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NEOPLASIA
Loss of Bcl-x in Ph⫹ B-ALL increases cellular proliferation and does not
inhibit leukemogenesis
Jason G. Harb,1 Brenda I. Chyla,1 and Claudia S. Huettner1,2
1BloodCenter
of Wisconsin, Blood Research Institute, Milwaukee; and 2Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin,
Milwaukee
The kinase inhibitors imatinib mesylate
and dasatinib are the preferred treatment
for Philadelphia chromosome–positive
(Phⴙ) leukemias, and they are highly successful in the chronic phase of chronic
myeloid leukemia (CML). However, they
are not efficient in Phⴙ B-cell acute lymphoblastic leukemia (B-ALL). Phⴙ leukemia cells are highly resistant to apoptosis, and evidence from cell lines and
primary cells suggest Bcl-xL as a critical
mediator of resistance to apoptosis: however, this concept has never been rigorously tested in an animal model. To clarify
the role of Bcl-xL in Phⴙ B-ALL, we generated 2 mouse models. In the first model,
Phⴙ B-ALL and loss of Bcl-xL expression
are coinduced; in the second model, leukemia is induced with expression of
Bcl-xL protein well above the levels found
in wild-type lymphoblasts. Deletion of
Bcl-xL did not inhibit leukemogenesis or
affect apoptosis, but increased cellular
proliferation. Consistent with this result,
overexpression of Bcl-xL led to decreased
cellular proliferation. These models reveal an unexpected role for Bcl-xL in
cell-cycle entry and the proliferation of
tumor cells. (Blood. 2008;111:3760-3769)
© 2008 by The American Society of Hematology
Introduction
The Philadelphia chromosome (Ph) arises from a translocation
between chromosomes 9 and 22 and results in formation of a
chimeric and constitutively activated tyrosine kinase known as
BCR-ABL, which is the cause of chronic myeloid leukemia (CML)
and is also expressed in cases of acute B-cell lymphoblastic
leukemia (B-ALL). CML initiates as a chronic disease that is
followed by an accelerated phase which eventually progresses to a
rapidly fatal blast crisis stage. Together, lymphoid blast crisis of
CML and Ph⫹ B-ALL account for 20% of adult patients and 5% of
pediatric patients with ALL.1,2 The abl-specific kinase inhibitor
imatinib mesylate is the first-line treatment for patients with CML,
and it is successful in patients in the chronic phase of the disease.
However, it does not provide lasting remission for patients in blast
crisis and patients with Ph⫹ B-ALL,2,3 which was also recently
demonstrated for the multitargeted kinase inhibitor dasatinib.4,5
Drugs that target signaling molecules downstream of BCR/ABL
may help to overcome this resistance. BCR/ABL is a potent
inhibitor of apoptosis, and cells expressing the oncogene are
stubbornly resistant to the induction of cell death by a variety of
apoptosis-inducing agents.6 Both the archetypical inhibitor of
apoptosis, Bcl-2, as well as a second member of this family of
antiapoptotic proteins, Bcl-xL, have been suggested as BCR/ABLregulated effector molecules. Transfection of the pro–B-cell line
BaF3 and of the myeloid 32D3 cell line with the oncogene rendered
them growth factor independent and led to increased levels of
Bcl-2, suggesting a role for this protein in circumvention of cell
death.7,8 These results were corroborated by another study, which
found a correlation between the amount BCR/ABL expression with
the level of Bcl-2 induction and resistance to apoptosis.9 However,
different investigators using the same pro–B-cell line to express
BCR/ABL reported an increase in the expression levels of the
antiapoptotic protein Bcl-xL.9 The relevance of this result is
strengthened by the fact that Bcl-xL is a target of the signal
transducer and activator of transcription STAT5, and it was
previously shown that BCR/ABL leads to constitutive activation of
STAT5.10,11 Furthermore, transfection of Ph⫹ K562 cells with a
dominant-negative isoform of STAT5 led to a decrease in Bcl-xL
expression and subsequent apoptosis of the cells, suggesting
Bcl-xL as an important factor in the prevention of programmed cell
death in the context of Ph⫹ leukemias.11
Given the well-characterized role of Bcl-xL in prevention of
apoptosis, cells that express high levels of this protein should have
an advantage under the growth-limiting conditions that are present
in the tumor microenvironment, thereby contributing to tumorigenesis. Evidence originating from studies with tyrosine kinase
inhibitors suggests that decreasing the expression level of Bcl-xL
will induce apoptosis. K562 cells express high levels of Bcl-xL,
while Bcl-2 is not detectable, and blocking of the tyrosine kinase
activity in this cell line as well as in cells isolated from patients
with CML in the chronic phase of the disease led to a decrease in
Bcl-xL followed by apoptosis.12,13 In agreement with these observations, it is a common finding that cancer cells expressing a
constitutively active tyrosine kinase are highly resistant to conventional antineoplastic drugs and concomitantly have high levels of
Bcl-xL.14 Thus, it is conceivable that inhibition of Bcl-xL could be
an effective treatment for patients with CML who have a resistance
to imatinib mesylate by suppressing the consequences of BCR/ABL
expression, as well as for patients with Ph⫹ acute B-cell leukemia.
In the present study, we used an inducible transgenic model of
acute B-ALL dependent on BCR/ABL to examine the role of the
Submitted August 22, 2007; accepted January 18, 2008. Prepublished online
as Blood First Edition paper, January 23, 2008; DOI 10.1182/blood-2007-08108803.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2008 by The American Society of Hematology
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
Bcl-x gene. Several proteins are generated from the Bcl-x gene by
alternative splicing, with antiapoptotic Bcl-xL being the most
abundant,15 while the shorter Bcl-xs that is not expressed in mice15
exerts proapoptotic signals opposing Bcl-2 and Bcl-xL.16 Using an
animal model that allowed us to combine cre/lox-mediated recombination with the tetracycline-inducible expression system, we
show that deletion of the Bcl-x gene, resulting in loss of expression
of all protein isoforms, does not impair initiation and progression
of the B-ALL–like phenotype but rather affects the cell cycle.
Bcl-x–deficient lymphoblasts progress faster through the cell cycle
than wild-type lymphoblasts, and we did not observe increased
apoptosis. Alternatively, we show that expression of Bcl-xL at
levels well above the amounts found in BCR/ABL-transformed
cells led to a significant decrease in cycling, thus confirming a role
for Bcl-xL in manipulating the cell cycle.
Methods
Generation of transgenic mice and genotyping
The human Bcl-xL cDNA17 was subcloned into the multiple cloning site of
pTRE2 (Clontech, Mountain View, CA), and the 2.9-kb transgenic construct was injected into the pronucleus of FVBN mice. A total of 3 founder
lines were established. Expression of the transgene was tested for by
polymerase chain reaction (PCR) and Western blot analysis.
The TRE-p210BCR/ABL transgenic construct and mouse line as well
as the Tet-O-cre mice and MMTVtTA transgenic mice were described
previously.18-20 Mice that carry loxP sites flanking the Bcl-x gene21,22 were
bred with TRE-BCR/ABL animals to generate BCR/ABL-Bcl-x f/f mice.
Tet-O-cre mice were bred with MMTVtTA-BCR/ABL mice as well as with
BCR/ABL-Bcl-x f/f animals. Cross breeding over several generations
produced MMTVtTA-BCR/ABL-cre-Bcl-x f/f animals. All breeding was
performed by continuous supplementation of the drinking water.18
Genotyping was performed by PCR with primers specific for the
transgenes. Sequences of primers are listed in Table 1. DNA was isolated
from tail snips following a standard protocol.18
Induction of transgenic expression and monitoring of disease
Induction of transgenic expression for p210BCR/ABL and cre recombinase
was performed by withdrawal of tetracycline from the drinking water
of mice. All animals described in this study were induced at an age of
6 to 8 weeks.
Peripheral blood was collected from the retro-orbital plexus, and total
white blood cell (WBC) and differential counts were performed starting on
day 10 after induction, followed by a biweekly schedule to monitor
development of the phenotype.
Tissue processing and histology
Mice were killed by CO2 inhalation, and cells from bone marrow, lymph
nodes, pleural effusion, and the spleen were isolated. All samples were
stained with Wright-Giemsa as indicated. Light microscopy was performed
with a Nikon Eclipse E600 microscope (Nikon, Melville, NY) using a 40⫻
Plan-Neofluar 0.80 or 100⫻ Plan-Neofluar 1.30 oil lens. Images were
captured with a Spot Insight FireWire 11.2 color mosaic camera and SPOT
software, version 4.1 (Diagnostic Instruments, Sterling Heights, MI), and
Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA) and
Microsoft Powerpoint 2003 (Microsoft, Redmond, WA).
Determination of efficiency of recombination
The efficiency of recombination leading to excision of the Bcl-x gene was
determined using a 3-primer PCR strategy.22 DNA was isolated using the
protocol also used to isolate DNA from tail snips. The recombined allele
generated an amplification product of 280 bp by amplification with primers
A and C, while the unrecombined allele gave rise to a product of 300 bp by
BCL-X IN PH⫹ B-ALL
3761
Table 1. Sequences of primers used for genotyping, real-time PCR,
and 3-primer PCR
Primers
Sequences
Primers used for genotyping
Bcl-x f forward
5⬘-GTCCTGGCCCTGTCACTTA-3⬘
Bcl-x f reverse
5⬘-CCCTTCCCACCTCACTTCCT-3⬘
Human Bcl-xL forward
5⬘-TATTGGTGAGTCGGATCGCAGCTT
␤-globin reverse
5⬘-GTGGTATTTGTGAGCCAGGGCAGG
BCR/ABL forward
5⬘-GAGCGTGCAGAGTGGAGGGAGAACA-3⬘
BCR/ABL reverse
5⬘-GGTACCAGGAGTGTTTCTCCAGACTG-3⬘
Cre forward
5⬘-ACCTGAAGATGTTCGCGATTATCT-3⬘
Cre reverse
5⬘-ACCGTCAGTACGTGAGATATCTT-3⬘
tTA forward
5⬘-GCTAGGTGTAGAGCAGCCTAC-3⬘
tTA reverse
5⬘-GCTAGGTGTAGAGCAGCCTAC-3⬘
Primers used for real-time PCR
A1 forward
5⬘-GATTGCCCTGGATGTATGTGCTTAC-3⬘
A1 reverse
5⬘-AGCCATCTTCCCAACCTCCATTC-3⬘
Bcl-x forward
5⬘-ACTTTTGTGGATCTCTACGGGAAC-3⬘
Bcl-x reverse
5⬘-CTGAAGAGTGAGCCCAGCAG-3⬘
Pim-1 forward
5⬘-GATCATCAAGGGCCAAGTGT-3⬘
Pim-1 reverse
5⬘-GATGGTTCCGGATTTCTTCA-3⬘
L19 forward
5⬘-TCTGGTTGGATCCCAATGAGA-3⬘
L19 reverse
5⬘-GTCACAGGCTTGCGGATGAT-3⬘
BCR/ABL forward
5⬘-CGTCCACTCAGCCACTGGAT-3⬘
BCR/ABL reverse
5⬘-GGCTTCACTCAGACCCTGAGG-3⬘
3-primer PCR (recombination)
Primer A
5⬘-CGGTTGCCTAGCAACGGGGC-3⬘
Primer B
5⬘-CTCCCACAGTGGAGGACCTCG-3⬘
Primer C
5⬘-TCAGAAGCCGCAATATCCCC-3⬘
amplification with primers A and B. Primers A and C could not form an
amplification product of the unrecombined allele.
Isolation of proteins and Western blot analysis
Proteins were isolated using 10% trichloracetate (TCA) as described.23
Primary antibodies used in this study were anti–Bcl-x (BD PharMingen,
San Diego, CA), anti–Bcl-2 (C-2), anti–c-abl (C- 24-11), anti-actin (C-11;
all Santa Cruz Biotechnology, Santa Cruz, CA), and anti–Mcl-1 (Rockland
Immunochemical, Gilbertsville, PA).
Isolation of RNA, first-strand cDNA synthesis, and
real-time PCR
Total RNA was isolated with Trizol reagent (Invitrogen, Carlsbad, CA)
following the manufacturer’s guidelines. Each sample was subjected to
DNAse treatment (Turbo DNAse; Ambion, Austin, TX), and first-strand
synthesis was performed with random hexamer primers and Moloney
murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen).
The expression of BCR/ABL, A1, Pim-1, and Bcl-x was measured as a
percentage of L19 expression using a SYBR Green assay (Power SYBR
Green PCR Master Mix; Applied Biosystems, Foster City, CA) with
gene-specific primers (Table 1). All reactions were performed in duplicate.
Flow cytometry analysis and staining for Annexin
Bone marrow cells were isolated as described,23 and cells were incubated
with the appropriate antibodies. All analyses were performed on a
dual-laser fluorescence-activated cell sorter (FACS; Becton Dickinson,
Franklin Lakes, NJ). The following anti-murine antibodies were used:
B220, CD34, CD41, Ter119, CD71, Gr-1, Mac-1, c-kit, Sca-1, and BP-1
(BD Pharmingen), CD43, immunoglobulin M (IgM), light chain, and
IL7R␣ (all from eBioscience, San Diego, CA). For detection of apoptosis,
cells were stained with APC-annexin-V and 7-AAD (BD Biosciences, San
Jose, CA) following manufacturer’s directions.
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
HARB et al
Table 2. Genotypes of mice used in this study
Genotype
Experimental group
MMTVtTA-TRE-cre-BCR/ABL-Bcl-x f/f
Bcl-x–deficient B-ALL
MMTVtTA-BCR/ABL
B-ALL wild-type control
MMTVtTA-TRE-cre-BCR/ABL
*
MMTVtTA-Bcl-x f/f
*
MMTVtTA
*
TRE-BCR/ABL
*
Bcl-x f/f
*
TRE-cre
*
Wild-type
MMTVtTA-TRE-cre-Bcl-xf/f
Milwaukee, WI). Animals were euthanized after 24 hours, and the
percentage of annexin-V⫹ cells in bone marrow and blood was determined
by FACS analysis.
Statistical analysis
Data are expressed as means plus or minus SEM unless otherwise indicated
and were compared using a paired Student t test, as described in “Results.”
P values less than .05 are considered significant.
*
Control for deletion of Bcl-x in B-cell lineage
* indicates control group.
Multicolor FACS sorting and analysis of developmental
B-cell stages
Bone marrow cells were isolated from MMTVtTA-cre mice and T cells,
myeloid cells, and erythroid cells were removed by staining with antibodies
against CD3, Gr-1, and Ter119, followed by removal of positive cells with
magnetic beads to enrich for the B-cell fraction. The remaining cells were
stained with antibodies against B220, CD19, CD43, BP-1, and IgM. Sorting
was performed according to the staging for mouse bone marrow B
lymphopoiesis suggested by Hardy.24,25 Fraction A, also described as
pre-pro–B-cell, was sorted as AA4.1⫹/B220⫹/CD19⫺/BP-1⫺; pro–B cells
corresponding to fraction B/C were sorted as B220dim/c-kit⫹/CD19⫹/BP1⫺. Pre–B cells corresponding to stage C⬘ were isolated as B220⫹/CD43⫹/
BP-1⫹; fraction D was sorted as B220⫹/CD43⫺/BP-1⫹; and fraction E/F
was sorted as CD19⫹/B220high/IgM⫹. Total RNA was isolated with Trizol
followed by DNAse treatment (Turbo DNAse; Ambion). Real-time PCR
was performed with primers specific for cre recombinase19 and L19 as
internal control. All experiments were performed in duplicates.
BrdU labeling and cell-cycle analysis
Animals were injected 16 hours prior to euthanization with 5 mg BrdU. A
total of 106 cells were stained with fluorescently tagged antibodies
(eBioscience) specific for B220 and CD19. Staining for BrdU was
performed according to the manufacturer’s suggestions provided with the
BrdU flow kit (BD PharMingen).
In vivo cytotoxicity assay
Animals received one intraperitoneal injection with 100 mg/kg body weight
cyclophosphamide (obtained from the Pharmacy of Children’s Hospital,
Results
Bcl-xL is highly expressed in P210 BCR/ABL–B-ALL
transgenic mice
To explore if expression of Bcl-xL is required for development
and maintenance of acute B-cell leukemia caused by BCR/ABL,
we made use of a p210 BCR/ABL-inducible transgenic expression system18 and combined it with cre/lox-mediated recombination to delete the Bcl-x gene in mice that carry loxP sites
framing the gene (Bcl-x f/f).22 We had previously shown that the
MMTV–long terminal repeat (LTR) driving the expression of
the tetracycline-regulatable transactivator protein tTA targets
the B-cell lineage within the murine bone marrow. 18,26
MMTVtTA-BCR/ABL transgenic mice succumb to acute pre–Bcell leukemia within 4 to 5 weeks after induction of BCR/ABL
expression.18 Analysis of bone marrow cells, lymph node cells,
and splenocytes of diseased animals by Western blot analysis
demonstrated increased levels of Bcl-xL protein when compared
with wild-type control animals, with highest levels found in
bone marrow and spleen (Figure 1A,B) and in cells isolated
from pleural effusion, which represents an almost homogenous
population of cells as determined by staining for cell-surface
markers (CD19⫹/B220⫹/CD43⫹/BP-1⫹/IgM⫺).
Crossbreeding of transgenic lines generated the MMTVtTABCR/ABL-Cre-Bcl-x f/f genotype that allows for expression of the
BCR/ABL oncogene and recombination of the Bcl-x allele within
the same target cell.
Figure 1. Expression levels of Bcl-xL protein in
mice suffering from B-ALL. Protein lysates from
(A) bone marrow (wild-type, lanes 1 and 2; B-ALL,
lanes 3-5) and (B) spleen (wild-type, lanes 1-3; B-ALL
mice, 4-6) from B-ALL mice were compared with
wild-type littermate controls after immunoblotting with
Bcl-x–, Bcl-2–, and actin-specific antibodies. Expression of p210 BCR/ABL was detected with an antibody
against c-abl that also recognizes the fusion protein.
Vertical bars indicate repositioned lanes.
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
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Figure 2. Development of B-ALL–like disease in the absence of Bcl-xL. (A) Macroscopic phenotype of Bcl-x–deficient B-ALL mouse 17 days after induction. Enlarged
lymph nodes are denoted by black arrows and spleen by the yellow arrow. (B) Top panel: lymphoblasts from diseased mice are B220dim. Bone marrow cells were isolated from
wild-type (purple), B-ALL (green), and Bcl-x–deficient B-ALL (red) and stained for expression of B220. Bottom panels: lymphoblasts from Bcl-x–deficient B-ALL mice are
arrested at a pre–B-cell stage of development. Bone marrow cells stained for coexpression of B220 and CD19, CD43, and BP-1. (C) Wright-Giemsa staining of peripheral blood
(PB; top panel) 14 days after induction and lymph node (LN) cells (bottom panel). Mitotic figures are denoted by black arrows. (D) DNA isolated from tissues of tissues from
Bcl-x–deficient B-ALL mice was subjected to 3-primer PCR to determine efficiency of recombination of Bcl-x f/f alleles. DNA was isolated from bone marrow (BM; lanes 1 and
2), lymph node (LN; lanes 3 and 4), pleural effusion (PE; lanes 5 and 6), and spleen (SPL; lanes 7 and 8). Arrows indicate floxed and recombined alleles. Lane 9 represents no
recombination. Vertical bars indicate repositioned lanes. (E) Result of Western blot analysis from pleural effusion of B-ALL (lanes 1-3) and Bcl-x–deficient B-ALL (lanes 4-6)
mice. Lysates were immunoblotted against c-abl– (loading control, also recognizes BCR/ABL oncoproteins), Bcl-x–, and actin-specific antibodies.
Phⴙ B-ALL is initiated and maintained in the absence of
Bcl-x expression
At 6 to 8 weeks of age, tetracycline was withdrawn from the
drinking water of experimental MMTVtTA-BCR/ABL-cre-Bcl-x
f/f animals (which shall be referred to as Bcl-x–deficient BCR/
ABL transgenic mice for brevity), MMTV-BCR/ABL mice that
were carried along as control for the development of the B-ALL–
like phenotype in the presence of wild-type Bcl-x alleles, and
single-transgenic and wild-type animals (Table 2). The disease
progressed rapidly, with animals being moribund as early as day
14. By day 29, all mice with the Bcl-x–deficient BCR/ABL
genotype had died or had to be killed due to moribund condition.
Necropsy of these mice demonstrated massive splenomegaly (up to
10-fold of the size of control animals), pleural effusion filling
almost the entire thoracic cavity, and enlargement of the majority of
lymph nodes (Figure 2A). FACS analysis demonstrated the transformed cells to be arrested at the pre–B-cell stage of development,24,25 with cells identified as B220dim/CD19⫹/CD43⫹/BP-1⫹,
similar to the cell-surface markers identified in Ph⫹ B-ALL patient
samples and MMTV-BCR/ABL transgenic animals (Figure 2B).
Although the severity of the disease appeared to be exaggerated in
Bcl-x–deficient animals with massive lymph node involvement and
rapid onset of pleural effusion, no statistically significant reduction
of the survival time was found when littermates were compared
(Figure 3). The most prominent difference between the 2 groups of
animals was the presence of mitotic figures in the peripheral blood
and other tissues isolated from Bcl-x–deficient BCR/ABL transgenic mice (Figure 2C).
Figure 3. Survival time of mice suffering from B-ALL and Bcl-x–deficient B-ALL
after induction of p210 BCR/ABL expression. Kaplan-Meier plot showing survival
time of mice suffering from B-ALL and Bcl-x–deficient B-ALL. Individual mice in each
arm are indicated by symbols.
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HARB et al
BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
Figure 4. Consequences of recombination of Bcl-x alleles on B lymphocytes in the bone marrow and spleen. (A) FACS analysis of bone marrow and spleen cells
isolated from MMTVtTA cre bcl-x f/f (Bcl-x⫺/⫺), and littermate controls (Bcl-x⫹/⫹) 4 or 8 weeks after induction. Cells were stained with antibodies against B220 and CD19.
(B) 3-primer PCR performed on DNA isolated from bone marrow (BM; lanes 1 and 2) and spleen (SPL; lanes 4 and 5) of MMTVtTA cre bcl-x f/f mice 4 weeks (lanes 1 and 4) or
8 weeks (lanes 2 and 5) after induction. Arrows indicate floxed and recombined alleles. Lanes 3 and 6 are examples of complete recombination, lane 7 is an example of no
recombination, and M denotes 100-bp ladder. Vertical bars indicate repositioned lanes. (C) Recombination of Bcl-x f/f alleles in B-cell development. DNA prepared from whole
bone marrow (lane 1), fraction A B lymphocytes (lane 2), and fraction B lymphocytes (lane 3) was subjected to 3-primer PCR. Lane 4 demonstrates no recombination; lane 5,
nontemplate control; M, 100-bp ladder.
Blast cells have undergone complete recombination of the
Bcl-x alleles
MMTVtTA-cre–mediated recombination occurs in early B-cell
progenitor cells
To ensure that B220dim/CD19⫹/CD43⫹/BP-1⫹ lymphoblasts had
undergone complete recombination of both Bcl-x alleles, we
performed 3-primer PCR and found almost complete recombination in cells from bone marrow and pleural effusion, while
incomplete recombination was observed in the spleen (Figure 2D).
Incomplete recombination can be accounted for by the presence of
cell lineages, which are not targeted by the MMTVtTA. Even a
grossly enlarged spleen consisting mostly of B220dim/CD19⫹ B
cells still contained some mature cells demonstrated by FACS
analysis as B220high/CD19⫹ as well as T lymphocytes, which are
not targeted by the MMTVtTA transactivator strain.18 We performed real-time PCR to test for Bcl-xL mRNA as a more sensitive
means to validate successful recombination resulting in absence of
Bcl-xL expression. Bcl-xL mRNA was not detectable or significantly reduced in cells isolated from Bcl-x–deficient BCR/ABL
mice (data not shown), while high levels were found in MMTVBCR/ABL control mice consistent with the protein data presented
in Figure 1. Furthermore, we verified our data by Western blot
analysis for expression of p210 BCR/ABL and lack of Bcl-xL in
tissues of diseased mice (n ⫽ 10; Figure 2E). We conclude that loss
of Bcl-xL in the context of BCR/ABL leukemia is not sufficient to
either halt the development or reduce the severity of the disease.
The development of the B-ALL phenotype is all the more
surprising in light of the role of Bcl-xL during B-cell development.
Expression of the Bcl-x gene is tightly regulated from the
pre-pro–B-cell stage (fraction A as defined by Hardy25) throughout
B-cell development,27 and germ-line deletion of Bcl-x resulted in
greatly reduced numbers of cells that were defined as small
B220dim/IgM⫺ pre–B cells.22,28 In our model, deletion of Bcl-x
targeted by the MMTVtTa transactivator strain in the absence of
BCR/ABL resulted in a 60% reduction of the CD19⫹/B220⫹ bone
marrow cell population compared with controls after 4 weeks, and
80% fewer CD19⫹/B220⫹ cells after 8 weeks (Figure 4A).
Consistent with these data, we detected recombination by 3-primer
PCR in DNA isolated from whole bone marrow and spleen cells
(Figure 4B). Based on these observations, we concluded that the
pool of cells available for transformation by the BCR/ABL oncogene should be diminished if recombination occurs at the stage of
pre-pro–B cell or pro–B cell, when differentiation is more dependent on survival signals mediated by Bcl-xL than in mature
populations that are known to express lower amounts of the
protein,27 making them perhaps more tolerant to complete deficiency.
In the following experiment, we sough to determine the
developmental stage at which recombination occurs in this model.
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
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Figure 5. Expression of Bcl-2, Mcl-1, A1, and Pin-1 kinase in Bcl-x–deficient B-ALL. (A) Western blot analysis of protein lysates from pleural effusion isolated from B-ALL
(lanes 1-3) and Bcl-x–deficient B-ALL mice (lanes 4-6). *Actin response from previous stain. (B) mRNA expression levels of A1 and pim1 in cells isolated from pleural effusion
were determined by real-time PCR (results are given as mean ⫾ SEM; n ⫽ 4).
We isolated pre-pro–B cells (fraction A), pro–B cells (fraction B),
and early pre–B cells (fraction C) from the bone marrow of
MMTV-Tet-O-cre mice and determined mRNA expression of the
cre recombinase gene by real-time PCR. Expression was detected
in fraction A, followed by a 5-fold increase of expression in
fraction B and subsequent decrease in fraction C cells, comparable
with the level observed in fraction A (data not shown). To verify
that expression of cre resulted in successful deletion of Bcl-x, we
isolated fraction A and fraction B cells from Bcl-x–deficient B-ALL
mice 3 and 4 weeks after induction and tested for recombination by
3-primer PCR. Again, we found a high level of recombination in
fraction A and a further increase in fraction B (Figure 4C).
Expression of antiapoptotic proteins and target genes of STAT5
is not affected by deletion of Bcl-x in B-cell lymphoblasts
The presence of recombined alleles in fractions A and B taken
together with reduction of CD19⫹/B220⫹ cells in the absence of
BCR/ABL expression should compromise the pool of cells available for transformation by the oncogene. Following this line of
reasoning, an increase in the latency period or complete lack of the
B-ALL phenotype would have been the expected result of loss of
Bcl-xL expression, as was recently described for STAT5 deficiency.29 This raises the question of whether a compensatory
mechanism provides a survival signal otherwise mediated by
Bcl-xL. We concentrated on expression of Bcl-2, mcl-1, A1, and
pim1 because they either belong to the Bcl-2 family of antiapoptotic genes or are directly downstream of the STAT5 signaling
pathway. In addition, they have been linked to BCR/ABL leukemogenesis in in vitro studies using cell lines or primary patient
samples.30-32 We determined protein expression levels of Mcl-1 and
Bcl-2 by Western blot analysis and mRNA expression by real-time
PCR for A1 and Pim-1. Cells from pleural effusion were chosen for
this experiment, as they represent an almost homogenous cell
population with very few contaminating cells (Figure 2B). Both
assays did not show changes in expression of any of these genes in
BCR/ABL⫹ B lymphoblasts (Figure 5A,B).
Cell-cycle analysis of Bcl-x–deficient
lymphoblasts
BCR/ABLⴙ
B
Microscopic evaluation of blast cells from tissues and peripheral
blood of Bcl-x–deficient BCR/ABL mice revealed a high number of
mitotic figures (Figure 2C). Increased mitosis is indicative of rapid
proliferation; in light of the rapid progression of the phenotype, we
wondered if loss of Bcl-xL might have an effect on the cell cycle.
Previous studies using overexpression of Bcl-2 or of Bcl-xL in
fibroblasts and lymphocytes had suggested that these genes can
affect the cell cycle in vitro in addition to their biological roles as
inhibitors of apoptosis.33,34
We sought to determine if proliferation and cell-cycle status of
cells in mice with the Bcl-x–deficient phenotype was different from
MMTVtTA-BCR/ABL control animals. Using BrdU incorporation
to perform analysis of the cell cycle, we found a consistent increase
in the percentage of cells in S/G2/M phases of the cell cycle in both
pleural effusion (29.1% ⫾ 6.5%) and spleen (31.9% ⫾ 4.0%) in
Bcl-x–deficient B-ALL cells compared with B-ALL control cells.
The difference was significant in both tissues (Student t test,
P ⬍ .05; Figure 6A,B). These results suggest that although Bcl-xL
is dispensable for BCR/ABL-dependent B-ALL development, the
protein plays a role in regulating the proliferation rate of
lymphoblasts.
Loss of Bcl-x expression does not increase apoptosis in
Bcl-x–deficient B-ALL
Bcl-xL is a potent inhibitor of apoptosis; loss of this gene might
result in higher numbers of cells undergoing cell death, which is
negated by increased cycling and proliferation. Cells from the bone
marrow, spleen, and pleural effusion of Bcl-x–deficient BCR/ABL
transgenic mice were labeled with Annexin, and the number of
APC-Annexin⫹ cells in the B220dim/CD19⫹ cell population was
compared with the same cell population isolated from MMTV-BCR/
ABL control mice. There was no difference in Annexin⫹ cells
isolated from spleens and pleural effusion, but we observed a slight
increase of cell death in the bone marrow of Bcl-x–deficient B-ALL
(1.4-fold) over that of control cells; however, this difference was
statistically not significant (Figure 5C).
Overexpression of Bcl-xL in BCR/ABL disease delays
progression through the cell cycle
As a corollary study, we tested the effect of overexpression of
Bcl-xL on the development of BCR/ABL⫹ B-ALL by generating
mice which carry the cDNA for human Bcl-xL under the control
of a tetracycline-responsive element (Figure 7A). Of 3 founder
lines that were obtained, one expressed the transgene in bone
marrow, lymph nodes, and spleen after withdrawal of tetracycline (data not shown). Analysis of the bone marrow demonstrated that expression of hBcl-xL resulted in a 2-fold increase
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HARB et al
tion, which was detected at day 10 (WBC ⱖ 15 000 cells/␮L).
Overall, the development and progression of the phenotype between MMTV-BCR/ABL control and MMTVtTA-BCR/ABLhBcl-xL mice was not significantly different, and all animals were
killed due to moribund condition within 4 weeks of induction.
Expression of the hBcl-x transgene was readily detected by Western blot
analysis (Figure 7C) in triple-transgenic mice. Based on our data that
suggest that Bcl-xL affects the proliferation of BCR/ABL⫹ lymphoblasts, we performed cell-cycle analysis of B220dim/CD19⫹
B lymphoblasts isolated from the spleen of MMTVtTA-BCR/ABLhBcl-xL and control mice. We found a significant reduction in the
number of cells in S/G2/M phases in triple-transgenic mice
compared with controls, which is in agreement with our hypothesis
that Bcl-xL does affect the proliferation of cells transformed by
BCR/ABL (Figure 7D).
Similar to what we observed in the MMTV-hBcl-xL model
without BCR/ABL, we found an increase in fraction B, while
fraction A was not affected (data not shown). The effect on the cell
cycle underlines our observation made in the Bcl-x–deficient
B-ALL mice and suggests that expression of Bcl-xL in the context
of BCR/ABL expression in the B-cell lineage rather affects the cell
cycle then providing an essential prosurvival signal. Overexpression of the Bcl-x gene leads to reduced cycling of cells, while
reduction in the number of B-cell progenitors due to loss of Bcl-x in
deficient animals is overcome by the oncogene, leading to an
increase in lymphoproliferation.
Apoptotic response after antineoplastic treatment
Figure 6. Effect of Loss of Bcl-x on cycle entry and apoptosis in BCR/ABLⴙ
lymphoblasts. (A) Pleural effusion isolated from B-ALL and Bcl-x–deficient B-ALL
mice 16 hours after BrdU injection. Cells were stained with fluorescently labeled
antibodies against B220, CD19, and BrdU. Cell-cycle analysis, BrdU versus 7-AAD,
is shown for the gated B220dim, CD19⫹ population. Shown are representative results
of 4 experiments. (B) Graph displaying difference in B220dim, CD19⫹ S ⫹ G2M status
in either pleural effusion (PE) or spleen (SPL) cells isolated from B-ALL and
Bcl-x–deficient B-ALL diseased mice (average ⫾ SEM; n ⫽ 4 for PE and n ⫽ 3 for
SPL). (C) Bone marrow, pleural effusion, and splenocytes isolated from B-ALL and
Bcl-x–deficient B-ALL mice were stained with fluorescently labeled anti-B220,
anti-CD19, and anti–annexin-V. Percentage of annexin⫹ cells are reported for
B220dim, CD19⫹ population only (results are given as means ⫾ SEM; n ⫽ 4).
of cells corresponding to fraction B (CD19⫹/B220dim/c-kit⫹/
BP1⫺), while other fractions were not significantly affected
(data not shown), as it was reported using an E␮-Bcl-x
transgene.27,35 Cell-cycle analysis of bone marrow cells from
double-transgenic animals compared with littermate controls
showed reduced cycling of cells in S phase and reduced number
of cells in G2 and M phases of the cell cycle (Figure 7B). This
difference was highly significant (Student t test, P ⬍ .001).
In order to test the effect of high levels of Bcl-xL in the context
of BCR/ABL leukemogenesis, we generated mice with the
MMTVtTA-BCR/ABL-hBcl-xL genotype. Development of an aggressive lymphoproliferative disease of the B-cell lineage was
detected as measured by the number of white blood cells in the
peripheral blood and the appearance of the B220dim/CD19⫹ popula-
The number of lymphoblasts undergoing apoptosis did not differ
between Bcl-x–deficient B-ALL and control B-ALL, indicating that
expression levels of Bcl-xL do not affect apoptosis in Ph⫹ B-ALL
progenitors. However, it is conceivable that Bcl-xL does provide a
level of protection apparent only after exposure to potent apoptotic
stimuli, such as antineoplastic treatment. To test this hypothesis, we
evaluated the apoptotic response after a single administration of
cyclophosphamide, a drug that is also used for the treatment of
patients with Ph⫹ B-ALL.36 To assess the number of apoptotic
cells, mice were killed 24 hours after administration of the drug,
and B220dim/CD19⫹ cells from bone marrow and spleen and the
percentages of annexin-V⫹ cells were determined by FACS
analysis. There was a statistically significant increase in the number
of apoptotic cells in both tissues from Bcl-x–deficient B-ALL mice
compared with B-ALL controls, while less apoptotic cells were
detected in the hBcl-xL B-ALL model (Figure 7E), indicating a
direct correlation between Bcl-xL protein levels and susceptibility
to apoptosis following a potent stimulus.
Discussion
Disruption or dysregulation of genes that control apoptosis cannot
only initiate tumor progression but also enhance resistance to
cancer treatment. Previous studies suggest that BCR/ABL⫹ cells
express high levels of Bcl-xL, making them resistant to the
induction of apoptosis by a variety of agents.37 Here, we demonstrate that expression of Bcl-xL is not required for leukemogenesis
by the BCR/ABL oncogene, and that the disease progresses
unimpeded in the absence of this protein. This result is surprising
given the fact that expression of Bcl-xL is tightly regulated during
B-cell development, and deficiency of this protein leads to a
decrease in the number of pro–B cells as shown previously22,28 and
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BLOOD, 1 APRIL 2008 䡠 VOLUME 111, NUMBER 7
BCL-X IN PH⫹ B-ALL
3767
Figure 7. Effect of overexpression of Bcl-xL on cell-cycle entry and response to antineoplastic treatment. (A) Schematic of hBcl-xL transgene. TRE is the responsive
element with minimal promoter upstream of the human Bcl-xL cDNA followed by intervening sequence (IVS) and poly A site. (B) Graph displaying difference in B220dim/CD⫹
S ⫹ G2M status in splenocytes isolated from MMTV hbcl-xL and littermate controls (WT; mean ⫾ SEM). For wild-type, n ⫽ 5; MMTV hbcl-xL, n ⫽ 3. * indicates statistically
significant; and **, statistically highly significant. (C) Splenocytes from B-ALL and hbcl-xL B-ALL mice were isolated 16 hours after BrdU injection. Cells were stained with
fluorescently labeled antibodies to B220, CD19, and BrdU. Cell-cycle analysis, BrdU versus 7-AAD, is shown for the B220dim/CD19⫹ population. (D) Western blot analysis of
protein lysates from the spleen of B-ALL (lanes 1 and 2), hbcl-xL B-ALL (lanes 3 and 4), and Bcl-x–deficient B-ALL diseased mice. (E) Percentage of cells undergoing apoptosis
in bone marrow and spleen after treatment with cyclophosphamide. Animals were killed after a single administration of the drug (results are given as mean ⫾ SEM; n ⫽ 3).
substantiated in this study. Our data demonstrate that the MMTVtTA
transactivator targets expression of transgenes to the pre-pro–Bcell developmental stage. We did observe a decline in the number
of early pro–B cells when deleting the Bcl-x gene in the absence of
BCR/ABL, which suggests that the pool available for transformation by the oncogene was diminished (J.G.H., B.I.C., C.S.H.,
unpublished data, March 2007). Nevertheless, the phenotype
developed within the same time frame. Several scenarios can
explain this phenomenon. Bcl-xL may not be required for the
survival of lymphoblasts, and BCR/ABL provides an alternative
survival signal sufficient for early progenitors to progress to the
pre–B-cell stage. We have tested this theory and determined the
expression levels of Bcl-2, mcl-1, Pim-1 kinase, and A1 in
lymphoblasts from Bcl-xL–deficient B-ALL mice and B-ALL
control mice. These genes were selected because they either belong
to the same family of antiapoptotic proteins as Bcl-xL or are known
targets of STAT5. While we did not find changes in expression, it is
possible that the expression levels are already sufficient for
survival independent of Bcl-xL. Alternatively, an entirely different
signaling pathway may be used, and BCR/ABL has been demonstrated to target myriad signaling pathways.38
Last, we cannot exclude the possibility that expression of
BCR/ABL and deletion of Bcl-x occurs at different time points.
This allows for a sequence of events in which cells first express
the oncogene, allowing them to proceed in development before
deletion of Bcl-xL expression takes place. Alternatively, recombination of Bcl-xL is the first event leading to reduction of
B cells, and the phenotype develops from a small pool of cells.
Retroviral transduction of bone marrow cells with BCR/ABL can
establish B-ALL as a monoclonal disease suggestive of transformation of a single cell.39
STAT5 is a major activator of Bcl-xL transcription and a
regulator of cellular growth and differentiation in a wide variety of
tissues.40-43 It was recently shown that complete deletion of STAT5
renders bone marrow cells resistant to transformation and leukemia
development induced by BCR/ABL.29 The lack of transformation in
this model also raises the question about the role of Bcl-xL. STAT5
is required for a plethora of signaling pathways, including but not
limited to immunoglobulin heavy chain variable gene rearrangement,44 which is absolutely necessary for B cells to avoid elimination by apoptosis. Thus, BCR/ABL is either entirely deprived of the
B-cell population in the absence of STAT5, or it does not provide a
strong enough survival signal to overcome this impediment in the
absence of STAT5. However, this STAT5-mediated effect is
upstream of Bcl-xL, as we have confirmed in our model system that
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HARB et al
rearrangement of IgH variable genes is not affected by loss of
Bcl-xL (data not shown).
Regardless, these considerations are only important from a
developmental point of view; they are not relevant with respect to
maintenance of BCR/ABL⫹ leukemia, as B lymphoblasts in our
model have undergone complete recombination and targeting
expression of the Bcl-x gene appears to exacerbate development of
the leukemia phenotype with significantly more cells in S and G2/M
phases of the cell cycle than lymphoblasts from control animals. In
contrast to this result and consistent with the ability of Bcl-xL to
manipulate the cell cycle, we found G1 arrest and reduced cycling
in lymphoblasts that express amounts of the protein well above the
levels of wild-type cells transformed by the BCR/ABL oncogene in
our model (Figure 7C; J.G.H., B.I.C., C.S.H., unpublished results,
June 2007). The difference in cell-cycle progression and generation
time between control lymphoblasts and deficient cells becomes all
the more apparent by theoretic calculation of the number of tumor
cells that can arise from one single cell within 1 week: we report a
difference in cell cycle of 29.1% plus or minus 6.5%; by taking a
difference of 23% into calculation, 256 cells will arise as progeny
from a wild-type tumor cell compared with 1024 cells from a
Bcl-x⫺/⫺ cell.
Known for its survival promoting function, our results suggest
an additional function for Bcl-xL, an antiapoptotic protein as a
regulator of the cell cycle; this concept has support in the literature.
Transgenic expression of Bcl-2 in normal fibroblasts and lymphocytes delayed entry into the cell cycle,33,45,46 while Bcl-2–deficient
T lymphocytes exhibited accelerated cell-cycle progression.34 One
molecular mechanism by which loss of Bcl-xL might affect cycling
in our model system could be through degradation of the cyclindependent kinase inhibitor p27. A previous study using transgenic
expression of Bcl-xL in NIH-3T3 cells revealed a delay in
cell-cycle entry through elevation of p27,46 and cell lines transfected with BCR/ABL have shown that the oncogene targets p27
protein leading to its proteasomal degradation, thereby enhancing
proliferation of cells.47,48
There is also evidence that support the idea that antiapoptotic
proteins manipulate the cell cycle in the context of neoplastic
disease. Transgenic expression of Bcl-2 delayed tumor development and reduced proliferation in a murine model of breast
tumors.49 In hematologic malignancies, high levels of Bcl-2 were
found to correlate with lower proliferative capacity in non-Hodgkin
lymphoma,50,51 and the majority of follicular lymphoma carry a
chromosomal translocation t(14;18) that places Bcl-2 under control
of the immunoglobulin heavy chain enhancer, leading to high
expression of the protein.52-55 Nevertheless, these tumors are
indolent and have a low proliferative index and clinical aggressivity. Another classic example is chronic lymphoid leukemia (CLL).
The hallmark of this disease are nondividing cells with increased
expression of Bcl-2 in almost all patients and of another antiapoptotic protein, Mcl-1, in approximately 50% of patients.56-59
Here, we report a novel role for Bcl-xL in the context of
BCR/ABL-associated leukemia as a regulator of the cell cycle that
tempers disease progression. In contrast to the prevailing concept
that suggests that levels of Bcl-xL are important for BCR/ABLtransformed cells to evade apoptosis, we found that Bcl-x–deficient
cells proliferate at a greater rate, while high levels of this protein
correlated with slower growth rate. Taken together, we conclude
that Bcl-x is not essential for Ph⫹ B-ALL, but its expression limits
tumor burden.
Acknowledgments
We thank Dr Lothar Hennighausen (National Institute of Diabetes
and Digestive and Kidney Diseases, National Institutes of Health
[NIH], Bethesda, MD) for providing Bcl-x f/f mice, Dr Ulrich
Steidl (Beth Israel Deaconess Medical Center, Boston, MA) for
critically reading the manuscript, the Transgenic Facility of the
Medical College of Wisconsin for performing microinjections, and
Hope Albertz and Corbett Reinbold from the FACS Core Facility at
the Blood Research Institute for assistance with cell sorting.
This work was supported by grants from the Blood Center
Research Foundation and the Lauri Strauss Leukemia Foundation
to C.S.H. J.G.H. was supported by NIH training grant HL-07209.
Authorship
Contribution: J.G.H. designed and performed experiments; B.I.C.
performed experiments; and C.S.H. designed the study, supervised
J.G.H. and B.I.C., and wrote the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Claudia S. Huettner, The Blood Research
Institute, Rm 210, 8727 Watertown Plank Rd, Milwaukee, WI
53226; e-mail: claudia.huettner@bcw.edu.
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From www.bloodjournal.org by guest on September 29, 2016. For personal use only.
2008 111: 3760-3769
doi:10.1182/blood-2007-08-108803 originally published
online January 23, 2008
Loss of Bcl-x in Ph+ B-ALL increases cellular proliferation and does not
inhibit leukemogenesis
Jason G. Harb, Brenda I. Chyla and Claudia S. Huettner
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