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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3780-3792
Efficient and Rapid Induction of a Chronic Myelogenous
Leukemia-Like Myeloproliferative Disease in Mice Receiving P210
bcr/abl-Transduced Bone Marrow
By
Warren S. Pear,
Juli P. Miller,
Lanwei Xu,
John C. Pui,
Benny Soffer,
Robert C. Quackenbush,
Ann Marie Pendergast,
Roderick Bronson,
Jon C. Aster,
Martin L. Scott, and
David Baltimore
From the Department of Pathology and Institute for Medicine and
Engineering, University of Pennsylvania, Philadelphia, PA; the
Department of Internal Medicine (Hematology/Oncology), Pharmacology,
and Cancer Biology, Duke University School of Medicine, Durham, NC; the
Department of Veterinary Medicine, Tufts University, Boston, MA; the
Department of Pathology, Brigham and Women's Hospital, Boston, MA; and
the Department of Biology, Massachusetts Institute of Technology,
Cambridge, MA.
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ABSTRACT |
Expression of the 210-kD bcr/abl fusion oncoprotein can cause a
chronic myelogenous leukemia (CML)-like disease in mice receiving bone
marrow cells transduced by bcr/abl-encoding retroviruses. However,
previous methods failed to yield this disease at a frequency sufficient
enough to allow for its use in the study of CML pathogenesis. To
overcome this limitation, we have developed an efficient and reproducible method for inducing a CML-like disease in mice receiving P210 bcr/abl-transduced bone marrow cells. All mice receiving P210
bcr/abl-transduced bone marrow cells succumb to a myeloproliferative disease between 3 and 5 weeks after bone marrow transplantation. The
myeloproliferative disease recapitulates many of the hallmarks of human
CML and is characterized by high white blood cell counts and extensive
extramedullary hematopoiesis in the spleen, liver, bone marrow, and
lungs. Use of a retroviral vector coexpressing P210 bcr/abl and green
fluorescent protein shows that the vast majority of bcr/abl-expressing
cells are myeloid. Analysis of the proviral integration pattern shows
that, in some mice, the myeloproliferative disease is clonal. In
multiple mice, the CML-like disease has been transplantable, inducing a
similar myeloproliferative syndrome within 1 month of transfer to
sublethally irradiated syngeneic recipients. The disease in many of
these mice has progressed to the development of acute lymphoma/leukemia
resembling blast crisis. These results demonstrate that murine CML
recapitulates important features of human CML. As such, it should be an
excellent model for addressing specific issues relating to the
pathogenesis and treatment of this disease.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
clonal, biphasic disease that accounts for approximately 20% of all
leukemias.1,2 The initial or chronic phase of the disease
lasts 3 to 5 years, on average, and is characterized by extremely high
peripheral white blood cell (WBC) counts and
extramedullary hematopoiesis leading to splenomegaly. These
characteristics result from expression of the bcr/abl fusion gene that
is the product of the (9;22) chromosomal translocation. Expression of
the bcr/abl oncoprotein in a hematopoietic progenitor cell is thought
to be rate-limiting in the pathogenesis of chronic-phase CML. Almost
all patients are diagnosed during chronic phase and this phase of the
disease can be successfully palliated with relatively nontoxic drugs.
Chronic-phase CML is followed by a terminal blast phase, during which
the patient develops an acute leukemia. In contrast to chronic-phase
CML, the blast phase is refractory to treatment and median survival is
2 to 4 months. The only cure for CML is a bone marrow transplant
performed during chronic phase.
Identification of the consistent (9;22) translocation has led to a
relatively detailed understanding of some of the molecular events in
CML.3 The molecular consequence of the t(9;22) is juxtaposition of the bcr and abl genes with the consequent expression of an abnormal fusion protein created by the joining of the amino terminus of the bcr protein with the carboxyl-terminus of the abl
protein.4 Creation of the P210 bcr/abl fusion protein via chromosomal translocation juxtaposes several protein domains that are
thought to contribute to the transforming ability of the fusion protein. Bcr contributes an oligomerization domain, serine-threonine kinase domain, grb2 binding site, Dbl homology domain, and PH domain.5-9 Important abl protein domains that are retained
in the bcr/abl fusion protein include an SH3 domain, which may
negatively regulate transformation, an SH2 domain that binds
phosphotyrosine, an SH1 or tyrosine kinase domain, whose presence is
absolutely essential for transformation, and sequences for nuclear
localization, actin binding, and DNA binding.5,9-12
Approximately 92% of patients with CML express the 210-kD bcr/abl
fusion protein, whereas 3% express the shorter 190-kD fusion protein.
The 190-kD bcr/abl fusion protein lacks the Dbl-homology and PH domains
that are present in the P210 kD bcr/abl fusion protein.
There are several important differences between the bcr/abl fusion
protein and its normal cellular counterparts (c-abl, c-bcr) that may
explain the transforming ability of bcr/abl. The abl tyrosine kinase
activity of the bcr/abl fusion protein is much greater than that of
c-abl.13 Unlike c-abl, which is primarily localized in the
nucleus in transfected cells, bcr/abl is localized in the
cytoplasm.11 The cytoplasmic localization and increased tyrosine kinase activity of bcr/abl induces the formation of multimeric protein complexes that may affect cellular proliferation, adhesion, and
apoptosis, eventually leading to transformation.9 These complexes with bcr/abl include the adapter proteins, grb2 and crkL, the
focal adhesion proteins, paxillin and talin, and the ras-activating
proteins, shc and p62 dok.6,14-18
Most of our understanding of abl transformation is based on results
obtained through in vitro transformation assays using fibroblasts or
factor-dependent hematopoietic cell lines. It is not clear if these
assays are relevant to CML, because this disease arises in an early
hematopoietic progenitor cell. In addition, the in vitro assays have
often given contradictory results. For example, the Y177F mutation in
bcr/abl abrogates fibroblast transformation but confers factor
independence on factor-dependent cell lines.6,7,19 Results
such as these have made it difficult to evaluate the contribution of
certain bcr/abl domains to transformation.
One approach for investigating CML that is relevant to the disease is
to use primary human tissue for study. Studies of this sort have shown
that primary tissue has fewer phosphorylated proteins than cell lines
and have turned the focus to several proteins that are highly
phosphorylated in patients with CML, such as p62 dok and
crkL.18,20 A major deficiency in using patient tissue to
study the pathogenesis of CML is that relevant functional assays do not
exist. One promising methodology for identifying the CML reservoir cell
is to transfer human cells into SCID-NOD mice, as has been successfully
performed to identify the cell type causing AML.21 A recent
report shows that CML cells engraft at low frequency in NOD/SCID
mice22; however, the utility of this assay for studying the
pathogenesis of CML has not been proven.
The deficiencies of the in vitro assays and the lack of a functional
assay using human cells has driven the search for a murine CML model
that accurately recapitulates this disease. Although creation of
transgenic mice that express bcr/abl would be an excellent system, many
of these transgenic strains develop acute leukemias without evidence of
a myeloproliferative disease.23-25 A recent report suggests
that expression of P210 bcr/abl via the tec promoter in transgenic mice
may cause a myeloproliferative disease and blast
transformation.26 One major drawback to this potentially useful system is that the latency period of the disease is greater than
1 year.26 In addition, the penetrance of either the
myeloproliferative disease or blast crisis has not been determined.
To date, the only approach that successfully recapitulates both the
myeloproliferative and blast crisis phases of CML is a retroviral
transduction model.27 In this model, introduction of
P210-infected bone marrow into lethally irradiated
syngeneic recipients caused a myeloproliferative disease resembling
human CML.28-32 The mice developed very high WBC counts
consisting primarily of cells of the granulocytic lineage,
splenomegaly, and extramedullary hematopoiesis (EMH). In a small number
of cases, it was possible to serially passage disease to syngeneic
recipients. Some of these recipients developed neoplasms resembling
blast crisis. Analysis of the proviral integration patterns in the mice
with myeloproliferative syndromes and mice with acute
leukemias/lymphomas showed identical proviral integration sites, which
suggested that the disease arose in a hematopoietic progenitor cell.
Although it was hoped that these earlier retroviral transduction models
would be useful for studying the pathogenesis of CML, limitations
became apparent.27 First, the murine disease was not
reliably induced because of difficulties in generating high-titer bcr/abl-expressing retroviruses. Second, the incidence of the myeloproliferative disease in transplant recipients was low, typically 20%, even when viral titers were relatively high (>5 × 105). Third, serial transfer of the myeloproliferative
disease occurred at a low frequency.30,32 The combination
of these deficiencies greatly limited the use of these earlier murine
CML models as a functional assay.
In this report, we describe a murine CML model that successfully
overcomes the limitations of previous retroviral transduction models.
We are able to induce a myeloproliferative disease that resembles many
aspects of human CML in 100% of recipient mice by 4 weeks after
transfer of P210 bcr/abl-transduced bone marrow cells. In many cases,
it is possible to serially passage the disease for up to 3 rounds. In
all cases of serial passage, the myeloproliferative disease transforms
into a lymphoid neoplasm, whose proviral integration pattern is
identical to that in the primary myeloproliferative disease. This
suggests that the disease arises in a myeloid/lymphoid progenitor cell.
The consistency and rapidity of disease onset together with the ability
to induce blast transformation should allow this murine model to be
used for studying the pathogenesis of CML in a setting that is relevant
to the human disease.
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MATERIALS AND METHODS |
Retroviral vectors and constructs.
MSCV 210 was constructed by ligating the 7.2-kb EcoRI fragment
containing the bcr/abl cDNA from pGD21028 into
MSCV2.2.33 MSCV Grb2 was constructed by amplifying the
myc-tagged grb2 cDNA34 to which an Xba I site was
added at the 3 end and subcloning this into the Nco
I-Xba I site of pCITE (Novagen, Madison, WI). The
1.5 kB EcoRI-Sal I fragment containing the
encephalomyocarditis virus (EMCV), internal ribosomal entry site
(IRES), and grb2 cDNA was then cloned into the identical restriction
fragment sites in MSCV2.2. The enhanced green fluorescent protein
(eGFP)-expressing retroviral vector, MigR1, was constructed by
three-way ligation with the 5-kb EcoRI-Sal I cleaved
MSCV2.2, the 0.5-kb IRES containing EcoRI-Nco I
fragment from pCITE, and the 0.8-kb Nco I-Sal I
fragment containing a GFP with solubility and red shift mutations
(J. Jacob, unpublished data). Mig210 was
constructed by ligating the 7.2-kb EcoRI bcr/abl
cDNA28 into MigR1.
Retroviral production and bone marrow infection protocols.
Transfection of the retroviral vectors, cocultivation with
5-fluorouracil (FU)-treated bone marrow, and injection
into lethally irradiated Balb/c recipients were performed as previously
described.35 For experiments using MSCV 210, MSCV 210/grb2,
and MSCV grb2, transduction of bone marrow cells was performed by
cocultivation with transfected Bosc23 cells. Cocultivation of the
transfected Bosc23 cells and 5-FU-treated bone marrow was performed in
a cocktail consisting of Dulbecco's Modified Eagle medium
(DME), 10% heat-inactivated fetal bovine serum
(JRH, Lenexa, KS), 5% WEHI-conditioned medium, 6 U/mL
recombinant mouse interleukin-3 (IL-3; Genzyme, Cambridge, MA), 10,000 U/mL recombinant mouse IL-6 (Genzyme), 5 U/mL
recombinant mouse stem cell factor (SCF; Amgen, Thousand Oaks,
CA), 4 µg/mL polybrene (Sigma, St Louis,
MO), 100 U/mL streptomycin (GIBCO, Grand Island,
NY), 100 U/mL penicillin (GIBCO), and 2 mmol/L
L-glutamine (GIBCO). Between 2.5 × 105 and 5 × 105 nonadherent cells were injected into a tail vein of
each lethally irradiated syngeneic recipient mouse. 5-FU-treated bone
marrow cells were transduced by spinoculation36 for the
experiments using the MigR1 and Mig210-derived retroviral supernatants.
For these experiments, bone marrow cells, which were obtained
4 days after 5-FU treatment, were cultured for 24 hours in the
above-described cocktail at a density of 1 × 106
cells/mL. The following day, 4 × 106 cells were added
to a well of a 6-well dish containing 3 mL of the infection cocktail
and 1 mL of thawed, previously frozen retroviral supernatant.
Spinoculation was performed as described,37 after which the
cells were returned to the incubator. Twenty-four hours later, a second
round of spinoculation was performed and the cells were returned to the
incubator for 8 to 16 hours. At this point, 1.25 × 105 to 2.5 × 105 cells were injected into
the tail veins of lethally irradiated syngeneic recipient mice. For
serial transplants, spleen or bone marrow cells from diseased mice were
injected into sublethally (450 rad) irradiated syngeneic mice. Balb/C
mice were obtained from Taconic Farms (Germantown, NY).
Comparison of cocultivation and spinoculation for bone marrow
transduction (assayed using MSCV 210/grb2) showed that there was no
difference in disease latency and phenotype between these two methods
(not shown).
Flow cytometry.
Cells obtained from peripheral blood, spleen, bone marrow, liver, or
lymph node were analyzed for forward scatter (FSC), side scatter (SSC),
and expression of GFP, Gr1, Mac1, Ter119, B220, Thy1.2, CD4, and CD8 by
multiparameter flow cytometry as described.38 Antibodies
used were as follows: CD8a-biotin (53-6.7; Pharmingen, San Diego,
CA), CD4-phycoerythrin (PE) (RM4-5; Pharmingen),
TCR -PE (H57-597; Caltag, South San Francisco, CA),
Thy1.2-biotin (53-2.1; Pharmingen), B220-biotin (RA3-6B2; Pharmingen),
MAC1-biotin (M1/70; Pharmingen), Ter119-biotin (Pharmingen), and
Gr1-biotin (RB6-8C5; Pharmingen). Dead cells were identified by
propidium iodide staining as described.38 Fluorescence was
analyzed on a FACScan flow cytometer with CellQuest software (Becton
Dickinson, San Jose, CA).
DNA analysis.
High molecular weight DNA was isolated from fresh or snap-frozen
tissues, digested with appropriate restriction enzymes, and analyzed by
Southern blot hybridization as previously described.35 Hybridization probes were the cDNA for neomycin resistance or the
592-bp ECMV IRES.
Immunoblotting and immunohistochemistry.
Tissues were minced and single-cell suspensions were prepared in
phosphate-buffered saline (PBS) in the presence of aprotinin (Sigma),
benzamadine (Sigma), and Pefabloc SC (Boerhinger Mannheim, Indianapolis, IN). Cells were lysed in 2× sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample
loading buffer and separated by electrophoresis. The abl-specific
antibody was 8E9 (Pharmingen). Cytospin slides were prepared from
peripheral blood using standard hematologic techniques. Briefly, the
slides were fixed in 3% paraformaldehyde/PBS for 10 minutes, rinsed
twice with PBS, and then permeabilized with 0.3% Triton X-100/PBS for
10 minutes. After rinsing twice with PBS, the slides were blocked for
30 minutes with blocking buffer consisting of 0.05 mol/L Tris-HCl (pH
7.6)/0.14 mol/L NaCl/1% bovine serum albumin (BSA)
fraction V/5% normal goat serum. The blocking solution was then
drained off the slide and a 1:100 dilution of primary antibody against
c-abl (24-11; Santa Cruz, Santa Cruz, CA) was applied for
1 hour. The slides were then washed in PBS for 10 minutes and drained,
and secondary antibody was applied (1:100 dilution of AP conjugated
goat antimouse IgG1 [Jackson Immunochemicals, West Grove,
PA] in blocking buffer) for 30 minutes. The slides were
then washed in PBS for 10 minutes and drained, and a 1:1 mixture of
stable fast red and stable napthol phosphate (Research Genetics,
Huntsville, AL) was applied for 10 minutes. The slides
were rinsed in deionized water and counterstained with hematoxylin. The
slides were visualized under bright field and fluorescence microscopy
using the rhodamine filter set. Positive and negative controls
consisted of cytospins of FL512 cells previously transduced with Mig210
retrovirus and untransfected FL512 cells, respectively.
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RESULTS |
MSCV210 induces a myeloproliferative disease in 100% of recipient mice
within 6 weeks of bone marrow transfer.
We believed that the low induction frequency and possibly the long
latency in existing mouse models of CML were caused by low transduction
rates of appropriate hematopoietic progenitors. Multipotent
hematopoietic progenitor cells are rare in the bone marrow
population,39 and retroviruses must be of sufficient titer
to infect these rare cells. To consistently generate high-titer P210
bcr/abl-expressing retroviruses, we used Bosc23 retroviral packaging
cells. Bcr/abl retroviral supernatants obtained after transfection of
Bosc23 cells had infectious titers in excess of 1 × 106 infectious units/mL, as assayed by either
G418 resistance or GFP expression.40 To improve the
likelihood that the transduced hematopoietic progenitors would express
the provirus, the MSCV retroviral vector was used to express the P210
bcr/abl cDNA (Fig 1A).33 For
transduction of hematopoietic progenitors, this vector offers the
advantages of (1) expressing cloned cDNAs from the murine stem
cell retroviral LTR that has been shown to express at high levels in
embryonic carcinoma and embryonic stem cells and (2) the presence of an
extended packaging site for maximal retrovirus production. In
conjunction, the bone marrow transduction conditions were altered by
including murine stem cell factor in the transduction
medium,41 preincubating the donor bone marrow cells for 24 hours before retroviral transduction,42 and either including additional retroviral supernatant in the coculture
medium32 or transducing the bone marrow cells by spin
infection (spinoculation).36 These latter changes were made
to enhance transduction of increased numbers of cycling hematopoietic
progenitors.
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| Fig 1.
(A) Structure of the retroviral vectors used to transduce
murine bone marrow. The LTRs and vector backbone for all constructs was
MSCV2.2. The EcoRI and Xba I restriction enzyme sites
used for determining proviral integration are indicated. (B) Protocol
for retroviral transduction of FU-treated bone marrow and subsequent
reconstitution. During the 48-hour cocultivation, retroviral
supernatants were added to the media (1 mL every 24 hours) or cells
were transduced by spin infection without cocultivation (see Materials
and Methods).
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High-titer retroviral supernatants (at least 1 × 106
G418 resistant NIH3T3 colonies/mL) were prepared by transfecting Bosc23 cells with MSCV 210. In our initial experiments (and all experiments using MSCV 210, MSCV 210/grb2, and MSCV grb2), bone marrow cells obtained from 5-FU-treated Balb/c mice were cocultured with the transfected Bosc23 cells for 48 hours, beginning 24 hours after transfection (Fig 1B). For the entire 72-hour period, the donor bone
marrow cells were cultured in medium containing IL-3, IL-6, SCF, and
5% WEHI-conditioned medium. Freshly thawed high-titer retroviral
supernatant was also added to the transduction medium at 24 and 48 hours after transfection. After the 48-hour cocultivation, nonadherent
cells were removed and injected into lethally irradiated syngeneic
recipients. Between 21 and 31 days, each mouse developed cachexia,
decreased movement, and poor grooming (Fig
2). Peripheral WBC counts were also elevated (>35 × 106/mL) in the recipient mice (Table 1 and
Fig 3A and B). In all mice, differential counts
showed that the cells in the peripheral blood were largely composed of
mature granulocytes, consisting of polymorphonuclear neutrophils (PMNs)
and metamyelocytes (Fig 3C). Blasts were rare and, when present, did
not account for greater than 3% of the WBC. Basophils were rarely
present in the peripheral blood. In some mice, a significant percentage
of nucleated red blood cells were present. In contrast to mice
receiving P210-transduced bone marrow cells, the peripheral WBC of mice
receiving bone marrow cells transduced with a control retroviral vector
was largely composed of mature lymphocytes. With the onset of these
triad of changes (elevated WBC, decreased activity, and cachexia), the mice were euthanized and analyzed for gross and histopathologic changes
and the presence of provirus.
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| Fig 2.
Survival of mice receiving transduced bone marrow cells.
The survival data are cumulative from two separate experiments for all
retroviral constructs, except MSCV 210, which are from one
experiment.
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| Fig 3.
Characteristics of the P210-induced myeloproliferative
disease. (A) Peripheral blood from a mouse transduced with a control
retrovirus (MigR1). Original magnification × 100. (B) Typical
appearance of peripheral blood from mouse with myeloproliferative
disease. Original magnification × 100. (C) Higher power view of (B).
Original magnification × 1,000. (D) Splenomegaly associated with
myeloproliferative disease. The diseased spleen is on top and the
spleen from a MigR1 control animal is below. (E) Hematoxylin and eosin
(H&E) section of spleen from (D). The red pulp is
replaced by sheets of granulocytes. Original magnification × 400. (F)
Hypercellular murine CML bone marrow. The great majority of cells are
mature granulocytes. Original magnification × 400. (G) Liver in
murine CML shows infiltration of mature myeloid cells and EMH in
sinusoids. Original magnification × 100. This mouse did not have the
macrophage expansion. (H) Murine CML liver with macrophage expansion
(arrow). Note infiltrating hematopoietic cells in sinusoids. Original
magnification × 400. (I) Pulmonary infiltrates of EMH in P210 mice.
Original magnification × 100. (J) T-cell lymphoma associated with
blast transformation (from an abdominal mass). Original magnification × 400. This tumor developed after 2 rounds of serial transplant of
the myeloproliferative disease from mouse H2. (K) Wright-Giemsa
staining of cells from peripheral blood of mouse receiving
Mig210-transduced bone marrow cells. Original magnification × 400. (L) Abl expression in the cells from (K) as detected by the abl
monoclonal antibody 24-11. Original magnification × 400. (M)
Wright-Giemsa staining of cells from peripheral blood of mouse
receiving Mig210-transduced bone marrow cells. Original magnification × 400. (N) Staining with an isotype control. Original magnification × 400.
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The pathologic changes found at necropsy of mice receiving
P210-transduced bone marrow were remarkably consistent, principally stemming from increased numbers of maturing hematopoietic cells within
the bone marrow and extramedullary tissues. The spleens were red, firm,
and enlarged, with weights 3 to 5 times greater than control animals
(Tables 1 and 2
and Fig 3D). Histologic examination showed marked expansion of the
splenic red pulp and partial to complete replacement of the splenic
white pulp by hematopoietic elements, mostly consisting of mature and
immature granulocytes admixed with variable numbers of immature
nucleated erythroid cells and megakaryocytes (Fig 3E). In some spleens,
foci of necrosis of up to 0.5 mm in size were present that appeared to
represent areas of infarction. The bone marrow was hypercellular (no
residual fat cells), with most of the cellularity consisting of
maturing granulocytes and scattered megakaryoctyes (Fig 3F). The livers were enlarged and firm, and histologic examination showed extensive EMH
within sinusoids and around portal tracts and central veins (Fig 3G).
Similar to the spleen, the liver contained immature and mature
granulocytes, foci of erythropoiesis, and megakaryocytes. In addition,
10 of 11 animals receiving MSCV 210 bone marrow cells exhibited a focal
perivascular expansion of macrophages, primarily confined to the liver
(Fig 3H). In addition, there was marked pulmonary hemorrhage in all of
the MSCV 210 mice. Microscopically, areas of hemorrhage were frequently
associated with foci of extramedullary hematopoiesis, predominantly
consisting of maturing granulocytes (Fig 3I). There was no evidence of
thymic enlargement or lymphadenopathy in the P210 animals.
Two other P210-expressing retroviral vectors, MSCV 210/grb2 and Mig210,
were also used to transduce bone marrow cells (Fig 1A). Like MSCV 210, both of these vectors expressed P210 from the MSCV LTR, but, in
contrast, both vectors expressed a second protein via the ECMV IRES
rather than an internal promoter. The use of the ECMV IRES results in a
bicistronic message that is independently translated. This strategy
increases the likelihood that both proteins will be expressed in
transduced cells by alleviating the effects of promoter competition,
which frequently causes downregulation of protein expression in
internal promoter vectors.43 MSCV 210/grb2 coexpresses P210
and grb2, whereas Mig210 coexpresses P210 and GFP. The former vector
was originally constructed to assess the effects of coexpressing P210
and grb2 on development of the murine disease (Pear et al, manuscript
in preparation) and the latter vector was developed to
use GFP as a surrogate marker to identify P210-transduced cells.
Coexpression of P210 and grb2 appears to have little effect on the
P210-induced disease, because MSCV 210/grb2 causes a myeloproliferative
disease with an identical phenotype to Mig210 and MSCV210 (not shown).
The disease latency was slightly shorter in experiments using either
the Mig210 or MSCV 210/grb2 vectors (Table 1 and Fig 2).
This difference may be due to the lack of promoter competition in the
bicistronic vectors.
P210 is expressed in involved tissues.
To evaluate expression of the introduced bcr/abl protein,
immunofluorescent and immunoblotting studies were performed. Cytospin preparations from involved organs showed P210 expression in myeloid cells, nucleated erythroid cells, and megakaryocytes using an indirect
immunofluorescent assay with an abl-specific monoclonal antibody (Fig
3K through N and not shown). To show that the correctly sized protein
was expressed, lysates prepared from spleen, liver, bone marrow, and
peripheral blood were analyzed by Western blot with an abl-specific
antibody. All tissues showed high levels of expression of the expected
210-kD bcr/abl protein (Fig 4).
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| Fig 4.
P210 bcr/abl is expressed in tissues involved in the
myeloproliferative process. Cells (5 × 106) were loaded
per lane and detected with the 8E9 abl-specific monoclonal antibody
(Pharmingen). Many of the lower molecular weight bands are most likely
the result of proteolysis due to the high percentage of neutrophils in
the cell lysates.28 Lanes 1 through 4, bone marrow,
peripheral blood, spleen, and liver cells from a Mig210 mouse. Lane 5, day-12 cells from bone marrow cultures transduced with Mig185. Mig185
is similar to Mig210, except Mig185 expresses P185 bcr/abl (Miller and
Pear, unpublished data). Lane 6, Balb/c bone marrow
cells. The band at approximately 140 kD in all lanes is c-abl. Arrows
indicate the 210- and 185-kD proteins.
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Analysis of P210-transduced cells in vivo demonstrates that the
majority of cells are myeloid in origin.
Further evaluation of the effects of P210 expression was facilitated by
use of GFP as a surrogate marker. In Mig210, GFP is coexpressed with
P210 as a bicistronic message. Thus, tissues that express GFP should
also express P210. This was confirmed by cytospin preparations in which
bcr/abl-expressing cells also expressed GFP (not shown). Use of the
Mig210 retroviral vector has allowed immunophenotyping of the involved
tissues, as described below (Fig 5). The
majority of cells in peripheral blood, spleen, bone marrow, and liver
expressed GFP. In all Mig210 mice analyzed by flow cytometry, the
GFP-expressing cells were contained within a single sharp peak on the
GFP histogram (Fig 5, column 2), suggesting the possibility that many
of the cells were derived from a single clone (see below). In
peripheral blood, spleen, and bone marrow, the great majority of
GFP-expressing cells coexpressed the myeloid-specific markers, Gr1 and
Mac1 (Fig 5, columns 3 and 4). Most of the cells in the peripheral
blood expressed high levels of both Gr1 and Mac1, features of mature
granulocytes. In contrast, cells in the spleen, bone marrow, and liver
expressed Gr1 over a wide range, consistent with the presence of both
mature and immature myeloid cells in these tissues.44 A
small percentage of B220lo/GFP+
cells were present in all tissues (Fig 5, column 5). There were very
few Thy1.2/GFP-positive cells in any organ, possibly because these mice
died before the occurrence of T-cell reconstitution (Fig 5, column 6).
Both liver and spleen contained a significant number of Ter119/GFP
positive cells, consistent with the histologic observation that the
bulk of erythropoiesis occurred in these tissues (Fig 5, column 7).
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| Fig 5.
Immunophenotypes of cells obtained from peripheral blood,
spleen, bone marrow, and liver of mice receiving either P210 (Mig210)
or control (MigR1) bone marrow cells. Mig210 expresses both P210
bcr/abl and GFP, whereas MigR1 expresses only GFP. GFP fluoresces in
the FL1 channel. The lineage-specific antibodies (Gr1, Mac1, B220,
Thy1.2, and Ter119) were directly or indirectly labeled with PE and
fluoresce in the FL2 channel. Hematopoietic cells were not present in
the livers of mice receiving MigR1. Ter119 staining was not performed
on the cells derived from MigR1 peripheral blood. The Mig210 data were
obtained from mouse BB12 and are a representative Mig210 phenotype. The
MigR1 data were obtained from mouse FF4 and are a representative MigR1
phenotype.
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The immunophenotypes of cells obtained from mouse tissues after
reconstitution with bone marrow cells transduced by the control GFP-expressing retroviral vector, MigR1, were very different than the
immunophenotypes described above (Fig 5). A lower percentage of cells
in all organs expressed GFP (Fig 5, column 2). In addition, unlike the
single peak present in the GFP histograms of the Mig210 mice, there was
a much broader pattern of GFP expression in tissues from the MigR1 mice
(Fig 5, column 2). In comparison to Mig210 mice, there was a much lower
percentage of myeloid cells in the spleen and a greater percentage of
B220+/GFP+ cells (Fig 5, columns 3, 4, and 5).
B220 expression was higher in the B cells in both the spleen and bone
marrow as compared with the P210 mice.
All affected tissues contain P210 proviral DNA.
To demonstrate that the tumors contained an intact provirus, DNA was
digested with Xba I and probed with either IRES sequences (MSCV
210/grb2 or Mig210) or the neo gene (MSCV 210). All tumors contained
the single expected 9-kb (MSCV 210) or 8.8-kb (Mig210 or MSCV 210/grb2)
band demonstrating an intact provirus (not shown). To enumerate the
proviruses, DNA was digested with EcoRI and probed with either
IRES sequences (Fig 6A through D and F) or
neo sequences (Fig 6E). The number of integrated proviruses ranged from
one (Fig 7B) to seven (Fig 6E, lane 3). The
number of proviral integration sites did not appear to influence the
disease, because all primary mice had a very similar disease latency
and disease phenotype. In some mice with multiple proviruses, the
proviral integrants were at levels comparable to a single copy of each
per genome (G22, G40, G41, G42, G56, H27, BB14, and BB16). This finding
suggests that the disease tissue arose from a single cell that had
sustained multiple retroviral infections. Moreover, DNA prepared from
different tissues from the same animal showed a similar proviral
integration pattern (G40, G42, BB14, and BB16). In other mice with
multiple proviruses, there were proviral integrants that did not show
equal contribution from all clones (G21, G50, and H28). Because DNA was
prepared from the entire tissue, it is possible that some of the
tissues contained multiple different clones but that the majority of
the cells were derived from a single clone.
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| Fig 6.
Proviral integration in P210 mice. (A through D) Tissues
from mice receiving MSCV 210/grb2. (E) Tissues from mice receiving MSCV
210. (F) Tissues from mice receiving Mig210. All DNA preparations were
digested with EcoRI except for lanes 1 and 3 in (F), which were
digested with BamHI, which also cleaves once in the provirus.
Digestion with Xba I showed the presence of intact proviral DNA
for all samples (not shown). All samples are from primary mice and
labeled with the tissue from which the DNA was derived, except for the
following in (B): lane 2, G46A1 spleen-secondary recipient of spleen
cells from G46, this mouse developed the myeloproliferative disease;
lane 1, G46A1B2 spleen: recipient of spleen cells from G46A1, this
mouse developed T-ALL; lanes 9 and 10, G41A1 spleen and peripheral
blood, G41A2 spleen, and G41A3 peripheral blood: recipient of spleen
cells from G41; these mice developed the myeloproliferative disease.
Abbreviations: PB, peripheral blood; B, BamHI; R1,
EcoRI. Sizes of the  HindIII marker are shown.
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| Fig 7.
Development of blast transformation after serial passage.
(A) Cartoon showing disease development in recipients of cells from
serial passage. Secondary recipients (A1 through 5) received a 1:1
mixture of spleen and bone marrow cells from mouse H2. Spleen or bone
marrow cells were transferred to tertiary recipients. Only mice
receiving the spleen cells developed the myeloproliferative disease.
All mice receiving bone marrow cells developed T-cell lymphomas with
characteristics similar to H2A4B1. Cells derived from the spleen of
tertiary recipients that developed the myeloproliferative disease were
transferred to quaternary recipients. All of these mice developed
T-cell lymphomas. (B) Proviral integration pattern shows a common
single proviral integration site in all H2 mice. Lanes 1 and 2 are from
primary mice. Lanes 3 through 8 are from secondary mice. (C) Both the
myeloproliferative disease and T-cell lymphomas show an identical
proviral integration pattern. Lanes 2 through 4 are derived from T-ALL.
Lanes 5 through 7 are secondary and tertiary recipients. (D) Tissues
from the H2 mice show the presence of an intact integrated provirus.
For (B) and (C), genomic DNA was digested with EcoRI and
genomic DNA shown in (D) was digested with Xba I. The blots
were hybridized with the cDNA expressing the neomycin resistance
gene.
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|
Murine CML can be serially passaged before blast transformation
occurs.
Serial passage is important for studying disease progression in the
murine CML model, because the myeloproliferative disease is rapidly
fatal due to the extraordinary hematopoietic expansion. In addition,
the ability to serially passage the disease allows determination of
clonality and also gives further proof that the disease is a
malignancy. In fact, there have been several cases in which murine
myeloproliferative defects have been called CML models; however, the
myeloid cells did not contain the provirus, suggesting that the
granulocyte accumulation was a reactive process.45
In primary mice that develop murine CML, serial passage was performed
by transferring either spleen or bone marrow cells (or both) to
sublethally irradiated syngeneic mice. This resulted in the occurrence
of hematopoietic malignancies in mice receiving 7 of 8 different
primary tumors (Table 3 and
not shown). For six of the primary tumors, it was possible to transmit
the myeloproliferative disease for at least one round and the disease
in the secondary recipients closely resembled the primary
myeloproliferative disease, both in latency and phenotype. Recipients
of G4 spleen and bone marrow cells developed a T-cell lymphoma with a
latency of 12 to 16 weeks (Table 3). The T-cell lymphoma
occurred in either the thymus or abdominal lymph nodes and its cells
were Thy1.2+/CD4+/CD8+ (not shown).
Some of the secondary recipients of cells from G28 developed the
myeloproliferative phenotype (3 of 4 secondary recipients), whereas one
recipient developed acute lymphocytic leukemia (ALL) at 14 weeks after
transfer as determined by histology. We were unable to immunophenotype
this tumor, because the mouse died before workup.
In our most successful series of serial transplantation, it was
possible to transfer the myeloproliferative disease to three generations of mice (Fig 6A). The myeloproliferative disease occurred with a latency period of 4 to 6 weeks in all mice and the phenotype of
the disease was similar to that shown in Fig 3. In the third round of
serial passage, recipients of mouse H2A4 developed T-cell lymphomas
presenting as solitary abdominal masses that were
Thy1.2+/CD4+/CD8+ (not shown). In
contrast, all of the recipients of spleen cells from H2A3 developed the
myeloproliferative disease. Serial passage of spleen cells from the
H2A3B mice resulted in the development of T-cell lymphomas in tertiary
serial transplant recipients (Fig 3J). The T-cell lymphomas were either
thymic masses or abdominal masses and expressed the immature T-cell
markers Thy1.2/CD4/CD8. Although serial passage of spleen cells
resulted in the myeloproliferative disease in the H2A3B tertiary
recipients, serial passage of H2A3 bone marrow cells caused only T-cell
lymphomas in tertiary recipients (not shown).
The proviral integration pattern was analyzed to determine whether the
myeloproliferative disease and T-cell lymphomas in the H2 mice were
derived from the same precursor. To enumerate the proviruses present in
the tissue samples, genomic DNA was digested with EcoRI and
probed with the neo gene (Fig 7B and C). The H2 primary mouse contained
a single proviral integrant of approximately 15 kb (Fig 7B, lanes
1 and 2). Affected tissues from secondary and tertiary mice that
developed the myeloproliferative disease contained a single identical
15-kb proviral integration (Fig 7B, lanes 3 through 8, and 7C, lanes 5 through 7). A single, identical proviral integration was also present
in the T-cell lymphomas (Fig 7C, lanes 2 through 4). This demonstrates
that murine CML arises in a pluripotential hematopoietic cell that is
capable of giving rise to both the myeloid and lymphoid lineages. To
demonstrate that the tumors contained an intact provirus, DNA was
digested with Xba I (which cleaves in both retroviral LTRs) and
probed with the neo gene. The expected 9-kb band from MSCV 210 is
present in all affected tissues from H2 mice (Fig 7D and not shown).
Analysis of the proviral integration pattern in other serial passage
recipients showed that the identical proviral integration pattern that
was present in the primary mice is also present in secondary and
tertiary recipients of serially transplanted spleen and bone marrow
cells (Fig 6B, lanes 1 through 4 and 7 through 11). In addition, spleen
cells from G56 were transferred to lethally irradiated recipients for a
day-12 colony-forming unit-spleen (CFU-S) assay and all
individual spleen colonies contained intact, integrated proviral DNA
(not shown). This suggests that the P210-transduced cell that caused
the G56 myeloproliferative disease was also capable of giving rise to
day-12 spleen colony-forming units.
| |
DISCUSSION |
We have developed an efficient and reproducible method for inducing a
murine disease that recapitulates the cardinal features of human CML.
This has resulted in the ability to induce murine CML in 100% of
recipient mice with a latency of 4 to 6 weeks. Previous investigators
have induced murine CML after transfer of bcr/abl-transduced bone
marrow cells into lethally irradiated recipient mice. Although these
studies showed that bcr/abl induced a CML-like disease in mice, the
utility of the model was compromised by low induction frequencies
and/or long latency periods. To overcome these problems, we
introduced several changes into the transplant protocols. These
included (1) consistent and reproducible production of high-titer
bcr/abl retroviral supernatants, (2) use of the MSCV retroviral vector,
and (3) changes in the bone marrow transduction milieu. Although
high-titer retroviral production is a prerequisite for disease
induction, the use of the MSCV retroviral vector and addition of stem
cell factor to the transduction cocktail are likely to be important for
efficient induction of murine CML. In contrast to the Moloney leukemia
virus LTR, the MSCV LTR is capable of efficient gene expression in
embryonic stem and carcinoma cells.46 Our results suggest
that the MSCV LTR functions equally well in hematopoietic progenitors.
In addition to the use of the MSCV LTR as a promoter, the use of an
IRES rather than an internal promoter to express a second mRNA is
associated with more efficient disease induction.
The 4- to 6-week latency period for disease development in our model is
shorter than that described in previous CML models, in which the
average latency varied between 9 weeks and 1 year.28-32 Potential explanations for our decreased latency are (1) the changes in
the culture conditions are putting the appropriate cell into cycle; (2)
the increased retroviral titer increases transduction of the correct
target cell; and/or (3) use of the MSCV LTR results in more
efficient bcr/abl expression. One effect of the changes introduced into
the methodology is to increase the number of proviral integrants. In
previous reports, the majority of analyzed tumors had only a single
proviral integration.28-32 In contrast, many of the tumors
described in this report contain multiple proviral integrations.
Nevertheless, the number of proviral integrations is unlikely to
explain the difference in disease, because several of the tumors had
only a single proviral integration and the phenotype of the disease in
these mice was indistinguishable from the disease phenotype in mice
harboring multiple proviral integrations.
The murine myeloproliferative disease that we observe in mice receiving
P210 transduced bone marrow is very similar to that described by
previous investigators. The common features include (1) markedly
elevated WBC with granulocyte predominance, (2) splenomegaly, (3)
multiple organ involvement, and (4) the presence and expression of the
bcr/abl provirus in affected tissues.28,29,31,32 One difference in the disease phenotype in our model is the consistent occurrence of pulmonary hemorrhage, which likely contributes to morbidity and mortality in P210-transplanted mice. One potential explanation is that the high WBC causes stasis in the pulmonary microvasculature, leading to thrombosis and subsequent tissue damage
and hemorrhage. This is unlikely to be a complete explanation, because
the WBC counts in previous murine CML models were similar to the WBC
counts in our model.28,29,31,32 A second possibility is
platelet dysfunction. Immunohistochemistry showed that at least some
megakaryocytes express bcr/abl and abnormally large platelets were
observed in the peripheral blood (not shown). Although other investigators have not described pulmonary hemorrhage, evidence of
thrombotic complications, including splenic infarcts and apical necrosis of the ears and tail, have been reported.28
Pulmonary hemorrhage in mice induced by our protocols may be due to a
similar mechanism.
Similar to previous studies, we frequently observe an expansion of
macrophages in the liver in association with the myeloproliferative disease.28 One difference between the three different
retroviral constructs used to induce the myeloproliferative disease was
their propensity to cause macrophage proliferation in the liver
(Table 2). Twenty percent of mice that received Mig210
bone marrow cells developed the macrophage expansion, whereas 45% and
90% of mice receiving MSCV 210/grb2 or MSCV 210, respectively,
developed this expansion. Rather than being due to differences in the
retroviral vector, this variation may be due to the way in which the
bone marrow cells were transduced. In the MSCV 210 and MSCV 210/grb2 experiments, transduction was performed by cocultivation with transfected producer cells, whereas in the Mig 210 experiments, transduction was accomplished by spinoculation. This suggests that the
variable macrophage expansion may be explained by enhanced transduction
of macrophage progenitors when cocultivation is used, possibly because
macrophage precursors adhere more strongly to the adherent producer
cells. In spinoculation, this is not a factor, because transduction is
performed in the absence of producer cells. In both our model and
similar models,28,47 the macrophage expansion does not
appear to be an essential aspect of the P210 myeloproliferative disease, because the other characteristics of the myeloproliferative disease remain consistent whether or not the macrophage expansion is
present. This is further supported by our failure to identify the
macrophage expansion in any mice that developed secondary or tertiary
tumors after serial transplantation.
Our hybridization studies show that both the myeloproliferative disease
and T-cell blast transformation can arise from a single cell containing
a single bcr/abl proviral integration. The disease in mice harboring a
unique and single proviral integration are clearly monoclonal. Other
tumors also appeared to be monoclonal, as suggested by the equal
intensity of proviral integration sites on Southern blot. In addition,
in all cases of serial passage that we examined, the same proviral
integration present in the primary tumor was also present after
multiple rounds of serial passage. Together, these results suggest that
a single clone is sufficient for murine CML. This is in agreement with
previous reports from other groups, in which the myeloproliferative
disease and blast transformation was associated with a single clone
harboring one proviral integration.28,30-32 However, it is
not clear if the disease is the result of selection of a single clone
arising in the presence of multiply infected cells or if there is only a rare cell that engrafts and subsequently causes the
myeloproliferative disease. Previous reports suggested that the latter
was the case as a consequence of the transduction
conditions.28 With the protocols described in this report,
it is likely that we are more efficiently transducing bone marrow donor
cells than previously described. Consistent with this possibility is
our finding of multiple proviral integrations in a majority of the
tumors. Further support of our ability to transduce multiple cells is
the pattern of GFP expression in tissues of mice receiving
MigR1-transduced bone marrow cells. In all tissues, GFP expression is
spread over a broad range (Fig 5, column 2), suggesting that expression
occurs from multiple clones. In contrast, GFP expression in tissues
expressing Mig210 shows a sharp peak (Fig 5, column 2), suggesting that
GFP expression arises from one (or a few) clones.
Because the MigR1 and Mig210 retroviral supernatants were normalized
for GFP expression before transduction and the donor bone marrow cells
were treated identically, the two different viral supernatants should
transduce bone marrow cells with equal efficiency. Thus, the
differences in the patterns of GFP expression between MigR1 and Mig210
suggest that bcr/abl expression causes the outgrowth of a single (or
few) clone(s) with a proliferative advantage. It is unclear if
development of the myeloproliferative disease by this clone requires
events in addition to bcr/abl expression or if the level of bcr/abl
expression determines the ability to cause the myeloproliferative
disease. In the latter scenario, there may be multiply
bcr/abl-transduced cells that are injected into each recipient mouse
but only one or a few of these cells expresses bcr/abl at a level that
is sufficient to cause the myeloproliferative disease.
Our serial transplantation studies show that the myeloproliferative
disease is transformed into a lymphoma. All of the lymphomas that we
have immunophenotyped are immature T cells. These tumors arise in
either the thymus, mesenteric lymph nodes, or both. The progression of
the myeloproliferative disease to T-cell lymphomas is puzzling, because
transduced T cells are a very small proportion of cells in mice with
the myeloproliferative disease. In previous descriptions of blast
crisis in retroviral murine CML models, transformation occurred in
myeloid cells, B cells, and T cells.30,32 The propensity
for T-cell transformation in our model is unlikely to be due to a bias
in expression of the MSCV LTR. As demonstrated by the immunophenotyping
results in the MigR1 mice, the Mig retroviral vectors are capable of
driving expression in most hematopoietic cell types, including myeloid,
B- and T-lymphoid, erythroid, and megakaryocytic cells. A
partial explanation for the proclivity for T-cell transformation upon
serial passage is that serial transplant itself is biased towards
T-cell proliferation. Spangrude et al48 have shown that
enriched hematopoietic progenitors lose their ability for multilineage
reconstitution after three rounds o |
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Abstract
Introduction