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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2067-2075
Development of Acute Lymphoblastic Leukemia and Myeloproliferative
Disorder in Transgenic Mice Expressing p210bcr/abl:
A Novel Transgenic Model for Human Ph1-Positive
Leukemias
By
Hiroaki Honda,
Hideaki Oda,
Takahiro Suzuki,
Tsuyoshi Takahashi,
Owen N. Witte,
Keiya Ozawa,
Takatoshi Ishikawa,
Yoshio Yazaki, and
Hisamaru Hirai
From the Third Department of Internal Medicine, Faculty of Medicine,
and the Department of Pathology, University of Tokyo, Tokyo, Japan; the
Department of Microbiology and Molecular Genetics, Howard Huges Medical
Institute, University of California; and the Department of Molecular
Biology, Jichi Medical School, Japan.
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ABSTRACT |
The Philadelphia (Ph1) chromosome can be detected in
chronic myelogenous leukemia (CML) and a significant number of acute lymphoblastic leukemia (ALL) cases. Generation of
p210bcr/abl, a chimeric protein with enhanced
kinase activity, is thought to be involved in the pathogenesis of these
diseases. To elucidate the biological properties of
p210bcr/abl and to create an animal model for human
Ph1-positive leukemias, we generated transgenic mice
expressing p210bcr/abl driven by the
promoter of the tec gene, a cytoplasmic tyrosine-kinase preferentially expressed in the hematopoietic lineage. The founder mice
showed excessive proliferation of lymphoblasts shortly after birth and
were diagnosed as suffering from ALL based on surface marker and
Southern blot analyses. Expression and enhanced kinase activity of the
p210bcr/abl transgene product were detected in the
leukemic tissues. In contrast, transgenic progeny exhibited marked
granulocyte hyperplasia with thrombocytosis after a long latent period
and developed myeloproliferative disorders (MPDs) closely resembling
human CML. Expression of p210bcr/abl mRNA
in the proliferating granulocytes was detected by RT-PCR. In
particular, one MPD mouse showed remarkable proliferation of blast
cells in the lung, which might represent an extramedullar blast crisis.
The results demonstrate that the expression of
p210bcr/abl in hematopoietic progenitor cells in
transgenic mice can contribute to two clinically distinct hematopoietic
malignancies, CML and ALL, indicating that this transgenic system
provides a novel transgenic model for human Ph1-positive
leukemias.
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INTRODUCTION |
CHRONIC MYELOGENOUS leukemia (CML) is a
hematopoietic disorder of pluripotent stem cells characterized by
uncontrolled proliferation of the myeloid series in peripheral blood
and bone marrow.1,2 In the initial stage of the disease,
chronic phase, the leukemic cells proliferate but have the ability to
differentiate into mature granulocytes. However, the disease eventually
accelerates after several years' duration and ultimately progresses to
the terminal phase, blast crisis, which involves the accumulation of
monoclonal immature hematopoietic cells arrested at an early stage of
differentiation.3
The cytogenetic hallmark of the disease is the Philadelphia chromosome
(Ph1), a shortened chromosome 22.4,5 It is generated by a reciprocal translocation between chromosome 9 and 22, t(9;22)(q34;q11), where the c-abl proto-oncogene on chromosome 9 is translocated into a 5.8 kilobase (kb) region on chromosome 22, denoted the major breakpoint cluster region
(M-bcr).6 This translocation fuses the 5 exons of
the bcr gene to most of the 3 exons of the c-abl gene
in a head-to-tail manner, thereby producing a novel 8.5kb chimeric
bcr/abl mRNA encoding a 210 kD protein (p210bcr/abl).
The Ph1 chromosome is also observed in 10% to 20% of
acute lymphoblastic leukemia (ALL) patients.2 In
approximately half of the cases, the translocation occurs at the same
breakpoint as in CML patients and creates the
p210bcr/abl hybrid protein. However, in the
remaining Ph1-positive ALL patients, the breakpoint exists
within the first intron of the BCR gene, designated the minor
breakpoint cluster region (m-bcr). This event generates a 7.0 kb mRNA, which is transcribed to a 190 kD chimeric protein
(p190bcr/abl) with a smaller bcr
moiety.7-11 Both of the chimeric proteins (p210bcr/abl and p190bcr/abl)
possess enhanced kinase activity in comparison with the normal 145 kD
c-abl product and both have been considered to be implicated in
the pathogenesis of Ph1-positive human
leukemias.12
To investigate the biological function of the
p210bcr/abl in vivo, efforts have been made to
create a mouse model that mimics human Ph1-positive
leukemias. For this purpose, two different approaches have been used,
namely, virus-mediated gene transfer and generation of transgenic mice.
Daley et al performed bone marrow transplantation (BMT) in mice. They
reconstituted lethally irradiated mice with bone marrow cells infected
with p210bcr/abl-expressing retroviruses and showed
that some of the recipients developed hematologic malignancies
including granulocytic hyperplasias resembling human CML, ALLs, and
tumors of macrophage cell type.13 DNA analysis revealed
that a limited number of the infected cells had differentiated and
reconstituted the hematopoietic cell series. Other studies also using
syngeneic mice lethally irradiated and transplanted with
p210bcr/abl-expressing bone marrow cells
demonstrated development of various types of hematologic disorders such
as granulocytic leukemias, myelomonocytic leukemias, pre-B and T-cell
lymphomas, reticulum cell sarcomas, and erythroid
tumors.14,15 As an alternative approach of virus-mediated
gene transfer, Clark et al injected p190bcr/abl- or
p210bcr/abl-expressing retroviruses directly into
thymi of mice.16 The recipients expressing both types of
chimeric proteins developed thymomas after a relatively long latent
period. These results imply that hematopoietic progenitor cells
infected with p210bcr/abl-containing retroviruses
acquire a proliferative advantage and cause various types of
hematologic malignancies.
Generating transgenic mice is another attractive approach for
investigating the biological properties of
p210bcr/abl. Hariharan et al microinjected a
bcr and v-abl fusion gene coupled with either the
immunoglobulin heavy-chain enhancer (Eµ) or the part of the long
terminal repeat (LTR) of the myeloproliferative sarcoma virus (MPSV)
and generated transgenic mice expressing p210bcr/v-abl.17 Pre-B or T-cell
lymphomas developed in some of the animals bearing either of the
constructs after a variable latent period. These findings indicate that
bcr/v-abl has oncogenic activity in vivo. However,
bcr/v-abl differs from bcr/abl in that it lacks parts
of the bcr- and abl-derived regions and it has several
amino acid substitutions in the latter.17,18 Thus, the
results obtained with p210bcr/v-abl transgenic mice
may not accurately reflect the biological properties of the original
hybrid protein. To overcome this issue, we have generated transgenic
mice expressing p210bcr/abl using the
metallothionein (MT) enhancer/promoter element
(MT/p210bcr/abl).19 We found two of six
founder mice and transgenic progeny to develop ALLs, classified as
being of T-cell origin by flow cytometric and Southern blot analyses.
Expression and enhanced kinase activity of the
p210bcr/abl transgene product in the leukemic
tissues were detected. In addition, the tissue distribution of
bcr/abl mRNA expression indicated that the oncogenecity of
p210bcr/abl is restricted to hematopoietic organs.
Our results thus demonstrated that the p210bcr/abl
chimeric protein contributed a proliferative advantage selectively to
hematopoietic precursor cells and induced T-cell leukemia in transgenic
mice.19 Subsequently, Voncken et al also generated mice
transgenic for p210bcr/abl using two variations of
the MT promoter and described acute hematopoietic malignancies mainly
of lymphoid origin and rarely of myeloid origin.20
As described above, p210bcr/abl transgenic mice
provide good models for human Ph1-positive leukemias.
However, they develop exclusively ALLs, in contrast to mice
reconstituted with p210bcr/abl-expressing bone
marrow cells which exhibit myeloproliferative disorders (MPDs)
resembling human CML. To date, no transgenic system exists that models
human CML, raising the question of whether p210bcr/abl transgenic mice are capable of
exhibiting CML-like disease. To address this issue, we cloned and
analyzed the promoter region of mouse tec
gene,21,22 a cytoplasmic kinase preferentially expressed in
hematopoietic precursor cells23 and generated
p210bcr/abl transgenic mice using the tec
promoter as a regulatory sequence. While the founder mice developed
ALLs, the transgenic progeny indeed demonstrated MPDs closely
resembling human CML.
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MATERIALS AND METHODS |
Construction of the transgene and generation of transgenic mice.
The metallothionein-I enhancer/promoter of
pMT/bcr/abl19 was replaced by the mouse tec
promoter ( 1948 to +22, designated the major transcription initiation
site defined by RACE-PCR as +121,22). A DNA fragment
containing the mouse tec promoter, the bcr/abl (p210)
cDNA (b3a2 type), and the SV40 early splicing and polyadenylation signals was microinjected into pronuclei of eggs from C57BL/6XDBA/2 F2
mice essentially as described earlier.24 The schematic
structure of the transgene is shown in Fig
1. The transgenic mice were identified by
hybridizing tail DNA with the injected fragment as described earlier.19
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| Fig 1.
Schematic model of the injection fragment for generating
transgenic mice. The upper part of the figure shows the genomic
structure of the mouse tec gene. The 5 flanking region and the
first intron are represented by thick and thin bars, respectively and
the first exon is shown as a shaded box. Restriction enzyme sites are
P; PstI, A; ApaI, S; SalI, X; XhoI, Sm;
SmaI, Nc; NcoI, Nh; NheI, Ac; AccIII,
and Sa; SacI. The lower part illustrates the structure of the
injection fragment. The tec promoter (1948 to +22), the bcr/abl (p210) cDNA (b3a2 type), and the SV40 early splicing
and poly(A) signals are shown as white, black, and shaded boxes,
respectively. The primers used for RT-PCR (P1-P4) are also indicated.
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Histopathologic examination.
Autopsies were performed on dead or moribund animals. Smears of
peripheral blood and stamp specimens of tissues were stained with
Wright-Giemsa (WG). Tissues were also fixed in 10% neutral buffered
formaldehyde for routine light microscopy. All the organs were grossly
examined and representative slices were prepared for hematoxylin-eosin
(HE) staining.
Western blotting, immunoprecipitation, and in vitro kinase assays.
Tissues were homogenized in RIPA lysis buffer (150 mmol/L NaCl, 50 mmol/L Tris-Cl (pH 7.4), 1% Triton X-100, 0.05% sodium dodecyl
sulfate [SDS], 1% sodium deoxycholate) with 50 U/mL aprotinin. For
detecting the p210bcr/abl transgene product, 100 µg aliquots of total proteins were separated by 6%
SDS-polyacrilamide gel electrophoresis (PAGE), transferred to
polyvinylidene membranes (Immobilon; Millipore, Yonezawa, Japan), and
probed with 1:200 diluted anti-c-Abl monoclonal antibody (MoAb), AB3
(Oncogene Science, Manhasset, NY). For in vitro kinase
assays, 1 mg aliquots of total proteins were incubated with 1:200
diluted AB3 and antimouse rat IgG (Sigma, St Louis, MO) coupled with
protein A (Sigma). The immunoprecipitated proteins were washed five
times with the lysis buffer, followed by five times with the kinase buffer (50 mmol/L Tris-Cl (pH 7.4), 10 mmol/L MgCl2, and 10 mmol/L MnCl2), and incubated with 10 µCi of
 32P-ATP (Amersham, Arlington Heights, IL) at room
temperature for 15 minutes. The phosphorylated proteins were separated
by 6% SDS-PAGE, dried, and autoradiographed.
Flowcytometric analysis.
Leukemic cells were stained with fluorescein isothiocyanate
(FITC)-conjugated commercial MoAbs including Thy-1.2, B220, Mac-1, or
Gr-1 (Fujisawa, Osaka, Japan) according to the manufacturer's instructions. The stained cells were washed three times with PBS and
analysed on a FACScan (Becton Dickinson, Sunnyvale, CA).
DNA analysis of leukemic cells.
High molecular weight DNAs were extracted from thymi of leukemic mice
or nontransgenic controls as previously described.25 Ten
microgram aliquots of DNA were digested with appropriate restriction enzymes (Takara, Kyoto, Japan), separated in 0.7% agarose gels, transferred to Nylon membranes (Hybond-N; Amersham International plc,
UK), and hybridized with a mouse TCR probe. Hybridization and
washing conditions were as described earlier.19 The signals were detected using a Fuji image analyser (Fujix bas 2000; Fujifilm, Kanagawa, Japan).
RNA extraction and PCR amplification.
Total RNAs were extracted using the acid guanidine/phenol-chloroform
method.26 The reverse transcription and PCR amplification methods used were as previously detailed.27
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RESULTS |
ALLs and myeloproliferative disorders in transgenic mice expressing
p210bcr/abl.
We generated five founder mice carrying five to ten copies of the
transgene. Among them, two mice, numbers 4-3 and 4-5, died of leukemias
3 and 4 months after birth, respectively (Table
1). Macroscopically, both mice showed
thymic enlargement, marked splenomegaly, and lymphnode swelling as
observed in MT/p210bcr/abl leukemic
mice.19 Blood parameters for 4-5 were: WBC 30 × 103/µL (>90% blast cells), Hb 14.0 g/dL, and Plt 50 × 104/µL (Table 1). Smears of peripheral blood showed
massive proliferation of blast cells with no granules, having the
appearance of lymphoblasts (Fig 2A).Histopathologic examination revealed marked proliferation of blast
cells in the thymus (Fig 2C), spleen (Fig 2E) and all other tissues
sampled (data not shown). According to the clinical course and the
results of surface marker and DNA analyses (see below), ALLs were
diagnosed.
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| Fig 2.
WG staining of tissues from 4-5 (A, C, E) and 4-5-10 (B,
D, F) that developed ALL and MPD, respectively. (A) Peripheral blood of
4-5. Massive proliferation of lymphoblasts is apparent. (B) Peripheral
blood of 4-5-10. Granulocyte hyperplasia is evident. (C) Thymus of 4-5. Note the marked infiltration of lymphoblasts. (D) Bone marrow of
4-5-10. The bone marrow is hyperplastic and contains myeloid cells at
various stages of differentiation. (E) Spleen of 4-5. Note infiltrating
lymphoblasts. (F) Spleen of 4-5-10. Extensive infiltration of
granulocytes and immature myeloid cells is apparent.
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The transgenic progeny of 4-5, one of the two founder mice that
developed ALLs, were maintained under continual observation. No obvious
signs of illness were observed until approximately 1 year after birth,
when one transgenic mouse, number 4-5-4, was found dead. Autopsy
revealed splenomegaly without swelling of the thymus, in contrast to
the marked thymic enlargement found in the parental founder mouse.
Several weeks after the death of 4-5-4, two transgenic littermates,
4-5-2 and 4-5-10, became moribund. They demonstrated emaciation,
unsteady gait, and necrotic areas on the skin. Peripheral blood
parameter for 4-5-2 and 4-5-10 were: WBC 45 × 103/µL
(>90% granulocytes), Hb 3.1 g/dL, and Plt >400 × 104/µL and WBC 15 × 103/µL (>90%
granulocytes), Hb 4.1 g/dL, and Plt >400 × 104/µL,
respectively (Table 1). Smears showed remarkable hyperplasia of
granulocytes (Fig 2B). Pathologic analysis after death showed bone
marrow to be hypercellular with a predominance of myeloid cells at
various stages of differentiation and also with proliferation of
megakaryocytes (Fig 2D and Fig 3C).Extensive infiltration of granulocytes and immature myeloid cells was
observed in the spleen (Fig 2F) and in other organs including
mesenteric lymph nodes (data not shown). Thus, the transgenic progeny
of 4-5 developed myeloproliferative disorders having cardinal features
of human CML.
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| Fig 3.
HE staining of 4-5-2 that developed extramedullar crisis
in the lung. (A) Lung. Massive proliferation of blast cells is apparent around the vessels. (B) Higher magnification of (A). (C) Bone marrow.
In contrast to the findings for the lung, bone marrow shows a
predominance of myeloid cells with megakaryocyte proliferation.
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In addition, an aggressive proliferation of immature blast cells was
found around the vessels in the lung of 4-5-2 (Fig 3, A and B) with a
predominance of myeloid cells and megakaryocytes in the bone marrow
(Fig 3C), indicative of extramedullar blast crisis. Another transgenic
littermate, 4-5-6, also showed a hematologic picture of MPD (WBC
10 × 103/µL, (>90% granulocytes), Hb 8.0 g/dL, and
Plt 200 × 104/µL, see Table 1) but was healthy in
general appearance and survived for more than 2 months after the
diagnosis.
To confirm the reproducible development of the myeloproliferative
disorders and to further analyze the alteration in the hematologic parameters in the time course, the peripheral blood samples of the
progeny of 4-5-6 were subjected to hematologic examination. The changes
of Hb, WBC, and Plt in transgenic and nontransgenic littermates from 4 to 8 months after birth are shown in Fig 4A. The most remarkable
hematologic change observed in the initial phase of the transgenic
group was the elevation of Plt count. In contrast to the stable Plt
count in the nontransgenic group (40-50 × 104/µL),
that of the transgenic group began to increase at 5 months after birth
and reached approximately 100 × 104/µL (right panel in
Fig 4A). Although the WBC count of the
transgenic group was almost similar to that of the nontransgenic group
until 6 months after birth, it seemed to be gradually increasing
thereafter (middle panel in Fig 4A). In addition, a significant and
progressive change in WBC was detected in the peripheral blood smears
of the transgenic group (Fig 4B). At 4 months after birth, the
differential count of leukocytes was almost the same between the
transgenic and nontransgenic groups. It showed a slight predominance in
lymphoid lineage (left panel in Fig 4B). However, as the mice grew up, proliferation of myeloid cells became apparent in the transgenic group
(middle panel in Fig 4B). Moreover, at 8 months after birth, the major
population of the WBC in the peripheral blood of the transgenic group
was found to be mature granulocytes (right panel in Fig 4B). In
addition, the appearance of large cells morphologically resembling
megakaryocytes was frequently observed (shown by an arrowhead in the
right panel in Fig 4B). At this point, the transgenic progeny, numbered
4-5-6-2, 4-5-6-6, and 4-5-6-7 were diagnosed as CML based on the
proliferation of granulocytes in the peripheral blood (>80%) and
significantly increased Plt number (>100 × 104/µL,
see Table 1). Another transgenic mouse, 4-5-6-3, was strongly suggested
as suffering from CML, since the granulocyte ratio in the peripheral
WBC was over 60% and Plt number was up to 80 × 104/µL (Table 1). Along with the changes in WBC and Plt
counts, progressing anemia was observed in the transgenic group (left panel in Fig 4A), probably due to an excessive proliferation of myeloid
and megakaryocytic cells in the hematopoietic tissues such as bone
marrow and spleen. In spite of these hematologic abnormalities, the
transgenic mice looked healthy in general appearances. These results
demonstrate that the initial hematologic abnormality occurred at an
early age and that the disease has a long duration of chronic phase
with no obvious phenotypical changes. Therefore, we consider these
transgenic mice to be a model for human CML. The hematologic parameters
observed in the descendants of 4-5 at the diagnosis are summarized in
Table 1 and the family tree of 4-5 is shown in Fig 4C.
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| Fig 4.
(A) Comparison of hematologic parameters in the
transgenic mice (Tg,  ) and nontransgenic controls (C,  ) from 4 months to 8 months after birth. The data of mean values and standard
deviations for groups of four mice are shown. (B) WG-stained peripheral
blood smears of a transgenic mouse (Tg) and a nontransgenic control (C)
at 4, 6, and 8 months after birth. A cell morphologically resembling
megakaryocyte is indicated by an arrowhead. (C) Family tree derived
from a founder mouse, 4-5. Circles and squares indicate female and male
mice, respectively. Transgenic or nontransgenic mice are shown as
transgene + or . Mice that died of leukemia are shown as or  .
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Expression of the p210bcr/abl transgene in the
leukemic tissues.
To detect the p210bcr/abl transgene product in two
leukemic founder mice, 4-3 and 4-5, that developed ALLs, proteins
extracted from enlarged thymi, including massive leukemic cell
infiltration, were probed with anti-c-Abl MoAb, AB3. As shown in Fig
5A, the p210bcr/abl
protein product could be clearly detected. The expressed
p210bcr/abl was also proved to be highly
tyrosine-phosphorylated (data not shown). Furthermore, in vitro
kinase assays demonstrated that the p210bcr/abl
possessed enhanced kinase activity (Fig 5B). These results are similar
to those observed for MT/p210bcr/abl leukemic
mice.19
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| Fig 5.
Expression (A) and enhanced kinase activity (B) of the
p210bcr/abl transgene product in the thymi of the
two founder mice, 4-3 and 4-5 that developed ALLs. The expressed and
phosphorylated p210bcr/abl transgene products are
indicated by arrows and the positions of protein markers are shown on
the left. The thymi of nontransgenic mice were used as negative
controls (C).
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With regard to the transgenic offspring exhibiting MPDs, repeated
attempts to detect or immunoprecipitate the
p210bcr/abl protein in the spleen, containing
mature and immature myeloid cells, were unsuccessful, probably due to
the high protease activity existing in granulocytes.13 As
an alternative approach, RT-PCR was carried out using mRNA extracted
from peripheral blood cells of 4-5-6, in which >90% of the WBC cells
were mature granulocytes. The product of genomic amplification (230 bp)
can be distinguished from that of mRNA amplification (160 bp) since 70 bp should be removed by the splicing (Fig 1). As shown in Fig
6, without RT (-RT), the PCR product was
230 bp; this was reduced to 160 bp when RT was added (+RT). Thus, the
granulocytes in the peripheral blood of the MPD mouse were shown to
express transgene-derived mRNA.
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| Fig 6.
Expression of bcr/abl mRNA in peripheral
granulocytes of 4-5-6. The RT-PCR products generated with RT (+RT) or
without RT (RT) were electrophoresed in a 3% agarose gel and
stained with ethidium bromide. Products of genomic and mRNA
amplification are indicated by arrows. RT-PCR without RNA (No RNA) was
also performed as a negative control. The positions of DNA markers are
shown on the left.
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Cell lineage of the leukemic cells.
To identify the cell lineage of the leukemic cells, surface markers
were analyzed by flowcytometry. Blast cells disaggregated from the
enlarged thymus of 4-5 that developed ALL and white blood cells
prepared from the peripheral blood of 4-5-6 that developed MPD were
stained with MoAbs and analyzed by FACScan as described in the
Materials and Methods. As shown in Fig 7,the lymphoblasts in 4-5 expressed a T-cell antigen, Thy1.2, at high
intensity, whereas the proliferated granulocytes in 4-5-6 were positive
for a myeloid antigen, Gr-1, indicating commitment to the T-cell and myeloid lineages, respectively.
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| Fig 7.
Surface marker analysis of leukemic cells in 4-5 and
4-5-6 that developed ALL and MPD, respectively. Open histograms are for a negative control and marker staining is indicated by closed histograms. Lymphoblasts in the thymus of 4-5 expressed Thy1.2 at a
high intensity (left), whereas proliferating granulocytes in the
peripheral blood of 4-5-6 are positive for Gr-1 (right).
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To further demonstrate the clonality of the leukemic cells in the
founder mice, DNAs extracted from thymi of 4-3 and 4-5 were subjected
to Southern blot analysis. As shown in Fig
8, leukemic cells from both founder mice
carried rearrangements in the TCR- loci, confirming the T-cell
lineage and clonality in origin.
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| Fig 8.
TCR- rearrangement of the leukemic cells of 4-3 and
4-5. Rearranged bands are evident in both cases after each enzyme
digestion. Molecular markers are shown on the left.
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DISCUSSION |
To clarify the biological properties of p210bcr/abl
in vivo and to analyze the disease process caused by the chimeric
protein, it is necessary to establish a model animal that
expresses p210bcr/abl and eventually develops
hematopoietic disorders resembling human Ph1-positive
leukemias.
Earlier works focused attention on the BMT approach. Mice lethally
irradiated and reconstituted with bone marrow cells infected with
p210bcr/abl-expressing retroviruses exhibited
various hematologic disorders including granulocytic hyperplasias,
myelomonocytic leukemias, ALLs, macrophage cell tumors, reticulum cell
sarcomas, and erythroid tumors.13-15 This provided evidence
that p210bcr/abl indeed confers an oncogenic
potential on hematopoietic cells and that the precursors infected with
p210bcr/abl-expressing retroviruses acquire an
ability to form various hematologic malignancies. However, the outcome
of the experiments was markedly influenced by the infection conditions,
the structure of the viral construct, genetic background of the donor
and recipient mice, and the cell lineage and differentiation stage of
the targeted cells.28,29 In addition, since the integrated
sites of the retroviruses differed among the recipient mice, the
results lacked reproducibility. Furthermore, the possibility that
cytokines secreted with BMT might affect the reconstituted bone marrow
cells cannot be excluded. Thus, it has been emphasized that transgenic
models with stable transmission of the p210bcr/abl
transgene to the progeny and reproducible development of
Ph1-positive type leukemias are required.30
In human disease, p210bcr/abl is expressed under
the control of the bcr promoter. However, expression of a
bcr/abl construct driven by the bcr promoter was found
to result in a lethality during embryogenesis in transgenic
mice.31 Therefore, we and others have used alternative
promoters such as MT, Eµ, and LTR of MPSV.17,19,20 Although the resultant transgenic mice expressing
p210bcr/abl by these promoters developed
hematologic malignancies, they were diagnosed exclusively as ALLs and
no granulocyte hyperplasia resembling human CML was observed even when
the LTR of MPSV, which allowed exhibition of CML-like disease in BMT
experiments, was used as the regulatory element.13,17 Thus,
the question of whether p210bcr/abl transgenic mice
could exhibit CML-like disease and whether the transcriptional unit
regulating the bcr gene expression might be required for
causing CML-like disease in human and mice have remained to be
clarified.20
Although the MT promoter we earlier used to create
MT/p210bcr/abl transgenic mice has been proven to
work efficiently in the transgenic system,19,20,32,33 the
expression level is relatively low,34 the tissue
distribution is ubiquitous,35 and it is not clear what
lineage and developmental stage of hematopoietic cells are targeted.
Thus, to create a transgenic model for human CML, a promoter that
drives p210bcr/abl expression in the hematopoietic
progenitor cells at a high level is necessary. With this aim, we cloned
and analyzed the promoter of mouse tec gene, a cytoplasmic
protein-tyrosine kinase predominantly expressed in hematopoietic
precursor cells.21,22 We identified and sequenced the 5
flanking region of mouse tec gene, determined the
transcriptional initiation site by RACE-PCR, and confirmed the promoter
activity using tec-expressing hematopoietic cell lines.21 Subsequently, we cloned and sequenced mouse
tec promoter up to approximately 2 kb from the transcription
initiation site, identified regions critical for the transcriptional
activity, and demonstrated nuclear proteins including PU.1 and Sp1
selectively to bind to the promoter through the regions essential for
the promoter activity.22 These results suggest that the
promoter we cloned will be useful and applicable for expressing a gene of interest preferentially in hematopoietic progenitor cells in vivo. Use of the 1948 to +22 region to express
p210bcr/abl in transgenic mice allowed generation
of five founders. Two of them developed ALLs shortly after birth, while
the transgenic progeny exhibited MPDs closely resembling human CML
after a long latent period. These results clearly demonstrate that the
expression of p210bcr/abl by an appropriate
promoter in transgenic mice can induce not only ALL but also MPD
resembling human CML. Considering that both diseases developed in a
single line, the difference in disease phenotypes is obviously not due
to an integration site effect. Further studies will be required to
clarify the mechanism of how the expression of
p210bcr/abl in early hematopoietic progenitors
causes two clinically distinct Ph1-positive human
leukemias. In addition, our findings strongly suggest that the
transcriptional mechanism regulating the expression of the bcr
gene does not play an essential role in causing
Ph1-positive human hematopoietic disorders. This is not in
line with the hypothesis that regulatory sequences within the
bcr promoter would contribute to the occurrence of myeloid
leukemia in humans and presumably in mice,20 but rather
supports the conclusions drawn from the BMT experiments, in which
recipient mice expressing the p210bcr/abl driven by
retrovirus-derived sequences developed human ALL- and CML-like
diseases.13-15
The finding of marked thrombocytosis in the peripheral blood of mice in
the present study, as frequently shown by CML patients,1 implies that the tec promoter functions in megakaryocytic as
well as myeloid and lymphoid lineages. Alternatively, the
thrombocytosis might have been due to the structure of the
bcr/abl cDNA used (b3a2 type), since some reports suggested a
positive correlation between thrombocytosis and b3a2 type
bcr/abl transcripts in human CML patients.36,37 The
observed progressive anemia, frequently associated with deformity and
fragmentation of red blood cells, could be explained by the impaired
erythropoiesis due to the proliferation of myeloid and megakaryocytic
cells in the bone marrow and spleen. No obvious fibrosis was observed
in the bone marrow and/or spleen on staining with Azan (data
not shown). In addition, prominent infiltration of mature and immature
myeloid cells in the spleen and all other tissues examined might be
indicative of the terminal stage of chronic phase. Thus, a long
duration of chronic phase might be fatal without application of
antiproliferative agents. In particular, one mouse, 4-5-2, showed
massive proliferation of blast cells along the vessels in the lung.
Although the precise origin of the blast cells remains to be
identified, it is very likely that an extramedullar blast crisis
occurred after a duration of chronic phase in that mouse.
The transgenic model described in the present report has several
advantages over BMT experiments. (1) Since the latter are markedly
influenced by the experimental procedures such as infection and culture
conditions of the bone marrow cells, the results vary with the mouse
and the experimental group. With transgenic mice, each line contains
the transgene in the same integration site and expresses the transgene
product in the same manner so that the results are reproducible and not
affected by extrinsic factors. (2) Since the transgenic mice transmit
the transgene stably to the progeny, they can be cross-mated with other
transgenic or knockout mice for investigating the possible synergistic
or suppressive effects of different genes in vivo. (3) The
recipient mice in BMT experiments develop erythroleukemias, macrophage
tumors, or reticulum cell sarcomas that are virtually unknown as human
Ph1-positive leukemias. In contrast, our transgenic mice
develop ALL and CML-like diseases within the same disease spectrum as human Ph1-positive leukemias. In this respect, our
transgenic mice may more accurately reflect the leukemogenic property
of p210bcr/abl in vivo.
In summary, we present a novel transgenic model for
Ph1-positive human leukemias. This model will be valuable
not only for investigating the biological properties of
p210bcr/abl in vivo but also for analyzing
the mechanism involved in the progression from chronic phase to blast
crisis. In addition, they should find application for examination of
the effectiveness of therapeutic regimens for human
Ph1-positive leukemias.
| |
FOOTNOTES |
Submitted April 3, 1997;
accepted October 29, 1997.
Supported by Grants-in-Aids from the Ministry of Education, Science and
Culture of Japan.
Address reprint requests to Hisamaru Hirai, MD, Third
Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr T.W. Mak for providing the mouse TCR probe and thank
Motoya Katsuki and the other staff of the Central Institute of
Experimental Animal for technical instructions.
| |
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Abstract
Introduction
Abstract