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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-01-0043.
NEOPLASIA
From the Klinische Kooperationsgruppe Gentherapie,
GSF The clinical progression of chronic myeloid leukemia
(CML) from chronic phase to blast crisis is characterized by the
increasing failure of myeloid precursors to differentiate into mature
granulocytes. This study was undertaken to investigate the influence of
Bcr-Abl and of the small molecule Abl tyrosine-kinase inhibitor
imatinib mesylate on granulocyte colony-stimulating factor
(G-CSF)-induced neutrophilic differentiation. We show that
differentiation of 32Dcl3 cells into mature granulocytes is accompanied
by the increased expression of the antigens macrophage adhesion
molecule-1 (Mac-1) and Gr-1, of the G-CSF
receptor (G-CSFR), of myeloid transcription factors
(CCAAT/enhancer-binding protein- Chronic myeloid leukemia (CML) is a clonal malignancy of a
hematopoietic stem cell caused in most cases by a reciprocal
translocation between chromosomes 9 and 22 (Philadelphia
translocation),1 which results in the generation of a
fusion protein, Bcr-Abl, with constitutive tyrosine kinase and
transforming activity in hematopoietic cells.
At 3 to 5 years after onset, CML often progresses to a
fatal blast crisis, characterized by a profound block in
differentiation. It is unclear whether this differentiation block is
caused by Bcr-Abl or by secondary mutations acquired during disease
progression. Indeed, progression from chronic phase to blast crisis has
been linked to multiple secondary cytogenetic or molecular alterations. These include trisomy 8, isochromosome i(17q), trisomy 19, and an extra
Philadelphia chromosome.2 Among the molecular
abnormalities found during blast crisis are alterations in
p16INK4A, p53, pRB, Ras, and c-Myc.2 Moreover,
fusion genes resulting from reciprocal translocations such as AML/EVI-1
or NUP98/HoxA9 have been reported to be associated with some cases of
blast crisis.2,3
Bcr-Abl is the molecular target for imatinib mesylate (STI571), an
adenosine triphosphate (ATP)-competitive inhibitor of the Abl
tyrosine kinase.4 In several clinical trials, imatinib mesylate was shown to induce hematologic and cytogenetic remissions in
up to 98% of patients with chronic phase CML.5,6
Surprisingly, therapeutic effects were also seen in patients with
myeloid blast crisis or Bcr-Abl+ lymphoid disease,
suggesting that even at this late stage and despite the accumulation of
numerous secondary and tertiary genetic alterations, the disease was
still dependent on Bcr-Abl.7 However, in many cases of
late-stage disease, relapses occurred,7 and this
could be correlated to the development of direct resistance of Bcr-Abl
induced by gene amplifications or point mutations.8
Hematopoietic cell differentiation is regulated by a complex network of
growth and differentiation factors.9,10 Granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR)
are of pivotal importance for the differentiation of myeloid precursors into mature granulocytes. Mice carrying homozygous deletions of the
G-CSF or the G-CSFR genes show reduced levels of morphologically mature
neutrophils (about 20% of normal mice
levels).11,12 Among the signaling molecules
mediating maturation and differentiation of hematopoietic cells induced
by G-CSF and other growth factors are transcription factors such as
CCAAT/enhancer-binding protein- Bcr-Abl was recently shown to block G-CSF-induced granulocytic
differentiation in a murine hematopoietic progenitor cell line, 32Dcl3.18 We made use of this model (1) to elucidate the
effects of Bcr-Abl on the G-CSFR-dependent granulocytic
differentiation program and (2) to investigate the reversibility of
these effects by imatinib mesylate. We show that blockage of
differentiation induced by Bcr-Abl is accompanied by a deficient
up-regulation of C/EBP Antibodies
Cell lines and cell culture
Plasmids and generation of stably expressing cell lines Generation of the kinase-inactive mutant of Bcr-Abl was described previously.19 Generation of the imatinib mesylate-resistant mutant Bcr-AblThr315Iso will be described elsewhere. Wild-type (wt) and mutant cDNAs were cloned into pLXSN or pMSCV. Stably expressing cell lines were generated by electroporation. Cells were selected in the presence of 1 mg/mL neomycin (G418). Subclones were generated by limited dilution starting at day 4 after transfection. Two independent clones of each cell type were used for further experiments. The 2 clones had the same phenotype and gave rise to comparable results. Importantly, cells were grown in the presence of interleukin 3 (IL-3) during selection and prior to G-CSF-induced differentiation to avoid secondary alterations due to selection for growth factor independence. New frozen stocks of cells were used every 2 weeks. Generation of retroviral stocks using pMSCV vectors, infection of 32Dcl3 cells, and generation of Bcr-Abl-expressing mass populations are described in the accompanying article by Warmuth et al,20 beginning on page 664.Cell lysis For lysis, 32D cells were harvested at the indicated time points after G-CSF stimulation and washed twice in cold PBS. For experiments evaluating the activity profile of imatinib mesylate, cells were incubated with either inhibitor or dimethyl sulfoxide (DMSO) at a density of 5 × 106 cells per milliliter for 1.5 to 2 hours. Then, 107 cells were lysed in 100 µL lysis buffer containing 1% Nonidet P-40 (NP-40), 20 mM Tris (tris(hydroxymethyl)aminomethane) (pH 8.0), 50 mM NaCl, and 10 mM EDTA (ethylenediaminetetraacetic acid), 1 mM phenylmethyl sulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 2 mM sodium orthovanadate. After resuspension in lysis buffer, cells were incubated for 25 minutes on ice. Unsoluble material was removed by centrifugation at 15 000g. Lysates were checked for protein concentrations by means of a BioRad protein assay (Bio-Rad Laboratories, Muenchen, Germany).Gel electrophoresis and immunoblotting Gel electrophoresis and immunoblotting were performed as described previously.19Flow cytometry For flow cytometry, cells were washed twice and resuspended in 100 µL PBS containing 2% FBS. Cells were stained with fluorescein isothiocyanate (FITC)-conjugated -macrophage adhesion
molecule-1 (-Mac-1) (Pharmingen, Heidelberg, Germany) or
-Gr-1 (Caltag, Burlingame, CA) abs for 30 minutes at room
temperature. Fluorescence-activated cell sorter (FACS)
analysis was done with an EPICS XL 4-color cytometer.
For detection of G-CSFR surface expression, the Fluorokine human G-CSF phycoerythrin conjugate kit from R&D Systems (Minneapolis, MN) was used according to the manufacturer's guidelines. In brief, cells were collected, washed twice, and resuspended in PBS to a final concentration of 4 to 5 × 106 cells per milliliter. Then, 10 µL phycoerythrin (PE)-labeled G-CSF was added to 25 µL washed cell suspension in a 12 × 75 borosilicate tube. As a control, an identical sample of cells was stained with 10 µL PE-conjugated streptavidin. Cells were incubated for 1 hour at 4°C. Thereafter, cells were washed twice with 2 mL 1× rapid dissolution formula-1 (RDF1) buffer and resuspended in 200 µL RDF1 buffer for flow cytometric analysis. To control for specificity of the staining reaction, an aliquot of washed cells was preincubated with a 25-fold molar excess of unconjugated rhG-CSF for 30 minutes at room temperature prior to addition of 10 µL PE-labeled G-CSF. Detection of apoptosis by flow cytometry First, 1 × 105 cells per milliliter were incubated with imatinib mesylate at a concentration of 1 µM. Apoptosis was assessed by measuring the binding of FITC-conjugated annexin V to membranes of apoptosing cells. At the indicated time points, aliquots of cells were taken and washed once in PBS. Thereafter, cells were resuspended in 195 µL annexin V binding buffer, and 5 µL annexin V-FITC (Bender MedSystems Diagnostics, Vienna, Austria) was added. After incubation at room temperature for 10 to 20 minutes, cells were washed once and resuspended in 190 µL annexin V binding buffer. Then, 10 µL of a 20 µg/mL propidium iodide stock solution was added, and the ratio of annexin V+ to negative cells was determined by FACS analysis by means of a Coulter EPICS XL 4-color cytometer.Indirect immunofluorescence Cells were placed on poly-L-lysine-covered microscope slides for 1 hour in a humidified chamber at 37°C. Then nonadherent cells were washed off with Hanks buffered saline solution, and adherent cells were fixed and immobilized with freshly prepared 2% (wt/vol) paraformaldehyde in PBS for 1 hour at 4°C. Subsequently, cells were permeabilized for 15 minutes with 0.2% (vol/vol) Triton X-100 in PBS, blocked with 2% (wt/vol) glycine in PBS, and incubated with a G-CSFR antibody in PBS for 2 hours at room temperature. Slides were washed with PBS and incubated with FITC-labeled secondary antibody. After the final wash with PBS, slides were mounted on a 9:1 mixture of glycerol and 100 mM Tris/HCl, pH 9.0, containing n-propyl-gallate at 20 mg/mL as antifading reagent. Then samples were examined on a confocal laser scanning apparatus (Leica TCS-NT system; Leica, Bernsheim, Germany) attached to a Leica DM IRB inverted microscope with a PLAPO 63 × 1.32 oil immersion objective.RNA extraction, cDNA synthesis, and quantitative real-time PCR For isolation of total RNA and subsequent synthesis of cDNA, the RNeasy Mini kit and the Omniscript Reverse Transcriptase protocol (Qiagen, Hilden, Germany) were used according to the manufacturer's guidelines. Real-time PCR for G-CSFR, C/EBP, PU.1, and glucose-6
phosphate dehydrgenase (G6PD) was performed by means of light cycler
technology (Roche Diagnostics, Mannheim, Germany). Primers for
C/EBP were designed as published previously21: sense,
5'-CCAGCAAGCTGAGGAGCGGCG-3'; antisense, 5'-AACAGCTGAGCCGTGAACTG-3'. For
amplification of PU.1, a commercially available light cycler primer set
was used (Search LC, Heidelberg, Germany). Primers for G-CSFR and G6PD
were as follows: G-CSFR sense, 5'-GCTTGAGCCAACTCCATAGC-3'; G-CSFR
antisense, 5'-AAATGCAGGGAAGGACACAG-3'; G6PD sense,
5'-CCGGATCGACCACTACCTGGGCAAG-3'; G6PD antisense,
5'-GTTCCCCACGTACTGGCCCAGGACCA-3'G6PD. All primer sets gave rise to
specific DNA fragments of expected sizes. For real-time PCR, 2 µL
master mix (Light Cycler FastStart DNA Master SYBR Green I; Roche
Diagnostics), 2 µL cDNA, 4 mM MgCl2,
7.5 µM primer, and water to a final concentration of 20 µL were
used. For calculation of fold induction or for comparison of DNA levels in different cell types, DNA amounts were normalized to G6PD mRNA expression. For semiquantitative assessment of mRNA expression, real-time PCR reactions were stopped after the indicated number of
cycles, and aliquots of the reactions were loaded onto agarose gels.
Again, G6PD mRNA expression was used as a control.
The differentiation block of freshly established 32DBcr-Ablwt cells is not due to a loss of G-CSFR expression, but depends on Bcr-Abl kinase activity To investigate whether Bcr-Abl interferes with differentiation induced by G-CSF in the presence of G-CSFR expression, stable transfectants of 32Dcl3 cells were established expressing either Bcr-Ablwt (32DBcr-Ablwt) or a kinase-inactive mutant of Bcr-Abl (32DBcr-AblK1172R) (Figure 1A). In brief, 32Dcl3 cells were transfected by electroporation as described in "Materials and methods." At 48 hours after transfection, neomycin was added to the media for selection. At 4 days after transfection, cells were plated into 96-well plates by limited dilution to establish individual single cell clones. At 3 weeks after transfection, several subclones were tested for Bcr-Abl expression. Two subclones of each cell type expressing comparable amounts of Bcr-Ablwt or Bcr-AblK1172R were identified (Figure 1A), and aliquots of such cells were cryopreserved to be used for further experiments. Freshly thawed and expanded aliquots were used for each individual experiment. Importantly, 32DBcr-Ablwt cells had been grown in IL-3-containing media continuously during the selection process and prior to analysis of G-CSF-induced differentiation.
It was previously shown that prolonged culture of Bcr-Abl-transformed
32Dcl3 cells might lead to loss of G-CSF receptor
expression.21 However, Western blot analysis and light
cycler real-time PCR showed that the 2 clones of
32DBcr-Ablwt cells used for further experiments
expressed comparable levels of G-CSFR protein and mRNA (Figure
2A-B). In Western blot analysis, 3 major
G-CSFR species of 85 to 90 kDa, 105 to 110 kDa, and 130 to 135 kDa,
possibly representing different glycosylation states, were seen (Figure 2A, arrows). Moreover, 32Dcl3 cells and
32DBcr-Ablwt cells expressed G-CSFR at comparable
levels at the cell surface, as revealed by confocal microscopy
(Figure 2C top panels) and FACS analysis (Figure 2C bottom panels). No
G-CSFR expression was seen in L-GM cells used as a negative control.
FACS controls were performed for every freshly thawed aliquot of
32DBcr-Ablwt cells prior to induction of G-CSF-induced
differentiation to confirm appropriate G-CSFR expression.
Despite appropriate G-CSFR expression, 32DBcr-Ablwt cells, but not 32DBcr-AblK1172R cells, were defective in G-CSF-induced differentiation as assessed by monitoring morphologic differentiation (Figure 1B) as well as up-regulation of myeloid cell surface markers such as Mac-1 or Gr-1 (Figure 1C and data not shown). Imatinib mesylate restores G-CSF-induced granulocytic differentiation and C/EBP transcription-factor regulation in 32DBcr-Ablwt cells These results suggested that Abl kinase activity was essential for the inhibition of granulocytic differentiation. To further address this issue, we investigated the influence of the Abl kinase inhibitor imatinib mesylate on the differentiation of 32Dcl3 and 32DBcr-Ablwt cells in response to G-CSF. The 32DBcr-Ablwt cells were cultured in the absence of IL-3. These cell clones were readily IL-3 independent for growth and survival on IL-3 withdrawal without any further selection. Incubation of 32DBcr-Ablwt cells with 1 µM imatinib mesylate in the absence of growth factors rapidly decreased cell growth and survival (Figure 3A). Incubation of cells with IL-3 completely rescued 32DBcr-Ablwt cells from imatinib mesylate-induced apoptosis (Figure 3A), and G-CSF decreased the number of apoptotic 32DBcr-Ablwt cells upon imatinib mesylate treatment from 94.5% to 27.5%, as assessed by annexin V staining after 24 hours (Figure 3A). Moreover, proliferation of imatinib mesylate-treated 32DBcr-Ablwt cells in response to IL-3 or G-CSF was similar to the cell growth seen in parental 32Dcl3 cells (Figure 3B). These results demonstrated that 32DBcr-Ablwt cells expressed functional IL-3 and above allG-CSF receptors on
their surface and regained growth factor responsiveness after
inhibition of Bcr-Abl by imatinib mesylate.
To next investigate whether the block of granulocytic differentiation
observed in 32DBcr-Ablwt cells could be reversed by imatinib mesylate, cells were treated with imatinib mesylate and G-CSF
for 14 days. In the presence of imatinib mesylate,
32DBcr-Ablwt cells differentiated into morphologically
mature neutrophilic granulocytes within 8 to 11 days (Figure
4A). Morphologic differentiation was
accompanied by up-regulation of myeloid-specific surface markers Mac-1
and Gr-1 (Figure 4B). These data prove that the block of granulocytic
differentiation in 32DBcr-Ablwt cells depended on Bcr-Abl
kinase activity and could be reversed by the addition of the Abl kinase
inhibitor imatinib mesylate.
Bcr-Abl reversibly blocks G-CSF-induced up-regulation of
C/EBP , C/EBP , and
PU.1.9,10 To investigate whether Bcr-Abl influences the
induction of expression of these transcription factors after G-CSF
stimulation, parental 32Dcl3 cells and 32DBcr-Ablwt cells
were grown in the presence of G-CSF for 3 days. Moreover, an aliquot of
32DBcr-Ablwt cells was cultured in the presence of both
G-CSF and 1 µM imatinib mesylate. Cells grown in the
presence of IL-3 were used as a control (Figure 5 left panel, lane
1). Western blot and real-time PCR
analysis revealed that in parental 32Dcl3 cells, G-CSF stimulated the
expression of these transcription factors in a coordinated manner
(Figures 5-6). While considerable amounts of PU.1 were found expressed,
only low levels of C/EBP and no C/EBP expression were seen in
32Dcl3 cells grown with IL-3 (Figure 5 left panel, lane 1). G-CSF
induced a several-fold increase of C/EBP and a moderate
up-regulation of PU.1 protein and mRNA expression (Figure 5 left panel,
lanes 1-4, and 6). Moreover,
G-CSF stimulated the expression of C/EBP protein. On day 2, only a
faint signal of the 14-kDa C/EBP isoform missing the transactivation
domain was detected (Figure 5 left panel, lane 3). On day 3, 3 different isoforms of C/EBP were seen (Figure 5 left panel,
lane 4).
Analysis of 32DBcr-Ablwt cells revealed that baseline
expression of C/EBP Bcr-Abl reversibly blocks c-Myc down-regulation upon G-CSF stimulation C/EBP transcription factors and c-Myc are inversely regulated during differentiation of myeloid cells.17,24 We wished to investigate the influence of Bcr-Abl on the regulation of c-Myc by G-CSF. Therefore, 32Dcl3 and 32DBcr-Ablwt cells treated with G-CSF for 1 to 3 days were analyzed for c-Myc expression. In 32Dcl3 cells, G-CSF led to a decrease of c-Myc expression over a 3-day period (Figure 5 left panel, lanes 1-4). Interestingly, the time kinetics of c-Myc down-regulation correlated with the up-regulation of C/EBP and C/EBP (Figure 5 left panel). In
contrast, c-Myc expression was only minimally reduced in
32DBcr-Ablwt cells (Figure 5 left panel, lanes 5-8).
Appropriate c-Myc regulation could be restored by culturing cells in
the presence of 1 µM imatinib mesylate (Figure 5 right panel, lanes
5-8). Kinetics of C/EBP and C/EBP up-regulation and c-Myc
down-regulation of imatinib mesylate-treated 32DBcr-Ablwt
cells were similar to those of 32Dcl3 cells, suggesting that the
effects of Bcr-Abl on the regulation of these transcription factors
were reversed by imatinib mesylate immediately.
Bcr-Abl inhibits up-regulation of G-CSF receptor expression upon G-CSF stimulation Rapid up-regulation of the G-CSFR is part of the differentiation program induced by G-CSF,25 and G-CSFR mRNA expression is regulated by both PU.1 and C/EBP sites in the
G-CSFR promotor.25 Western blot analysis showed that
baseline expression of the G-CSF receptor was low but equivalent in
both 32Dcl3 and 32DBcr-Ablwt cells (Figures 2A, and 5 left
panel, lanes 1 and 5). Importantly, the obvious differences between the
expression levels of G-CSFR of unstimulated cells seen in Figures 2A
and 5 were due to different exposure times of blots. These were
necessary as a consequence of the massive up-regulation of G-CSFR
protein expression by G-CSF stimulation, but do not reflect a loss of
basal level expression of G-CSFR in 32D and 32DBcr-Ablwt
cells used for Figure 5 when compared with Figure 2. As expected, G-CSF
rapidly induced G-CSFR protein expression in 32Dcl3 cells within 24 hours (Figure 5 left panel, lanes 2-4), which was accompanied by
induction of G-CSFR mRNA expression as assessed by real-time PCR and
semiquantitative RT-PCR (Figure 6A right panel). In marked contrast,
G-CSFR up-regulation was completely disrupted in
32DBcr-Ablwt cells (Figure 5 left panel, lanes 5-8). Block
of up-regulation of G-CSFR expression correlated to a block in
up-regulation of G-CSFR mRNA (Figure 6). Up-regulation of both G-CSFR
protein and mRNA could be restored by growing cells in the presence
of 1 µM imatinib mesylate. Identical results were obtained for
both clones used in this study. Taken together, these results
demonstrate that Bcr-Abl reversibly blocks G-CSF-induced up-regulation
of G-CSF receptor expression at the transcriptional level, although
basal level of G-CSFR expression was not affected by Bcr-Abl.
Bcr-Abl disturbs the regulation of cyclin-dependent kinase inhibitors p21Waf1/Cip1 and p27Kip1 Coordinated cell cycle arrest is part of the differentiation program in different cell types. Therefore, we wished to investigate the influence of Bcr-Abl on the expression of cyclin-dependent kinase inhibitors (CDKIs) such as p21Waf1/Cip1 and p27Kip1. Both p27Kip1 and p21Waf1/Cip1 were expressed in 32Dcl3 cells, at least at low levels (Figure 7A left panel, lane 1), but only p27Kip1 was up-regulated transiently upon stimulation with G-CSF (Figure 7A left panel, lanes 1-4). In 32DBcr-Ablwt cells, baseline expression of p27Kip1 was slightly decreased compared with 32Dcl3 cells (Figure 7A left panel, lanes 1 and 5), but p27Kip1 was still up-regulated by G-CSF, albeit less so than in 32Dcl3 cells (Figure 7A left panel, lanes 5-8). Surprisingly, baseline expression of p21Waf1/Cip1 was markedly increased in 32DBcr-Ablwt cells when compared with 32Dcl3 cells (Figure 7A left panel, lanes 5-8). Because p21Waf1/Cip1 is known to be an inhibitor of cell cycle progression, we used immunofluorescence microscopy to investigate whether cell cycle progression in the presence of high levels of p21Waf1/Cip1 could be explained by altered subcellular localization. Surprisingly, no significant influence of Bcr-Abl on subcellular distribution of p21Waf1/Cip1 was seen (Figure 7B). As observed for transcription-factor regulation, addition of imatinib mesylate to 32DBcr-Ablwt cells also restored the appropriate regulation of expression of p21Waf1/Cip1 and p27Kip1 (Figure 7A right panel). The p27Kip1 was found transiently upregulated in imatinib mesylate-treated cells as well as in untreated cells in response to G-CSF (Figure 7A right panel). The p27Kip1 expression levels at day 3 after G-CSF stimulation seemed somewhat higher after imatinib mesylate treatment when compared with untreated 32DBcr-Ablwt cells and were approximately equivalent to the levels found in 32Dcl3 cells after G-CSF stimulation. Also, over a 3-day period of imatinib mesylate treatment, p21Waf1/Cip1 expression was down-regulated to levels found in parental 32Dcl3 cells (Figure 7A right panel, lane 8).
Restoration of G-CSF-induced granulocytic differentiation by imatinib mesylate is blocked by expression of an inhibitor-resistant mutant To investigate if restoration of G-CSF-induced differentiation by imatinib mesylate in 32D cells expressing Bcr-Abl was due to the inhibition of Bcr-Abl or instead represented inhibition of other relevant targets, mixed cell populations of 32Dcl3 cells were established by retroviral infection expressing either Bcr-Ablwt or a imatinib mesylate-resistant mutant of Bcr-Abl, Bcr-AblThr315Iso. This mutation has been isolated from patients with clinical imatinib mesylate resistance and has been shown to abolish imatinib mesylate binding to the ATP-binding site of Abl.8 Accordingly, tyrosine phosphorylation of cellular substrates could not be reversed by imatinib mesylate in cells expressing mutant Thr315Iso (Figure 8A). At 10 days after retroviral transduction, the 2 cell populations expressed comparable amounts of G-CSFR and C/EBP as assessed by Western blot analysis and real-time
PCR (data not shown). Yet, both Bcr-Ablwt and the imatinib
mesylate-resistant mutant Bcr-AblThr315Iso blocked granulocytic
differentiation induced by G-CSF as assessed by monitoring morphologic
differentiation (Figure 8B) and expression of granulocytic surface
markers (data not shown). However, in contrast to
32DBcr-Ablwt cells, a reversal of the block of
differentiation by imatinib mesylate was not seen in cells expressing
Bcr-AblThr315Iso, proving that granulocytic differentiation of
Bcr-Abl+ 32D cells in the presence of imatinib mesylate was
dependent on inhibition of Bcr-Abl but not of any other target.
As proof of this, we could show that imatinib mesylate did not reverse the block of differentiation induced by IL-3 (data not shown).
Although previous reports had suggested that in certain cell types
and experimental systems, Bcr-Abl would rather induce than inhibit myeloid differentiation,27,28 we and
others could show that in 32Dcl3 cells Bcr-Abl blocks G-CSF-induced
neutrophilic differentiation in a kinase-dependent
manner.18 Perrotti et al22 recently
reported that Bcr-Abl blocks G-CSF-induced differentiation of 32D
cells by down-regulating basal level expression of C/EBP Despite preserved basal level expression of C/EBP Our data imply that disturbed regulation of c-Myc expression might be
involved in blocking C/EBP transcription-factor up-regulation. It was
shown only recently that c-Myc expression is negatively regulated by
C/EBP However, disruption of c-Myc regulation might not be the only mechanism
by which Bcr-Abl interferes with differentiation. In our study,
imatinib mesylate restored up-regulation of G-CSFR expression in
Bcr-Abl+ 32Dcl3 cells within 24 hours, clearly preceding
restoration of C/EBP transcription-factor up-regulation and
down-regulation of c-Myc. Although it is possible that up-regulation of
G-CSF receptor expression in this system does not depend on C/EBP If Bcr-Abl directly interferes with myeloid transcription-factor regulation and differentiation, why do leukemic cells from CML patients in chronic phase show only discrete abnormalities of myeloid differentiation? One possible explanation is that secondary genetic alterations might be necessary to confer a full differentiation block to Bcr-Abl-expressing leukemic cells. The 32Dcl3 cells already harbor such mutations, and these cells have a profound proliferative defect as they grow continuously in culture with just the addition of IL-3 as a single growth factor. Although it is not clear whether 32D cells bear similar secondary genetic alterations such as are found in late-stage CML, this system might reflect blast crisis rather than chronic phase disease. Moreover, G-CSF is just one of several factors inducing myeloid and,
above all, neutrophilic differentiation. G-CSF Still, dysregulation of transcription factors such as C/EBP
We thank S. Reis for technical assistance, C. Kurzeder for advice on FACS analysis, E. Buchdunger (Novartis, Basel, Switzerland) for providing imatinib mesylate, and S. Nagata and U. Just for kindly providing reagents and cell lines.
Submitted August 16, 2002; accepted August 21, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-01-0043.
Supported by grants Ha 1680/2-3 and 1680/2-4 from the Deutsche Forschungsgemeinschaft (M.H.) and by grants from the Novartis Foundation for Therapeutic Research (M.H. and M.W.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Markus Warmuth, KKG Gentherapie,
GSF
1. Rowley J. A new consistent chromosomal abnormality in chronic myelogenous leukemia identified by quinacrine fluorescence and Giemsa staining. Nature. 1973;243:290-293[CrossRef][Medline] [Order article via Infotrieve].
2.
Faderl S, Talpaz M, Estrov Z, O'Brien S, Kurzrock R, Kantarjian HM.
The biology of chronic myeloid leukemia.
N Engl J Med.
1999;341:164-172 3. Yamamoto K, Nakamura Y, Saito K, Furusawa S. Expression of the NUP98/HOXA9 fusion transcript in the blast crisis of Philadelphia chromosome-positive chronic myelogenous leukaemia with t(7;11)(p15;p15). Br J Haematol. 2000;109:423-426[CrossRef][Medline] [Order article via Infotrieve]. 4. Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;2:561-566[CrossRef][Medline] [Order article via Infotrieve].
5.
Druker BJ, Talpaz M, Resta DJ, et al.
Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia.
N Engl J Med.
2001;344:1031-1037
6.
Kantarjian H, Sawyers C, Hochhaus A, et al.
Hematologic and cytogenetic responses to imatinib mesylate in chronic myelogenous leukemia.
N Engl J Med.
2002;346:645-652
7.
Druker BJ, Sawyers CL, Kantarjian H, et al.
Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.
N Engl J Med.
2001;344:1038-1042
8.
Gorre ME, Mohammed M, Ellwood K, et al.
Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification.
Science.
2001;293:876-880
9.
Tenen DG, Hromas R, Licht JD, Zhang DE.
Transcription factors, normal myeloid development, and leukemia.
Blood.
1997;90:489-519 10. Ward AC, Loeb DM, Soede-Bobok AA, Touw IP, Friedman AD. Regulation of granulopoiesis by transcription factors and cytokine signals. Leukemia. 2000;14:973-990[CrossRef][Medline] [Order article via Infotrieve].
11.
Lieschke GJ, Grail D, Hodgson G, et al.
Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.
Blood.
1994;84:1737-1746 12. Liu F, Wu HY, Wesselschmidt R, Kornaga T, Link DC. Impaired production and increased apoptosis of neutrophils in granulocyte colony-stimulating factor receptor-deficient mice. Immunity. 1996;5:491-501[CrossRef][Medline] [Order article via Infotrieve].
13.
Zhang DE, Zhang P, Wang ND, Hetherington CJ, Darlington GJ, Tenen DG.
Absence of granulocyte colony-stimulating factor signaling and neutrophil development in CCAAT enhancer binding protein alpha-deficient mice.
Proc Natl Acad Sci U S A.
1997;94:569-574
14.
Wang X, Scott E, Sawyers CL, Friedman AD.
C/EBPalpha bypasses granulocyte colony-stimulating factor signals to rapidly induce PU.1 gene expression, stimulate granulocytic differentiation, and limit proliferation in 32D cl3 myeloblasts.
Blood.
1999;94:560-571
15.
Yamanaka R, Barlow C, Lekstrom-Himes J, et al.
Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice.
Proc Natl Acad Sci U S A.
1997;94:13187-13192
16.
Scott LM, Civin CI, Rorth P, Friedman AD.
A novel temporal expression pattern of three C/EBP family members in differentiating myelomonocytic cells.
Blood.
1992;80:1725-1735
17.
Nakajima H, Ihle JN.
Granulocyte colony-stimulating factor regulates myeloid differentiation through CCAAT/enhancer-binding protein epsilon.
Blood.
2001;98:897-905 18. Perrotti D, Bonatti S, Trotta R, et al. TLS/FUS, a pro-oncogene involved in multiple chromosomal translocations, is a novel regulator of BCR/ABL-mediated leukemogenesis. EMBO J. 1998;17:4442-4455[CrossRef][Medline] [Order article via Infotrieve].
19.
Warmuth M, Bergmann M, Priess A, Häuslmann K, Emmerich B, Hallek M.
The Src family kinase Hck interacts with Bcr-Abl by a kinase-independent mechanism and phosphorylates the Grb2-binding site of Bcr.
J Biol Chem.
1997;272:33260-33270
20.
Warmuth M, Simon N, Mitina O, et al.
Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate-resistant Bcr-Abl kinases.
Blood.
2003;101:664-672
21.
Kumano K, Chiba S, Shimizu K, et al.
Notch1 inhibits differentiation of hematopoietic cells by sustaining GATA-2 expression.
Blood.
2001;98:3283-3289 22. Perrotti D, Cesi V, Trotta R, et al. BCR-ABL suppresses C/EBPalpha expression through inhibitory action of hnRNP E2. Nat Genet. 2002;30:48-58[CrossRef][Medline] [Order article via Infotrieve]. 23. Valtieri M, Tweardy DJ, Caracciolo D, et al. Cytokine-dependent granulocytic differentiation: regulation of proliferative and differentiative responses in a murine progenitor cell line. J Immunol. 1987;138:3829-3835[Abstract].
24.
Johansen LM, Iwama A, Lodie TA, et al.
c-Myc is a critical target for c/EBPalpha in granulopoiesis.
Mol Cell Biol.
2001;21:3789-3806
25.
Steinman RA, Tweardy DJ.
Granulocyte colony-stimulating factor receptor mRNA upregulation is an immediate early marker of myeloid differentiation and exhibits dysfunctional regulation in leukemic cells.
Blood.
1994;83:119-127
26.
Smith LT, Hohaus S, Gonzalez DA, Dziennis SE, Tenen DG.
PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells.
Blood.
1996;88:1234-1247 27. Cambier N, Zhang Y, Vairo G, et al. Expression of BCR-ABL in M1 myeloid leukemia cells induces differentiation without arresting proliferation. Oncogene. 1999;18:343-352[CrossRef][Medline] [Order article via Infotrieve].
28.
Era T, Witte ON.
Regulated expression of P210 Bcr-Abl during embryonic stem cell differentiation stimulates multipotential progenitor expansion and myeloid cell fate.
Proc Natl Acad Sci U S A.
2000;97:1737-1742 29. Pabst T, Mueller BU, Harakawa N, et al. AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med. 2001;7:444-451[CrossRef][Medline] [Order article via Infotrieve]. 30. Pabst T, Mueller BU, Zhang P, et al. Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet. 2001;27:263-270[CrossRef][Medline] [Order article via Infotrieve].
31.
Wang QF, Friedman AD.
CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor.
Blood.
2002;99:2776-2785
32.
Mink S, Mutschler B, Weiskirchen R, Bister K, Klempnauer KH.
A novel function for Myc: inhibition of C/EBP-dependent gene activation.
Proc Natl Acad Sci U S A.
1996;93:6635-6640
33.
Collins SJ, Ulmer J, Purton LE, Darlington G.
Multipotent hematopoietic cell lines derived from C/EBPalpha(-/-) knockout mice display granulocyte macrophage-colony-stimulating factor, granulocyte- colony-stimulating factor, and retinoic acid-induced granulocytic differentiation.
Blood.
2001;98:2382-2388
34.
Ross SE, Erickson RL, Hemati N, MacDougald OA.
Glycogen synthase kinase 3 is an insulin-regulated C/EBPalpha kinase.
Mol Cell Biol.
1999;19:8433-8441 35. Verbeek W, Wachter M, Lekstrom-Himes J, Koeffler HP. C/EBPepsilon -/- mice: increased rate of myeloid proliferation and apoptosis. Leukemia. 2001;15:103-111[CrossRef][Medline] [Order article via Infotrieve].
36.
Harris TE, Albrecht JH, Nakanishi M, Darlington GJ.
CCAAT/enhancer-binding protein-alpha cooperates with p21 to inhibit cyclin-dependent kinase-2 activity and induces growth arrest independent of DNA binding.
J Biol Chem.
2001;276:29200-29209 37. Wang H, Iakova P, Wilde M, et al. C/EBPalpha arrests cell proliferation through direct inhibition of Cdk2 and Cdk4. Mol Cell. 2001;8:817-828[CrossRef][Medline] [Order article via Infotrieve]. 38. Wang H, Goode T, Iakova P, Albrecht JH, Timchenko NA. C/EBPalpha triggers proteasome-dependent degradation of cdk4 during growth arrest. EMBO J. 2002;21:930-941[CrossRef][Medline] [Order article via Infotrieve].
© 2003 by The American Society of Hematology.
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  J. S. Chang, R. Santhanam, R. Trotta, P. Neviani, A. M. Eiring, E. Briercheck, M. Ronchetti, D. C. Roy, B. Calabretta, M. A. Caligiuri, et al. High levels of the BCR/ABL oncoprotein are required for the MAPK-hnRNP-E2 dependent suppression of C/EBP{alpha}-driven myeloid differentiation Blood, August 1, 2007; 110(3): 994 - 1003. [Abstract] [Full Text] [PDF]
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G. Ferrari-Amorotti, K. Keeshan, M. Zattoni, C. Guerzoni, G. Iotti, S. Cattelani, N. J. Donato, and B. Calabretta Leukemogenesis induced by wild-type and STI571-resistant BCR/ABL is potently suppressed by C/EBP{alpha} Blood, August 15, 2006; 108(4): 1353 - 1362. [Abstract] [Full Text] [PDF]
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C. Guerzoni, M. Bardini, S. A. Mariani, G. Ferrari-Amorotti, P. Neviani, M. L. Panno, Y. Zhang, R. Martinez, D. Perrotti, and B. Calabretta Inducible activation of CEBPB, a gene negatively regulated by BCR/ABL, inhibits proliferation and promotes differentiation of BCR/ABL-expressing cells Blood, May 15, 2006; 107(10): 4080 - 4089. [Abstract] [Full Text] [PDF]
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 J.-F. Geay, D. Buet, Y. Zhang, A. Foudi, P. Jarrier, M. Berthebaud, A. G. Turhan, W. Vainchenker, and F. Louache p210BCR-ABL inhibits SDF-1 Chemotactic Response via Alteration of CXCR4 Signaling and Down-regulation of CXCR4 Expression Cancer Res., April 1, 2005; 65(7): 2676 - 2683. [Abstract] [Full Text] [PDF]
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S. Gery, A. F. Gombart, Y. K. Fung, and H. P. Koeffler C/EBP{epsilon} interacts with retinoblastoma and E2F1 during granulopoiesis Blood, February 1, 2004; 103(3): 828 - 835. [Abstract] [Full Text] [PDF]
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F. E. Ouriaghli, E. Sloand, L. Mainwaring, H. Fujiwara, K. Keyvanfar, J. J. Melenhorst, K. Rezvani, G. Sconocchia, S. Solomon, N. Hensel, et al. Clonal dominance of chronic myelogenous leukemia is associated with diminished sensitivity to the antiproliferative effects of neutrophil elastase Blood, November 15, 2003; 102(10): 3786 - 3792. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
-tubulin were purchased from Oncogene Sciences (Uniondale, NJ) and
Roche Molecular Biochemical (Mannheim, Germany).
/