130-kd intact ßc was observed in both membrane and cytosol fractions in similar amounts as shown in Figure 6c . However, the 27-kd truncated isoform, which was present in the cytosol fraction, was almost absent in the membrane fraction (Fig. 6d) .



View larger version (32K):
[in this window]
[in a new window]
  		Figure 6. Protein localization. (a) Flow cytometric analysis of Ba/F3 transfectants. The cells incubated with rat mAb against human ßc or mouse mAb against human GMR {alpha} chain were treated with FITC-conjugated anti-rat or mouse IgG, respectively. Negative controls were treated with isotype-mached IgG instead of mAb. The histograms of thin lines and thick lines represent the results of negative controls and mAb-treated cells, respectively. (b) Western blotting using whole cell lysates of HEK transfectants. The 130-kd intact ßc (lane 1) and 27-kd ßc79 (line 2) proteins were detected as indicated by the solid and open arrowss, respectively. (c, d) Localization of ßc. Proteins from whole cells, membranes, and cytosol of HEK transfected with intact ßc (c) or ßc79 (d) were blotted with the antibody against ßc. The membrane and cytosol fractions were isolated from the same numbers of cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous data, showing that reduced ßc expression could result in neutrophil dysplasia [40 ], prompted us to investigate its etiology. We first examined whether structural abnormalities in ßc caused the reduction in GMR surface expression. RT-PCR-SSCP showed that one transcript was increased in MDS patients compared with healthy controls. This transcript was found to contain an intron-identical 79-base insertion between exons V and VI. Contamination of genomic DNA was excluded for the following reasons. When RNA samples were subjected to RT-PCR-SSCP, small introns, such as a 566-base intron VI and 161-base intron VII sandwiched by a primer pair 4SA, were not detected by 1.5% agarose gel or acrylamide gel electrophoresis. In addition, the sequenced clones, which had full-length ßc mRNA sequence using primers 1S and 12A, did not contain other introns (data not shown). These suggests that the intron V was transcribed to mRNA and was possibly exempted from splicing in neutrophils. Thus, the transcripts with the retained intron V appear to be mis-spliced variants.

The elevated level of ßc79 compared with total ßc in MDS was confirmed by RT-PCR. Unfortunately, limited amounts of neutrophil RNA isolated from cytopenic patients precluded direct quantification such as Northern blotting [44 , 49 ]. The ßc79 was specifically amplified using an intron V-specific sense primer. To amplify total ßc transcripts with or without intron V retention, we used the primer pair 10SA (Fig. 3a) that located within exon XIV, where splicing is evitable, and no aberrations were detected by SSCP. As described in Materials and Methods, all three patients with RAEB needed more concentrated cDNA template to obtain the same levels of 10SA fragments as controls, suggesting that total ßc transcription levels were reduced in these patients. In RA/RARS, the total ßc transcription levels were considered to be similar to those of controls, as similar levels of 10SA fragments were generated from the same concentration of cDNA as controls. Although patients with RAEB may have smaller ratios, more cases need to be studied to determine if there are significant differences among disease subtypes. The increased amount of ßc79 in all patient samples tested suggests that its accumulation is a universal feature of MDS. This is an important finding, as the other molecular and genomic abnormalities reported in MDS are not as consistent [50 ].

The intron V retention in ßc79 abrogated the receptor activity by reducing cell-surface GMR expression. Previous studies have shown that the ßc lacking its cytoplasmic domain was incapable of transducing a signal but not interacting with the {alpha} chain [12 13 14 15 16 17 ]; we therefore tested the possibility of functional interactions of the isoform with the {alpha} chain by binding assay. The results of flow cytometry analysis of Ba/F3 transfectants showed that ßc79 was not expressed on the cell surface. To confirm the localization of translated ßc79, we used transient HEK transfectants that were thought to express introduced genes more efficiently. Western blot analysis of HEK cells also demonstrated that the truncated isoform was not expressed on the cell surface. The elevated incidence of the variants without compensatory increase in total ßc transcripts in MDS patients may result in reduction of intact ßc and reduced cell-surface GMR ßc expression.

The identification and characterization of ßc79 are novel findings. Analysis of mRNA and genomic DNA encoding the ßc extracellular domain in patients with acute leukemia detected only four kinds of point mutations with no differences in their frequencies between patients and controls [51 ]. We detected one of them as "D" (Fig. 2) . Another study detected a deletion of the entire exon XIII corresponding to our "H" (Fig. 2) but not intron V-retention in blasts from acute myeloid leukemia patients [24 ]. The intron V retention in ßc mRNA is therefore rare in de novo acute leukemia but appears to be characteristic of MDS. Further studies will determine whether elevated ßc79 is a potential diagnostic marker for patients with MDS.

Aberrant splice products have been found in leukemia, including the skipping of exons in deoxycytidine kinase [52 ] and SMAD5 [53 ], in addition to the ßc without exon XIII [24 ], and an intron retention in protein tyrosine phosphatase, nonreceptor type 6 (PTPN6) [54 ]. Expression of these variants was restricted in leukemic blasts and CD34+ stem cells [24 ] or decreased during culturing in the presence of differentiation-stimulating cytokines [53 ], suggesting that the expression of alternative splice variants is differentially regulated during hematopoiesis. Our results may be characterized by the following: First, the abnormal ßc is detected in the terminally differentiated neutrophils. Second, the genomic mutations that typically cause abnormal splicing were not found in the patients with ßc79 overexpression, whereas the intron retention in PTPN6 occurs from a point mutation within the intron [54 ].

The biological significance of increased ßc79 in neutrophils is not fully understood at this time. It is to be studied if the variant has dominant-negative effects on intact ßc. It would be interesting to determine whether the reduction in functional ßc affects IL-3 receptor activity, which can be detected on GM-CSF-stimulated neutrophils.

In conclusion, we demonstrate that increased ßc79 expression results in the reduced GMR expression and the reduced GM-CSF receptor activity observed in neutrophils of MDS patients.


   ACKNOWLEDGEMENTS
 
We thank Dr. Ken Ishioka (Fukushima Medical University, Japan) for technical support with PCR-SSCP analysis; Dr. Sumiko Watanabe (University of Tokyo, Japan) for providing cDNAs of human GMR {alpha} and ßc chains and Ba/F3 cell lines; Dr. Atsushi Miyajima (University of Tokyo) for providing mAb against human ßc; and Drs. Yutaka Katsu-ura (Fukushima Saiseikai Hospital, Japan), Hideo Kimura, Yutaka Shiga (Hobara Central Hospital, Fukushima, Japan), Shin Matsuda, Tetsugoro Tanaka, Hiroshi Kanbayashi, Yurie Saitoh (Ohta Nishino-uchi Hospital, Koriyama, Japan), Rokuo Abe, Kazuei Ogawa (Taiyounokuni Hospital, Shirakawa, Japan), Hideyoshi Noji, Kazuhiko Ikeda, Masatoshi Okamoto (Fukushima Medical University), and Junichi Kameoka (Tohoku University, Sendai, Japan) for the blood samples from patients with MDS. This study was supported by a grant (10770512) from the Ministry of Education, Science and Culture (Japan), Fukushima Medical Research Fund (Japan), and NISHINOMIA Basic Research Fund (Japan).

Received September 2, 2004; revised January 13, 2005; accepted January 19, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Metcalf, D., Johnson, G. R., Burgess, A. W. (1980) Direct stimulation by purified GM-CSF of proliferation of multipotential and erythroid precursor cells Blood 55,138-147[Free Full Text]
  2. Donahue, R. E., Wang, E. A., Stone, D. K., Kamen, R., Wong, G. G., Sehgal, P. K., Nathan, D. G., Clark, S. C. (1986) Stimulation of hematopoiesis on primates by continuous infusion of recombinant human GM-CSF Nature 321,872-875[CrossRef][Medline]
  3. Mayer, P., Lam, C., Obenaus, H., Liehl, E., Besemer, J. (1987) Recombinant human GM-CSF induces leukocytosis and activates peripheral blood polymorphonuclear neutrophils in nonhuman primates Blood 70,206-213[Abstract/Free Full Text]
  4. Antman, K. S., Griffin, J. D., Elias, A., Socinski, M. A., Ryan, L., Cannista, S. A., Oette, D., Whitley, M., Frei, E., III, Schnipper, L. E. (1988) Effects of recombinant human granulocyte-macrophage colony-stimulating factor on chemotherapy-induced myelosuppression N. Engl. J. Med. 319,593-598[Abstract]
  5. Fleischmann, J., Golde, D. W., Weisbert, R. H., Gasson, J. C. (1986) Granulocyte-macrophage colony-stimulating factor enhances phagocytosis of bacteria by human neutrophils Blood 68,708-711[Abstract/Free Full Text]
  6. Weisbart, R. H., Golde, D. W., Clark, S. C., Wong, G. G., Gasson, J. C. (1985) Human granulocyte-macrophage colony-stimulating factor is a neutrophil activator Nature 314,361-363[CrossRef][Medline]
  7. Vadas, M. A., Nicola, N. A., Metcalf, D. (1983) Activation of antibody-dependent cell-mediated cytotoxity of human neutrophils and eosinophils by colony-stimulating factors J. Immunol. 130,795-799[Abstract]
  8. Weisbart, R. H., Kwan, L., Golde, D. W., Gasson, J. C. (1987) Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to major physiological chemoattractants Blood 69,18-21[Abstract/Free Full Text]
  9. Cosman, D., Lyman, S. D., Izerda, R. L., Beckmann, M. P., Parks, L. S., Goodwin, R. G., March, C. J. (1990) A new cytokine receptor superfamly Trends Biochem. Sci. 15,265-270[CrossRef][Medline]
  10. Gearing, D. P., King, J. A., Gough, N. M., Nicola, L. A. (1989) Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor EMBO J. 8,3667-3676[Medline]
  11. Kitamura, T., Sato, N., Arai, K., Miyajima, A. (1991) Expression cloning of the human IL-3 receptor cDNA reveals a shared ß subunit for the human IL-3 and GM-CSF receptors Cell 66,1165-1174[CrossRef][Medline]
  12. Hayashida, K., Kitamura, T., Gorman, D. M., Arai, K., Yokota, T., Miyajima, A. (1990) Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): reconstitution of a high-affinity GM-CSF receptor Proc. Natl. Acad. Sci. USA 87,9655-9659[Abstract/Free Full Text]
  13. Kitamura, T., Sato, N., Arai, K., Miyajima, A. (1991) Expression cloning of the human IL-3 receptor cDNA reveals a shared ß subunit for the human IL-3 and GM-CSF receptors Cell 66,1165-1174
  14. Sakamaki, K., Miyajima, I., Kitamura, T., Miyajima, A. (1992) Critical cytoplasmic domains of the common ß subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation EMBO J. 11,3541-3549[Medline]
  15. Weiss, M., Yokoyama, C., Shikama, Y., Naugle, C., Druker, B., Sieff, C. A. (1993) Human granulocyte-macrophage colony-stimulating factor receptor signal transduction require the proximal cytoplasmic domains of the {alpha} and ß subunits Blood 82,3298-3306[Abstract/Free Full Text]
  16. Sato, N., Sakamaki, K., Terada, N., Arai, K-I., Miyajima, A. (1993) Signal transduction by the high-affinity GM-CSF receptor: Two distinct cytoplasmic regions of common ß subunit responsible for different signaling EMBO J. 12,4181-4189[Medline]
  17. Quelle, F. W., Sato, N., Witthuhn, B., Inhorn, R. C., Eder, M., Miyajima, A., Griffin, J. D., Ihle, J. N. (1994) JAK2 associates with the ßc chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane proximal region Mol. Cell. Biol. 14,4335-4341[Abstract/Free Full Text]
  18. Begley, C. J., Woodcock, J. M., Stomski, F. C., Lopez, A. F. (1997) The structural and functional basis of cytokine receptor activation: lessons from the common ß subunit of the granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), and IL-5 receptors Blood 90,3005-3017[Abstract/Free Full Text]
  19. Rossjohn, J., McKinstry, W. J., Woodcock, J. M., McClure, B. J., Hercus, T. R., Parker, M. W., Lopez, A. F. (2000) Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common ß-chain bound to an antagonist Blood 95,2491-2498[Abstract/Free Full Text]
  20. D’Andrea, R., Rayner, J., Moretti, P., Lopez, A., Goodall, G. J., Gonda, T. J., Vada, M. (1994) A mutation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5 that leads to ligand independence and tumorigenicity Blood 83,2802-2808[Abstract/Free Full Text]
  21. Jenkins, B. J., D’Andrea, R., Gonda, T. J. (1995) Activating point mutations in the common ß subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of ß subunit dimerization and cell type-specific molecules in signaling EMBO J. 14,4276-4287[Medline]
  22. Hannemann, J., Hara, T., Kawai, M., Miyajima, A., Ostertag, W., Stocking, C. (1995) Sequential mutations in the interleukin-3 (IL-3)/granulocyte-macrophage colony-stimulating factor/ IL-5 receptor ß-subunit genes are necessary for complete conversion to growth autonomy mediated by a truncated ßc subunit Mol. Cell. Biol. 15,2402-2412[Abstract]
  23. D’Andrea, R. J., Barry, S. C., Moretti, P. A. B., Jones, K., Ellis, S., Vadas, M. A., Goodall, G. J. (1996) Extracellular truncations of the h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5 lead to ligand-independent activation Blood 87,2641-2648[Abstract/Free Full Text]
  24. Gale, R. E., Freeburn, R. W., Khwaja, A., Chopra, R., Linch, D. C. (1998) A truncated isoform of the human ß chain common to the receptors for granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3), and interleukin-5 (IL-5) with increased mRNA expression in some patients with acute leukemia Blood 91,54-63[Abstract/Free Full Text]
  25. Weisdorf, D. J., Oken, M. M., Johnson, G. J., Rydell, R. E. (1983) Chronic myelodysplastic syndrome: short survival with or without evolution to acute leukemia Br. J. Haematol. 55,691-700[Medline]
  26. Tricot, G., Boogaerts, M. A., De Wolf-Peters, C., Vlietinck, R., Verwilghen, R. L. (1984) Bone marrow histology in myelodysplastic syndromes. I. Histological findings in myelodysplastic syndromes and comparison with bone marrow smears Br. J. Haematol. 57,423-430[Medline]
  27. Foucar, K., Langdon, R. M., II, Arnitage, J. O., Olson, D. B., Carrol, T. J. (1985) Myelodysplastic syndromes: a clinical and pathologic analysis of 109 cases Cancer 56,553-561[CrossRef][Medline]
  28. Kouides, P. A., Bennett, J. M. (1997) Understanding the myelodysplastic syndromes Oncologist 2,389-401[Abstract/Free Full Text]
  29. Cunningham, I., Maccallum, S. J., Nicholas, M. D., Byth, K., Hewson, J. W., Arnold, B., Motum, P. I., Mulligan, S. P., Crane, G. G. (1995) The myelodysplastic syndromes: an analysis of prognostic factors in 266 cases from a single institution Br. J. Haematol. 90,602-606[Medline]
  30. Boogaerts, M. A., Nelissen, V., Roelant, C., Goossens, W. (1983) Blood neutrophil function in primary myelodysplastic syndromes Br. J. Haematol. 55,217-227[Medline]
  31. Verhoef, G., Boogaerts, M. (1991) In vivo administration of granulocyte-macrophage colony-stimulating factor enhances neutrophil function in patients with myelodysplastic syndromes Br. J. Haematol. 79,177-184[Medline]
  32. Lowe, G. M., Dang, Y., Watson, F., Edwards, S. W., Galvani, D. W. (1994) Identification of a subgroup of myelodysplastic patients with a neutrophil stimulation-signaling defect Br. J. Haematol. 86,761-766[Medline]
  33. Fuhler, G. M., Drayer, A. L., Vellenga, E. (2003) Decreased phosphorylation of protein kinase B and extracellular signal-regulated kinase in neutrophils from patients with myelodysplasia Blood 101,1172-1180[Abstract/Free Full Text]
  34. Vadhan-Raj, S., Keating, M., LeMaistre, A., Hittelmann, W. N., McCrediw, K., Trujillo, L. M., Broxmeyer, H. E., Henney, C., Gutterman, J. U. (1987) Effects of recombinant human granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes N. Engl. J. Med. 317,1545-1552[Abstract]
  35. Ganser, A., Volkers, B., Greher, J., Ottmann, O. G., Walther, F., Becher, R., Bergmann, L., Shulz, G., Hoelzer, D. (1989) Recombinant human granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndromes-A phase I trial Blood 73,31-37[Abstract/Free Full Text]
  36. Thompson, J. A., Lee, D. J., Kidd, P., Rubin, E., Kaufmann, J., Bonnem, E. M., Fefer, A. (1989) Subcutaneus granulocyte-macrophage colony-stimulating factor in patients with myelodysplastic syndrome: toxicity, pharmacokinetics, and hematological effects J. Clin. Oncol. 7,629-637[Abstract]
  37. Estey, E. H., Kurzrock, R., Talpaz, M., McCredie, K. B., O’Brien, S., Kantarjian, H. M., Keating, M. J., Deisseroth, A. B, Guttertman, J. U. (1991) Effects of low doses of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) in patients with myelodysplastic syndromes Br. J. Haematol. 77,291-295[Medline]
  38. Kurzrock, R., Talpaz, M., Gomez, J. A., Estey, E. H., O’Brien, S., Hirsch-Ginsberg, C., Koller, C., Freireich, E. J., Gutterman, J. U. (1991) Differential dose-related hematological effects of GM-CSF in pancytopenia: evidence supporting the advantage of low-over high-dose administration in selected patients Br. J. Haematol. 78,352-358[Medline]
  39. Rose, C., Wattel, E., Bastion, Y., Berger, E., Bauters, F., Coiffier, B., Fenaux, P. (1994) Treatment with very low-dose GM-CSF in myelodysplastic syndromes with neutropenia Leukemia 8,1458-1462[Medline]
  40. Shikama, Y., Shichishima, T., Ohto, H., Jubinsky, P., Maruyama, Y. (2000) Neutrophil-specific reduction in the expression of granulocyte-macrophage colony-stimulating factor receptor subunits in myelodysplastic syndromes Br. J. Haematol. 111,863-872[CrossRef][Medline]
  41. Bennett, J. M., Catovsky, D., Daniel, M. T., Flandrin, G., Galton, D. A., Gralnick, H. R., Sulton, C. (1982) Proposals for the classification of the myelodysplastic syndromes Br. J. Haematol. 51,189-199[Medline]
  42. Kremer, E., Barker, E., D’Andrea, R. J., Skim, R., Phillips, H., Moretti, P. A. B., Lopez, A. F., Petit, C., Vadas, M. A., Sutherland, G. R., Coodall, G. J. (1993) A cytokine receptor gene cluster in the X-Y pseudo autosomal region? Blood 82,22-28[Abstract/Free Full Text]
  43. Dooley, D. C., Sinpson, J. F., Meryman, H. T. (1982) Isolation of large numbers of fully viable human neutrophils: a preparative technique using percoll dendity gradient centrifugation Exp. Hematol. 10,591-599[Medline]
  44. Nakayama, H., Yokoi, H., Fujita, J. (1992) Quantification of mRNA by non-radioactive RT-PCR and CCD imaging system Nucleic Acids Res. 20,4939[Free Full Text]
  45. Matsuoka, I., Suzuki, Y., Defer, N., Nakanishi, H., Hanoune, J. (1997) Differential expression of type I, II, and V adenylyl cyclase gene in the postnatal developing rat brain J. Neurochem. 68,498-506[Medline]
  46. Watanabe, Y., Kitamura, T., Hayashida, K., Miyajima, A. (1992) Monoclonal antibody against the common ß subunit (ßc) of the human interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor receptors shows upregulation of ßc by IL-1 and tumor necrosis factor-{alpha} Blood 80,2215-2220[Abstract/Free Full Text]
  47. Jubinsky, P. T., Laurie, A. S., Nathan, D. G., Yetz-Aldepe, J., Sieff, C. A. (1994) Expression and function of the human granulocyte-macrophage colony-stimulating factor receptor {alpha} subunit Blood 84,4174-4185[Abstract/Free Full Text]
  48. Shikama, Y., Barber, D. L., D’Andrea, A., Sieff, C. A. (1996) A constitutively activated chimeric cytokine receptor confers factor-independent cell growth in hematopoietic cell lines Blood 88,455-464[Abstract/Free Full Text]
  49. Yokoi, H., Nastuyama, S., Iwai, M., Noda, Y., Mori, T., Mori, K. J., Fujita, K., Nakayama, H., Fujita, J. (1993) Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development Biochem. Biophys. Res. Commun. 195,769-775[CrossRef][Medline]
  50. Fenaux, P. (2001) Chromosome and molecular abnormalities in myelodysplastic syndromes Int. J. Hematol. 73,429-437[Medline]
  51. Freeburn, R. W., Gale, R. E., Linch, D. (1998) Activating point mutations in the ßc chain of the GM-CSF, IL-3, IL-5 receptors are not a major contributory factor in the pathogenesis of acute myeloid leukemia Br. J. Haematol. 103,66-71[CrossRef][Medline]
  52. Veuger, M. J. T., Honders, M. W., Landegent, J. E., Willemze, R., Barge, R. M. (2000) High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia Blood 96,1517-1527[Abstract/Free Full Text]
  53. Jiang, Y., Liasng, H., Guo, W., Kottickal, L. V., Nagarajan, L. (2000) Differential expression of a novel C-terminal truncated splice form of SMAD5 in hematopoietic stem cells and leukemia Blood 95,3945-3950[Abstract/Free Full Text]
  54. Beghini, A., Ripamonti, C. B., Peterlongo, P., Roversi, G., Cairoli, R., Morra, E., Larizza, L. (2000) RNA hyperediting and alternative splicing of hematopoietic cell phosphatase (PNPN6) gene in acute leukemia Hum. Mol. Genet. 9,2297-2304[Abstract/Free Full Text]
  55. Smith, W. B., Guida, L., Sun, Q., Korpelainen, E. I., van den Heuvel, C., Gills, D. D., Hawrylowicz, M. A., Vadas, M. A., Lopez, A. F. (1995) Neutrophils activated by granulocyte-macrophage colony-stimulating factor express receptors for interleukin-3 which mediate class II expression Blood 86,3938-3940[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0904488v1
77/5/811    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Shikama, Y.
Right arrow Articles by Maruyama, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Shikama, Y.
Right arrow Articles by Maruyama, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS