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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2913-2921
NEOPLASIA
From the Department of Pharmacology and Cancer Biology and the
Divisions of Hematology and Oncology, Department of Medicine, Duke
University Medical Center, Durham, NC; and the Department of Pathology
and Institute for Medicine and Engineering, University of Pennsylvania,
Philadelphia, PA.
The reciprocal translocation between chromosomes 9 and 22 that fuses
coding sequences of the Bcr and Abl genes is responsible for a
remarkably diverse group of hematologic malignancies. A newly described
230-kd form of Bcr-Abl has been associated with an indolent
myeloproliferative syndrome referred to as chronic neutrophilic
leukemia. We have cloned the corresponding gene and examined the
biologic and biochemical properties of p230 Bcr-Abl after
retroviral-mediated gene transfer into hematopoietic cell lines and
primary bone marrow cells. p230 Bcr-Abl-expressing 32D myeloid cells
were fully growth factor-independent and activated similar signal
transduction pathways as the well-characterized p210 and p185 forms of
Bcr-Abl. In contrast, primary mouse bone marrow cells expressing p230
required exogenous hematopoietic growth factors for optimal growth,
whereas p185- and p210-expressing cells were independent of growth
factors. The 3 Bcr-Abl proteins exerted different effects on
differentiation of bone marrow cells. p185 induced outgrowth of
lymphoid precursors capable of tumor formation in immunodeficient mice.
In contrast, p210- and p230-expressing bone marrow cells caused limited
outgrowth of lymphoid precursors that failed to form tumors in
immunodeficient mice. Removal of cytokines and autologous stroma from
Bcr-Abl-expressing bone marrow cultures produced the expansion of
distinct lineages by the various Bcr-Abl proteins. p185 drove expansion
of cytokine-independent lymphoid progenitors, while p210 and p230
generated cytokine-independent monocyte/myeloid cells. These findings
suggest that the different Bcr-Abl fusion proteins drive the expansion
of different hematopoietic populations, which may explain the
association of the various Bcr-Abl oncoproteins with different spectra
of human leukemias.
(Blood. 2000;95:2913-2921)
The Bcr-Abl oncogene is formed by the
reciprocal translocation between chromosomes 9 and 22 and is associated
with diverse human leukemias.1 The Bcr-Abl oncogene
product, Bcr-Abl, is a chimeric protein with deregulated tyrosine
kinase activity. Bcr-Abl exerts its leukemogenic effects via both
autophosphorylation and phosphorylation of cellular substrates. This
ultimately leads to activation of signal transduction pathways involved
in the altered biologic behavior of Bcr-Abl-containing cells. These
include the Ras, Raf, Erk, Jnk, Myc, Jak/Stat, PI3kinase-Akt, and
NF- The leukemia-associated Bcr-Abl oncogenes vary in the amount of
the Bcr gene that is included within the chimera.1
The structure of the various Bcr-Abl oncoproteins may influence the disease phenotype associated with their expression. p185 Bcr-Abl (also
referred to as p190 Bcr-Abl) contains the dimerization, binding, SH2
and serine/threonine kinase domains of Bcr and is associated with the
subset of acute lymphoblastic leukemias (ALL) that are Philadelphia
chromosome positive (Ph1+).14,15 ALL is
typically an acute, aggressive leukemia that requires prompt treatment
with high-dose chemotherapy.16 p210 Bcr-Abl contains
additional Bcr sequences coding for Pleckstrin homology (PH)
and Dbl-like domains and is associated with nearly all cases of chronic
myelogenous leukemia (CML).17 CML begins as a chronic
myeloproliferative disorder that is amenable to management with
chemotherapeutic or biologic agents, but inevitably progresses to a
fatal blast crisis of either myeloid or lymphoid phenotype.
Recently, Pane et al18 have shown that a subset of patients
with Ph1+ chronic neutrophilic leukemia (CNL) have a unique
breakpoint (designated µ) within the Bcr gene on chromosome
22.19 This breakpoint is 3' to the more common
breakpoints found in patients with CML and the subset of patients with
ALL that are Ph1+. Thus, p230 Bcr-Abl contains additional
Bcr coding sequences that are not found in the p185 or p210
variants. Specifically, p230 Bcr-Abl contains the calcium-phospholipid
binding (CalB) domain and the first third of the domain associated with
GTPase activating activity for p21rac (GAPrac). CNL, which
is also known in the literature as neutrophilic chronic myelogenous
leukemia, is a rare disorder marked by sustained mature neutrophilic
expansion with mild hepatosplenomegaly. This disease typically has a
more indolent course when compared with classical CML, and progression
to blast crisis is uncommon.18 However, a few patients have
been reported who exhibit typical CML and carry the µ breakpoint that
generates p230 Bcr-Abl.20-22
Thus, the unique protein domains that are contributed by the various
Bcr breakpoints, when translocated to the c-Abl gene can lead to diseases with remarkably different phenotypes. To gain
insight into the molecular pathology of the various Bcr-Abl-related leukemias, we have cloned the CNL-associated p230 Bcr-Abl
oncogene and expressed the protein product in hematopoietic cell lines as well as primary mouse bone marrow cells under conditions that favor
the outgrowth of either myeloid or lymphoid progenitors. Here, we
analyze the biochemical and biologic properties of p230 Bcr-Abl and
compare it with the p210 and p185 variants.
P230 Bcr-Abl cDNA
Cell culture
Gene transfer For gene transfer into tissue culture cell lines, retroviruses were generated by transient transfection of 293T cells, as previously described.25 Briefly, the various Bcr-Abl constructs were cotransfected with PSV 2 onto 293T
fibroblasts26 by calcium phosphate transfection in the
presence of 25 µmol/L chloroquine. Two days after transfection,
Bcr-Abl-containing and control retroviral supernatants were collected
and incubated with 1 million 32D, FL5-12, or DAGM cells in 10% WEHI-CM
and 4 µg/mL polybrene in a volume of 1 mL. Infections were performed
at 37°C and 5% CO2 for 3 to 6 hours. Cells were
pelleted and resuspended in growth medium. Two days after infection,
cells were selected by IL3 deprivation or resistance to G418 (0.3 to
0.7 mg/mL). IL3 deprivation was performed by washing the cells twice
with RPMI and resuspending them in RPMI containing 10% FBS and P/S.
Western blotting Primary mouse bone marrow cells were lysed at a concentration of 5 × 107 cells/mL directly in boiling 2 × sample buffer. Lysates were centrifuged at 100 000g for 25 minutes at 4°C and supernatants equivalent to 1 × 107 cells were loaded onto SDS polyacrylamide gels for analysis. After gel electrophoresis, proteins were transferred to nitrocellulose membranes, blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) and blotted with antibodies to Abl (8E9, Pharmingen, San Diego, CA) or phosphotyrosine (4G10, Upstate Biotechnologies, Lake Placid, NY) in 2% nonfat dry milk in TBS, followed by enhanced chemiluminescent detection (Amersham, Piscataway, NJ).Immunophenotyping Cultures of Bcr-Abl and GFP-expressing or control GFP-expressing bone marrow cells were resuspended in phosphate-buffered saline (PBS) and 1% FBS. Cells were incubated with anti-CD16/CD32 ("Fc Block," Pharmingen) at 1.25 µg/mL for 15 minutes on ice, followed by incubation with biotin-conjugated primary antibodies at 3.3 µg/mL for 30 minutes on ice. Cells were washed 3 times with PBS and 1% FBS, followed by incubation with a combination of streptavidin-cy-chrome (Pharmingen) at 1.3 µg/mL and phycoerythrin (PE)-conjugated primary antibodies at 3.3 µg/mL for 15 minutes on ice. Cells were washed twice with PBS and 1% FBS and analyzed on a Becton Dickinson FACStarPLUS, equipped with a 488 nm argon laser and tunable dye laser. For some analyses, dead cells were excluded by staining with 7AAD, in which case streptavidin-allophycocyan (Pharmingen) was used as the secondary reagent. Primary biotin and PE-conjugated antibodies used for this study were purchased from Pharmingen: B220 (clone RA3-6B2), Thy 1.2 (clone 53-2.1), Gr-1 (clone RB6-8C5), Mac-1 (clone M1/70), CD 43 (Ly-48), cKit (clone 2B8), CD 34 (clone RAM 34), CD9 (clone KMC8), and Sca1 (Ly-6A/E). FACS analysis was performed until 10 000 GFP positive events were acquired.Cell cycle analysis Cultures of Bcr-Abl and GFP-expressing or control GFP-expressing bone marrow cells were incubated with 10 µmol/L bromodeoxyuridine (Brdu) for 30 minutes in IMDM with 15% FCS, 0.1% BSA, 25 µmol/L -mercaptoethanol and P/S with or without cytokines at 37°C, and 5% CO2. One million cells were then washed twice with PBS
and 1% FBS and fixed with 5 mL 70% ethanol for 30 minutes on ice. This successfully extracted 100% of the GFP from the cells in control
experiments. The cells were then stained with FITC-conjugated anti-Brdu
antibody and propidium iodide as per the manufacturer's instructions
(Becton Dickinson) and analyzed by flow cytometry on a Becton Dickinson
FACStarPLUS with laser excitation at 488 nm.
Tumor challenge Cells were washed twice in RPMI only and resuspended at 1 × 107 cells/mL RPMI containing 0.1% FBS. A total of 1 × 106 cells (100 µL) were injected subcutaneously into the right flanks of 7- to 8-week-old male SCID mice (strain CB17SC-M, Taconic Farms, Germantown, NY).
Cloning and expression of p230 Bcr-Abl.
We used an overlapping PCR technique with Bcr and Abl
cDNA templates to create a Bcr-Abl chimeric cDNA that contained
the unique p230 µ breakpoint.1,18,19 After
retroviral-mediated gene transfer of the various leukemia-associated
Bcr-Abl isoforms into 32D cells, Western blot analysis with an anti-Abl
antibody demonstrated expression of proteins of the expected sizes
(Figure 1A). Western blotting with an
antibody that recognizes the PH domain of Bcr showed that this domain
is present in p210 and p230 Bcr-Abl, but not in p185 Bcr-Abl (data not
shown). The tyrosine kinase activity of the Bcr-Abl chimeric
oncoprotein is critical to its transforming ability.3,28
32D cells expressing the various leukemia-associated Bcr-Abl isoforms
contained roughly equivalent levels of tyrosine-phosphorylated Bcr-Abl,
along with additional phosphoproteins (Figure 1B). This finding
indicates that p230 Bcr-Abl is kinase active, and its tyrosine kinase
activity is similar to p210 and p185 Bcr-Abl oncoproteins in
vivo.
p230 Bcr-Abl renders 32D cells factor independent and activates
similar signaling pathways as p185 and p210 Bcr-Abl in these cells.
Expression of p185 and p210 Bcr-Abl in 32D cells leads to growth factor
independence and inhibition of apoptosis.3 The ability of
p230 Bcr-Abl to cause growth factor independence of 32D cells was
compared with the p210 and p185 Bcr-Abl variants. After IL3 withdrawal,
p230 cells grew at a rate equivalent to p210- and p185-expressing
cells, whereas control cells underwent apoptosis (data not shown). p230
as well as p210- and p185-expressing 32D cells were resistant to the
differentiating effects of GCSF, whereas control cells stopped cycling
and differentiated into granulocytes (data not shown). Furthermore,
p230 Bcr-Abl-expressing 32D cells were as potent as p185 and p210
Bcr-Abl-expressing 32D cells in their ability to form tumors in SCID
mice (Table 1). Thus, the biologic effects
of the p230 Bcr-Abl isoform were indistinguishable from those of the
more aggressive p210 and p185 isoforms of Bcr-Abl after expression of
the proteins in 32D cells. Consistent with these findings, analysis of
the signaling properties of p230 Bcr-Abl in 32D cells revealed that
p230 activated Ras, Erk, and Jnk to similar levels as those induced by
p185 and p210 Bcr-Abl in these cells3,6,29 (data not
shown). Similar results were obtained in FL5-12 and DAGM cells
expressing the 3 Bcr-Abl isoforms (data not shown). This data indicates
that in lineage-restricted hematopoietic cell lines, the p185, p210,
and p230 forms of Bcr-Abl activate Ras, Erk, and Jnk to similar extent,
and elicit similar survival, proliferative, and tumorigenic
effects.
Effect of expression of distinct Bcr-Abl proteins on the
differentiation of primary mouse bone marrow cultures expanded in the
presence of exogenous cytokines and autologous stroma.
The effects of p230 Bcr-Abl expression were next studied on primary
bone marrow cells, the natural target of Bcr-Abl related leukemias. We
used a bicistronic retroviral vector to coexpress the
leukemia-associated Bcr-Abl cDNAs with the gene for enhanced green fluorescent protein (GFP) as a selectable
marker.27 After retroviral-mediated gene transfer in
primary bone marrow cells and selection for GFP, we observed equivalent
expression of the 3 forms of Bcr-Abl (Figure
2A). Furthermore, the levels of in vivo
phosphotyrosine were similar for cell lysates expressing the p185,
p210, and p230 forms of Bcr-Abl (Figure 2B).
Effect of expression of distinct Bcr-Abl proteins on the
differentiation, proliferation, and tumorigenicity of primary mouse
bone marrow cultures expanded in the presence of exogenous cytokines
without stroma.
To test the effect of the different Bcr-Abl chimeric proteins on the
differentiation of primary mouse bone marrow cultures in the absence of
autologous stroma, primary bone marrow cells were transduced with the
Bcr-Abl- and GFP-coexpressing retroviruses, selected for GFP
expression, and expanded in the presence of cytokines without stroma.
After 10 days of growth in the presence of cytokines without autologous
stroma, primary bone marrow cultures transduced with Bcr-Abl
retroviruses encoding p185, p210, and p230 were either deprived of
cytokines or injected subcutaneously into SCID mice. As shown in Figure
6, outgrowths of cytokine-independent cells immunophenotypically consistent with pre-B cells were obtained with all
3 Bcr-Abl isoforms from cultures that were initially predominantly B220
negative. The outgrowth of pre-B cells differed markedly among the
various Bcr-Abl-expressing cultures. p185 and p210 Bcr-Abl-expressing
mouse bone marrow cultures expanded with cytokines alone rapidly
evolved into pre-B cells after cytokine withdrawal, whereas pre-B-cell
development of p230 Bcr-Abl-expressing cells was delayed (data not
shown).
p230 Bcr-Abl elicits a weaker proliferative response than p210
Bcr-Abl in the absence of cytokines and stroma.
To compare more directly the proliferative responses of p210 and p230
Bcr-Abl, we followed the growth of primary mouse bone marrow cultures
expressing p210 or p230 2 days after retroviral-mediated gene transfer
and selection for GFP expression in the absence of cytokines and
autologous stroma. Ten days after GFP selection, p210
Bcr-Abl-expressing primary mouse bone marrow cells had expanded 13 000-fold, whereas p230 Bcr-Abl-expressing primary mouse bone marrow cells had barely expanded 300-fold, a 40-fold difference that
was statistically significant (P < .05, Student t
test) (Figure 8). After 10 days of growth
without cytokines or stroma, p185, p210, and p230 Bcr-Abl-expressing
cultures were all B220 positive. p185 Bcr-Abl cells grew at the same
rate as p210 Bcr-Abl-expressing cells under these conditions, whereas
murine bone marrow MNCs infected with the GFP-expressing retrovirus
alone did not proliferate under these conditions (data not shown).
Thus, although all leukemia-related Bcr-Abl isoforms can cause cytokine
independence of primary murine bone marrow cells under a variety of
conditions, they are markedly different in their abilities to drive the
proliferation of these cells.
The deregulation of normal hematopoiesis by chimeric oncogenes may
result from dual effects on promoting cell growth and inhibiting cell
differentiation, as well as enhancing cell survival. CML, considered
the prototypical disease of the hematopoietic stem cell, is caused by
the protein product of the p210 Bcr-Abl chimeric oncogene. The
closely related p185 Bcr-Abl oncogene is the causative agent of
a subset of ALL, whereas p230 Bcr-Abl has been associated with
CNL, which has a clinical presentation that resembles the more typical
p210 Bcr-Abl disease, but may have a more benign natural
history.18 Whether patients with p230 Bcr-Abl-related leukemia represent a bona fide stem cell disorder versus a myeloid lineage restricted disease remains to be established.39,40 Furthermore, there is overlap between the disease spectrum associated with the different Bcr/Abl fusion proteins.1 For example,
p210 Bcr-Abl is associated with 40% of ALL, p185 Bcr-Abl with
approximately 2% to 3% of CML,39,40 and p230 Bcr-Abl has
been described in patients with typical CML, some of whom have
progressed to blast crisis.20-22 It is not clear whether
intrinsic differences in the activities of the 3 Bcr-Abl proteins
account for their association with different disease phenotypes or
whether the expression of each of the 3 Bcr-Abl forms is restricted to
a distinct hematopoietic lineage, thus explaining their association
with different leukemias. Data exist supporting the 2 possibilities.
We thank Dr Michael Cook for the expert assistance with flow cytometry
and cell sorting.
Submitted August 12, 1999; accepted January 5, 2000.
R.C.Q. and G.W.R. contributed equally to this work.
Supported by National Institutes of Health grants CA61033 and CA77570
to A.M.P. and W.S.P., respectively. R.C.Q. was supported by the 4 Schools Physician-Scientist Program, sponsored by the Lucille P. Markey
Charitable Trust. G.W.R. was supported by an Environmental Protection
Agency Fellowship. K.D.C. was supported by the Medical Scientist
Training Program and the Department of Defense Breast Cancer Research
Program. A.M.P. and W.S.P. are Scholars of the Leukemia Society of America.
Reprints: Ann Marie Pendergast, Department of Pharmacology & Cancer Biology, Box 3813, Duke University Medical Center, Durham, NC
27710; e-mail: pende014{at}mc.duke.edu.
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.
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
B pathways, among others.
MSVtkneo retroviral vector. The generation of
pSR
-irradiated GP + E-86 producer lines consistently improved gene
transfer efficiency into primary murine stem cells by 25- to 50-fold,
compared with infection with retroviral supernatants alone. Two days
after retroviral infection, designated as "day zero," the murine
bone marrow MNCs were used for experimentation. For in vitro
differentiation experiments in the presence of autologous stroma, day
zero murine bone marrow MNCs were plated directly after
retroviral-mediated gene transfer and grown in the presence of 100 IU/mL recombinant murine IL3, 100 IU/mL recombinant murine IL6, and
1000 IU/mL (100 ng/mL) recombinant murine stem cell factor. We found
that cocultivation, followed by plating in the presence of cytokines,
reproducibly resulted in the formation of an adherent, confluent
stromal layer and a nonadherent hematopoietic layer. Fresh growth media
with cytokines was added every 2 days. The nonadherent cells were not
passaged into new flasks to maintain contact with the adherent stromal compartment, which we found to be resistant to trypsinization. For in
vitro experiments in the absence of autologous stroma, day zero
GFP-expressing murine bone marrow MNCs were selected by FACS before
plating either in the presence or absence of cytokines. We found that
both control and GFP-containing cultures plated after GFP selection
developed few, if any, adherent cells.
b 
b 
b 
b phospholipases, GAPs, and perforin.
Protein Sci.
1996;5:162
