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NEOPLASIA
From the Rosenstiel Basic Medical Sciences Research
Center, Department of Biochemistry, and Department of Biology, Brandeis
University, Waltham, MA; and Department of Pathology and Institute for
Medicine and Engineering, University of Pennsylvania, Philadelphia, PA.
Bcr-Abl plays a critical role in the pathogenesis of chronic
myelogenous leukemia (CML). It was previously shown that expression of
Bcr-Abl in bone marrow cells by retroviral transduction efficiently induces a myeloproliferative disorder (MPD) in mice resembling human
CML. This in vivo experimental system allows the direct determination
of the effect of specific domains of Bcr-Abl, or specific signaling
pathways, on the complex in vivo pathogenesis of CML. In this report,
the function of the SH2 domain of Bcr-Abl in the pathogenesis of CML is
examined using this murine model. It was found that the Bcr-Abl SH2
mutants retain the ability to induce a fatal MPD but with an extended
latency compared with wild type (wt) Bcr-Abl. Interestingly, in
contrast to wt Bcr-Abl-induced disease, which is rapid and monophasic,
the disease caused by the Bcr-Abl SH2 mutants is biphasic, consisting
of an initial B-lymphocyte expansion followed by a fatal myeloid
proliferation. The B-lymphoid expansion was diminished in mixing
experiments with bcr-abl/ The bcr-abl oncogene is produced when
the break-point cluster region gene (c-bcr) sequences on
chromosome 22 are fused to c-abl sequences on chromosome 9 by a reciprocal translocation.1 The Bcr-Abl fusion protein
is present in nearly all patients with chronic myelogenous leukemia
(CML) and in some patients with acute lymphoblastic leukemia. Depending
on the nature of the translocation and exactly how the bcr
and abl sequences become spliced into a final
bcr-abl messenger RNA, various Bcr-Abl fusion proteins including p185, p210, and p230 can be generated, which show a preferential association with different types of
leukemia.2
CML is a clonal myeloproliferative disorder (MPD) resulting from the
neoplastic transformation of hematopoietic stem cells.1,3 The disease usually has a biphasic course, comprising a chronic phase
and a blast phase. The initial chronic phase is characterized by
accumulation of large numbers of myeloid-lineage cells predominated by
granulocytes in peripheral blood (PB), bone marrow (BM), and spleen. It
is not clear why CML manifests as an MPD, even though Bcr-Abl is
expressed in the hematopoietic stem cell compartment in humans and
Bcr-Abl has the capacity to transform nearly all hematopoietic
elements. Progression of the disease after 3 to 5 years to terminal
blast phase, often through an accelerated phase, is characterized by
accelerated accumulation of immature myeloid or lymphoid cells. Bcr-Abl
is important in both initiation and maintenance of neoplastic
transformation4; however, disease progression to blast
crisis likely requires additional mutations.1,5
Bcr-Abl contains many domains/motifs that regulate and mediate its
function. Abl-derived sequences from Bcr-Abl contain Src-homology-3 (SH3), SH2, and tyrosine kinase domains in its N-terminal half, as well
as a DNA binding domain, an actin binding domain, nuclear localization
signals, and SH3 binding sites in its C-terminal region.6
The Bcr region of Bcr-Abl/p210 contains a coiled-coil oligomerization
domain, a serine/threonine kinase domain, a pleckstrin homology domain,
a Dbl/CDC24 guanine-nucleotide exchange factor homology domain, and
several serine/threonine and tyrosine phosphorylation sites and binding
sites for the Abl SH2 domain, Grb2, Grb10, and 14-3-3 proteins.7,8 Defining the roles of these
domains/motifs of Bcr-Abl is critical for understanding the molecular
mechanism of Bcr-Abl leukemogenesis.
The SH2 domain is a modular unit present in a wide variety of signaling
molecules.9,10 It mediates specific protein-protein interactions by binding phosphotyrosine-containing peptides. Some SH2
domains can also bind peptides in a phosphotyrosine-independent manner
and some can even bind phospholipids.11-14 The SH2 domains of intracellular protein-tyrosine kinases play a role in the catalytic function of the kinases.15,16 They may contribute to the
kinase substrate specificity by protecting the substrates from
dephosphorylation, localizing them to a specific subcellular location,
or facilitating processive phosphorylation of multiple tyrosine
residues in the same protein.17 The SH2 domain plays an
important role in the Abl proteins in interacting with and
phosphorylating signaling proteins, including p62dok, c-Cbl, Rin-1,
Tub, and mDab1.18-24 It can also interact with Bcr
sequences and Shc through phosphotyrosine-independent interactions.11,25
The Abl SH2 domain is required for transformation of cultured
fibroblast cells by Bcr-Abl.26 However, mutations in the
Abl SH2 domain do not diminish the ability of Bcr-Abl to render
cytokine-independent growth of factor-dependent hematopoietic cell
lines.27-31 In addition, Bcr-Abl SH2 mutants can transform
primary lymphoid progenitors in vitro as well as wild-type (wt)
Bcr-Abl.28 These conflicting observations in cultured
cells suggest that the requirement for the SH2 domain is cell type- or
context-dependent. Attempts have also been made to assess the role of
the SH2 domain in Bcr-Abl leukemogenesis in vivo. It was shown that the
pre-B-lymphoid cells transformed by Bcr-Abl SH2 mutants were poorly
tumorigenic in immunodeficient mice.28 The SH2 domain of
Bcr-Abl was also shown to be required for developing a myeloid leukemia
in mice due to failure to activate phosphatidylinositol (PI)-3
kinase/Akt pathway.32 However, in earlier models, Bcr-Abl
does not effectively induce CML-like MPD,33-37 making it
difficult to conclude whether the SH2 domain plays a role in
pathogenesis of CML.
We and others recently have shown that expression of Bcr-Abl in BM
cells by retroviral transduction efficiently induces an MPD in mice
resembling human CML.38-40 This murine model for CML provides an effective in vivo experimental system to study the roles
and relative importance of domains of Bcr-Abl and of signaling events
affected by Bcr-Abl in leukemogenesis.41 In this report, we used our in vivo model to study the function of Bcr-Abl SH2 domain
in the pathogenesis of CML. We found that SH2 mutations slowed the
onset of, but did not prevent, Bcr-Abl-induced MPD. The phenotype of
the disease induced by Bcr-Abl SH2 mutants differed from wt Bcr-Abl
disease in that the SH2 mutants induced a B-lymphoproliferative disorder prior to the fatal MPD. The B-cell lymphocytosis could be
suppressed by wt Bcr-Abl-induced MPD in a mixing experiment, suggesting that Bcr-Abl-induced MPD suppresses B-lymphoid expansion. This may provide a clue to the specificity of MPD induction by Bcr-Abl
in CML.
DNA constructs
Cell culture and retrovirus preparation
BM transduction and transplantation BM cell transduction and transplantation were performed as previously described.38Flow cytometry and cell sorting Flow cytometry and cell sorting were performed as described.38,41Southern blot PB obtained from the orbital sinus or tail and dispersed cells from spleen were treated with red blood cell lysis solution ACK (150 mM NH4Cl, 1 mM KHCO3, 0.1 mM Na2 ethylenediaminetetraacetic acid, pH 7.3). High molecular weight DNA from the white blood cells (WBCs) and sorted splenocytes was isolated by using the QIAamp Blood Kit (Qiagen). For proviral integration analysis, up to 15 µg DNA was digested with either EcoRI, BamHI, or BglII, separated on a 1% agarose gel, transferred to Hybond-N+ membrane (Amersham, Arlington Heights, IL), and hybridized with a probe containing IRES-gfp sequences derived from the retroviral vector as described.38 For determining the contribution of bcr-abl- and bcr-abl/ SH2-transduced cells in BM mixing experiments,
genomic DNA from PB was digested with HindIII and separated
on a 0.7% agarose gel, transferred to Hybond-N+ membrane,
and hybridized with a probe corresponding to a 1.2-kilobase (kb)
Eco47III-BglII fragment of the 3' end of human
c-abl complementary DNA. The washed membrane was exposed to
X-ray film.
Immunoblotting NIH3T3 cells were transduced with titer-matched viruses as described above. Two days after transduction, cells were serum-starved with DMEM containing 0.1% calf serum for 12 hours. The cells were then collected, washed once in ice-cold phosphate-buffered-saline (PBS) (Gibco BRL), resuspended in certain volume in ice-cold PBS, and boiled for 5 minutes in an equal volume of 2 × sodium dodecyl sulfate (SDS) sample buffer as described.43 The ACK-treated spleen cells were resuspended in ice-cold PBS at 2 × 106 cells/mL, lysed by adding equal volume of 2 × SDS sample buffer, and heated at 100°C for 5 minutes. Cell debris was cleared by centrifugation. Equal amounts of total protein of each lysate were run on 6% to 15% SDS-polyacrylamide gradient gels and transferred to nitrocellulose filters. Protein blots were probed with antibodies as described previously,41 with the exception that the signal transducer and activator of transcription (STAT)5 antibody was purchased from Transduction Laboratories (Lexington, KY). Bound antibodies were visualized using horseradish peroxidase-conjugated antimouse or antirabbit immunoglobulin G (IgG) and enhanced chemiluminescence reagents as described by the manufacturer (Amersham).Immunokinase assay Retroviral constructs were transfected into BOSC 23 cells as described.42 Two days after transfection, cells were lysed in lysis buffer (50 mM HEPES, pH7.4; 150 mM NaCl; 10% glycerol; 1% Triton X-100; 1 mM EGTA; 1.5 mM MgCl2; 1 mM dithiothreitol [DTT]; 10 mM NaF; 1 mM sodium orthovanadate; 1 mM freshly made phenylmenthylsulfonyl fluoride; 1 × complete protease inhibitor cocktail [Boehringer Mannheim, Indianapolis, IN]). Cell lysates were quantified with the Coomassie Protein Assay Reagent (Pierce, Rockford, IL) and adjusted to equal concentration with the above lysis buffer. One-milligram total proteins (in 500 µL) was immunoprecipitated with anti-Abl antibody Ab-3 as described.43 Immunoprecipitates were washed 3 times in lysis buffer and twice in kinase buffer (10 mM MgCl2, 1 mM DTT, 50 mM HEPES) and were then aliquoted equally into 3 Eppendorf tubes: 1 for Western blotting and 2 for kinase assays with substrate glutathione-S-transferase (GST)-10a and GST-Crk-II (Long and Ren, unpublished data, 1996), respectively. The p10a was isolated as a Src-SH3 binding polypeptide and subsequently was shown to be part of a signal integrating protein, Sin.44,45 It contains 92 amino acids including a single tyrosyl residue followed by amino acids Asp-Val-Pro. We found that p10a can be phosphorylated by the Abl protein tyrosine kinase in vitro (Long and Ren, unpublished data, 1996). The kinase assay was performed in total volume of 30 µL containing 1 × kinase buffer, 1 µg substrate, 0.5 mM adenosine triphosphate (ATP), and 1.85 × 105 Bq (5 µCi) _[32P]ATP for 30 minutes at room
temperature. Kinase reactions were terminated by adding 30 µL 2 ×
SDS sample buffer and heating at 100°C for 10 minutes. Equal amounts
of supernatant were analyzed by SDS-polyacrylamide gel electrophoresis
and autoradiography.
Enzyme-linked immunosorbent assay for IL-3 and GM-CSF PB from the orbital sinus was collected into an Eppendorf tube, incubated at room temperature for 4 hours, and then incubated at 4°C overnight. The samples were spun at 16 000 rpm in a microcentrifuge at 4°C. The supernatant was transferred into a new tube and frozen at 70°C until use. The serum levels of interleukin (IL)-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were assayed
using mouse IL-3 and GM-CSF enzyme-linked immunosorbent assay
(ELISA) kit from R & D Systems (Minneapolis, MN) and Endogen (Woburn,
MA), respectively.
Bcr-Abl with mutations in its SH2 domain causes a transient B-lymphoproliferative disorder and a delayed MPD in mice Two Bcr-Abl SH2 mutants were made to study the role of the SH2 domain in Bcr-Abl-mediated leukemogenesis. Bcr-Abl/R1057K is Bcr-Abl with a single arginine-to-lysine mutation (R1057K) in the conserved FLVRES motif of the SH2 domain. This mutation has been shown to significantly reduce the ability of the SH2 domain to bind tyrosine-phosphorylated proteins but does not affect the overall NMR structure of the SH2 domain.46 Bcr-Abl/SH2 is Bcr-Abl with its SH2 domain deleted. To examine the effect of the SH2 mutations
on the expression of Bcr-Abl, NIH3T3 cells were transduced with
titer-matched retroviruses containing wt bcr-abl or its SH2 mutants. Western blot analysis of the lysates of these cells
showed that wt Bcr-Abl, Bcr-Abl/R1057K, and Bcr-Abl/SH2
expressed at the same level in transduced NIH3T3 cells (Figure
1A).
Bcr-Abl and SH2 mutants were transiently expressed in 293T cells, and
the kinase activity of the immunoprecipitated Bcr-Abl proteins were
measured by an in vitro kinase assay. As shown in Figure 1B, both
Bcr-Abl/R1057K and wt Bcr-Abl were equivalent in their ability to
phosphorylate themselves and exogenous substrates Crk-II (Figure 1Bi,
compare lanes 1 and 3) and 10a (Figure 1Bii, compare lanes 1 and 3). In
contrast, deletion of the SH2 domain showed a slight decrease in the
ability to phosphorylate exogenous substrates (Figure 1Bi, Bii, lane 4)
and a more significant reduction in autophosphorylation of
Bcr-Abl/ To examine the leukemogenicity of the SH2 mutants of Bcr-Abl in vivo,
we transduced 5-fluorouracil-treated mouse BM cells in vitro
with titer-matched retroviruses containing wt bcr-abl or its
SH2 mutants and transplanted these cells into lethally irradiated
syngeneic recipient mice as previously described.38 Mice
transplanted with BM cells transduced with wt bcr-abl virus (Bcr-Abl mice) died within 3 weeks after BM transplantation (BMT) with
characteristics of the CML-like syndrome previously
described38 (Figure 2).
Bcr-Abl/R1057K mice and Bcr-Abl/
The Bcr-Abl/R1057K and Bcr-Abl/
A detailed immunophenotypic analysis was performed on Bcr-Abl/ Analysis of Bcr-Abl/ To further reveal the kinetics of the expansion of myeloid and
B-lymphoid cells in wt Bcr-Abl mice and Bcr-Abl/
The average number of B-lymphoid cells in Bcr-Abl/ Bcr-Abl-induced MPD suppresses Bcr-Abl/ To test this possibility, we transduced BM cells with titer-matched
bcr-abl virus and bcr-abl/
At day 22 after BMT, Bcr-Abl/ There are 2 possibilities that can account for fewer B-lymphoid cells
in the 1:4 mix and 1:8 mix BMT mice. One is that the expansion of
myeloid cells suppresses or competes with the expansion of B-lymphoid
cells as suggested above. Alternatively, Bcr-Abl-induced disease
suppresses establishment of Bcr-Abl/ To distinguish between these possibilities, we investigated the
relative amounts of bcr-abl-transduced and
bcr-abl/ As shown in Figure 5C, only the upper band (4.6 kb) is detected in DNA
from a Bcr-Abl mouse (Figure 5C, lane 1) and only the lower band (4.3 kb) in DNA from a Bcr-Abl/ The same bcr-abl/ SH2 induces both B-lymphoproliferative disorder and
MPD in mice. We wondered whether the Bcr-Abl/SH2 myeloid and
lymphoid cells originated from the same progenitor cells. To address
this question, we purified GFP+CD19+ cells and
GFP+Mac-1+ cells from the same Bcr-Abl/SH2
mice by FACSorter. We then checked the proviral integration pattern in
these cells by Southern blot analysis after digestion with restriction
enzyme EcoRI (Figure 6A). Like wt Bcr-Abl
mice,38 multiple clones were expanded in primary
Bcr-Abl/SH2 mice (multiple bands with different intensities) (Figure
6A, lanes 3,6). Among the multiple clones from mouse BMT11.53, some
were contributed only by B-lymphoid cells (Figure 6A, compare lanes 6 and 8), and some were contributed only by myeloid cells (Figure 6A,
compare lanes 6 and 7). However, there was a common band in both
B-lymphoid and myeloid cells (Figure 6A, arrowhead). A common
band was also found in myeloid cells and B-lymphoid cells in
Bcr-Abl/SH2 mouse BMT11.34 (Figure 6A; lanes 1,2; asterisk). These
results suggest that bcr-abl/SH2 virus can be targeted into multipotential hematopoietic progenitor cells that give rise to both myeloid and B-lymphoid cells.
We also found that the Bcr-Abl/ Bcr-Abl SH2 mutants retain the ability to induce overproduction of IL-3 and GM-CSF in mice We have previously found that in mice with Bcr-Abl-induced myeloproliferative disease, the bcr-abl virus-infected cells expressed excess IL-3 and GM-CSF.38 This finding is consistent with the reports that Bcr-Abl can induce production of IL-3 and GM-CSF in human and mouse myeloid cell lines52-54 and that serum levels of GM-CSF in CML patients and gene expression of IL-3 in primitive CML progenitor cells are often increased.55-58 It is possible that overproduction of hematopoietic growth factors contributes to the neoplastic expansion of myeloid cells in CML. It has been reported that the Abl SH2 domain was required for Bcr-Abl-induced IL-3 production in FDCP-1 myeloid cell line.54 It is therefore possible that lack of the ability of induction of IL-3 and/or GM-CSF in Bcr-Abl SH2 mutants may be responsible for their changed disease latency and phenotype. To test this possibility, we examined the production of IL-3 and GM-CSF in Bcr-Abl SH2 mutant mice, compared with wt Bcr-Abl mice and vector control mice, at different disease developmental stages. Consistent with our previous results, wt Bcr-Abl induced overproduction of IL-3 and GM-CSF in mice (Table 1). Interestingly, we detected a similar amount of IL-3 and GM-CSF in either Bcr-Abl/R1057K or Bcr-Abl/SH2 mice as in Bcr-Abl mice (Table
1). This result demonstrated that the SH2 domain of Bcr-Abl is not
required to induce overproduction of IL-3 and GM-CSF in vivo.
As we showed earlier, Bcr-Abl SH2 mutants induced an initial B
lymphoproliferation followed by a fatal myeloproliferation. We wondered
whether there were different amounts of IL-3 and GM-CSF induced at
different disease development stages. Among the 12 It was shown that the SH2 domain of Bcr-Abl was required both for
activation of Akt and cellular transformation.32 To test whether the differences in the disease phenotype of Bcr-Abl SH2 mutants
versus wt Bcr-Abl are due to their ability to activate Akt, we examined
the activation of Akt in diseased mice using an antibody that
recognizes the activation-specific phosphorylated site of the protein.
Figure 7A shows that Akt is expressed and phosphorylated at a similar level in hematopoietic cells isolated from
wt Bcr-Abl, Bcr-Abl SH2 mutant, and vector mice. Similar results were
also seen in serum-starved NIH3T3 cells containing wt Bcr-Abl, Bcr-Abl
SH2 mutants, or vector alone (Figure 7B). Because Akt is activated in
vector control mice and NIH3T3 cells, it is not clear whether Akt can
be activated by Bcr-Abl SH2 mutants in these cells, but our results
suggest that the changed disease phenotype of Bcr-Abl SH2 mutants may
not be due to the ability of the mutants to activate Akt.
Activation of Ras and STAT5 has also been shown to be important for Bcr-Abl transformation.1 We went on to examine the activation of extracellular signal-regulated kinase (Erk)1/2, a major downstream signaling protein of the Ras pathway, and STAT5 using antibodies that recognize activation-specific phosphorylated sites of these signaling proteins. Figure 7A shows that p42/Erk2 is phosphorylated in cells isolated from both wt Bcr-Abl and Bcr-Abl SH2 mutant mice. Both p44/Erk1 and p42/Erk2 were activated in cells isolated from vector control mice (Figure 7B, lanes 1,2). Both p44/Erk1 and p42/Erk2 were also activated in NIH3T3 cells containing wt Bcr-Abl, Bcr-Abl SH2 mutants, or vector alone (Figure 7B). It is not clear why p44/Erk1 was not activated in cells isolated from diseased Bcr-Abl mice. Activation of STAT5 was detected in total spleen cells from both wt
Bcr-Abl and Bcr-Abl SH2 mutant mice but not in spleen cells from the
vector control mice (Figure 7A), confirming that Bcr-Abl proteins can
induce STAT5 activation directly or indirectly (ie, through induced
cytokine signaling) or both. To evaluate the ability of Bcr-Abl SH2
mutants to activate STAT5 directly, we measured the amount of activated
phospho-STAT5 in NIH3T3 cells infected with wt Bcr-Abl, Bcr-Abl/R1057K,
Bcr-Abl/
Our results show that the SH2 domain of Bcr-Abl is not required to induce a fatal MPD. However, the signaling pathways initiated by the SH2 domain of Bcr-Abl influence the disease latency and phenotype. Most strikingly, in contrast to wt Bcr-Abl-induced disease, which is rapid and monophasic, the disease caused by the Bcr-Abl SH2 mutants is biphasic, consisting of an initial B-lymphocyte expansion followed by a fatal myeloid proliferation. Although the degree of lymphocytosis is severe, it can be suppressed by Bcr-Abl-induced MPD. The delayed expansion of myeloid cells in Bcr-Abl SH2 mutant mice is unlikely caused by the preceding B-lymphoproliferative disorder for the following 2 reasons: (1) Bcr-Abl SH2 mutants induced the outgrowth of fewer myeloid colonies from 5-fluorouracil-treated BM cells than wt Bcr-Abl in BM colony assays (data not shown); and (2) the B-lymphoproliferative disorder did not delay the MPD induced by wt Bcr-Abl; instead it was suppressed by Bcr-Abl-induced MPD in BM mixing experiments (Figure 5). These observations indicate that Bcr-Abl SH2 mutants have a diminished ability to stimulate myeloid proliferation. The mechanism underlying the differences in the diseases caused by the
SH2 mutants and wt Bcr-Abl is not completely understood. It appears
unlikely to be caused by differences in tyrosine kinase activity or
differences in retroviral titer. In support of the former, both the SH2
point mutant and deletion mutant cause nearly identical clinical
diseases in mice. Despite the in vitro kinase activity being lower in
the deletion mutant, the activity in the point mutant is similar to wt
Bcr-Abl (Figure 1). The lower kinase activity in the deletion mutant
may only account for a further delay of disease onset in Bcr-Abl/ It has been reported that the Abl SH2 domain was required for
Bcr-Abl-induced IL-3 production in the FDCP-1 myeloid cell
line.54 However, we detected similar levels of IL-3 and
GM-CSF in serum of both Bcr-Abl and Bcr-Abl SH2 mutant mice (Table 1).
We also found that Bcr-Abl SH2 mutants induced expansion of a large
population of bystander (GFP The SH2 domain of Bcr-Abl has been shown to play an important role in
interacting with and regulating phosphorylation of signaling proteins, including p62dok, c-Cbl, Rin-1, and Shc.18-22,25
The SH2 mutations abrogate the ability of Bcr-Abl to either interact or
phosphorylate these proteins, which may affect multiple downstream signaling pathways. Of particular interest, the SH2 domain of Bcr-Abl
has been shown to be required both for activation of the PI-3
kinase/Akt pathway and cellular transformation.32 However, we found that Akt was activated in Bcr-Abl SH2 mutant cells in mice
(Figure 7A). Although we do not know whether Akt was activated by
Bcr-Abl SH2 mutants or by growth factor signaling within mice or by
both, the ability of Bcr-Abl SH2 mutants to activate Akt appears not to
account for the changed disease latency and phenotype in Bcr-Abl SH2
mutant mice. Similarly, there was no significant difference in Erk
activation in cells isolated from both wt Bcr-Abl and Bcr-Abl SH2
mutant mice (Figure 7A). In contrast to the aforementioned signaling
pathways, Bcr-Abl/ CML is a clonal multilineage MPD. Although hypercellularity occurs in
multiple lineages such as Ph+ erythroid and B-lymphoid
cells, granulocytosis is predominant in CML patients. A similar
phenomenon is observed in the murine model for CML.38-40
It is not clear why the disease is manifested as an MPD, even though
Bcr-Abl is expressed in the hematopoietic stem cell compartment in
humans and Bcr-Abl has the capacity to transform nearly all
hematopoietic elements. Our findings that SH2 mutants of Bcr-Abl
induced a transient B-lymphoproliferative disorder prior to a delayed
MPD and that the lymphocytosis can be suppressed by Bcr-Abl-induced
MPD (Figures 4,5) indicate that the Bcr-Abl-induced MPD suppresses
lymphoid expansion. Suppression of the B-lymphoid expansion by the
rapidly occurring MPD may be one of the mechanisms that account for the
lack of B-lymphoproliferative disorder observed in CML patients and in
Bcr-Abl mice. One possible mechanism underlying the suppression of B
lymphoproliferation by Bcr-Abl-induced MPD is that myeloid cells may
compete with lymphoid cells for hematopoietic space or cytokines
required for proliferation of both cell types. Alternatively, Bcr-Abl
may directly or indirectly induce production of inhibitors for
lymphoproliferation. A similar mechanism may prevent erythroid
expansion, because Bcr-Abl is capable of functionally replacing the
erythropoietin receptor (EpoR) and supporting proliferation,
differentiation, and maturation of fetal liver erythroid progenitors
from EpoR In several in vivo models, Bcr-Abl induces a pre-B (IgM The TEL/PDGF
We thank Ben Hentel and Jonathan Schatz for their help on flow cytometry.
Submitted May 1, 2000; accepted October 5, 2000.
Supported by ACS grant RPG-97-131-01-LBC (R.R.) and NIH RO1 CA77570-01 (W.S.P.). W.S.P. and R.R. are recipients of the Leukemia and Lymphoma Society Scholar Award.
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: Ruibao Ren, Rosenstiel Basic Medical Sciences Research Center, MS 029, Brandeis University, 415 South St, Waltham, MA; e-mail: ren{at}hydra.rose.brandeis.edu.
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© 2001 by The American Society of Hematology.
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  D. Ye, N. Wolff, L. Li, S. Zhang, and R. L. Ilaria Jr STAT5 signaling is required for the efficient induction and maintenance of CML in mice Blood, June 15, 2006; 107(12): 4917 - 4925. [Abstract] [Full Text] [PDF]
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 P. Ramaraj, H. Singh, N. Niu, S. Chu, M. Holtz, J. K. Yee, and R. Bhatia Effect of Mutational Inactivation of Tyrosine Kinase Activity on BCR/ABL-Induced Abnormalities in Cell Growth and Adhesion in Human Hematopoietic Progenitors Cancer Res., August 1, 2004; 64(15): 5322 - 5331. [Abstract] [Full Text] [PDF]
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S. Ghaffari, C. Kitidis, M. D. Fleming, H. Neubauer, K. Pfeffer, and H. F. Lodish Erythropoiesis in the absence of janus-kinase 2: BCR-ABL induces red cell formation in JAK2-/- hematopoietic progenitors Blood, November 15, 2001; 98(10): 2948 - 2957. [Abstract] [Full Text] [PDF]
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N. C. Wolff and R. L. Ilaria Jr Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571 Blood, November 1, 2001; 98(9): 2808 - 2816. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
2 = 23.97,
P < .0001 and 
, n = 13;
bcr-abl/R1057K, 
, n = 15; bcr-abl/
, n = 11. The
survival curves were generated through Kaplan-Meier survival
analysis.
(Figure 
a nearly 12-fold difference between the
expectation and observation. As mentioned previously, there was one 1:8
mix BMT mouse with a very high number of B-lymphoid cells (Figure 
DNA fragments were
used as DNA molecular weight markers and are shown on the right. (B)
DNA from mice BMT11.48.36 and BMT11.48.38 were digested with
BamHI (lanes 1,2) and BglII (lanes 3,4) and
analyzed by Southern blot with the same probe as in panel A. Standard
1-kb size marker is shown partially on the right.
R fusion protein, generated by t(5;12)(q33;p13)
translocation in a subset of chronic myelomonocytic leukemia, was also
shown to induce a fatal MPD in mice.