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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2780-2790
RAPID COMMUNICATION
Expression of E2A-HLF Chimeric Protein Induced T-Cell
Apoptosis, B-Cell Maturation Arrest, and Development of Acute
Lymphoblastic Leukemia
By
Hiroaki Honda,
Toshiya Inaba,
Takahiro Suzuki,
Hideaki Oda,
Yasuhiro Ebihara,
Kohichiro Tsuiji,
Tatsutoshi Nakahata,
Takatoshi Ishikawa,
Yoshio Yazaki, and
Hisamaru Hirai
From the Third Department of Internal Medicine, Faculty of Medicine,
University of Tokyo; the Department of Molecular Biology, Jichi Medical
School, Tochigi-ken; the Department of Pathology, University of Tokyo;
and the Department of Clinical Oncology, Institute of Medical Science,
University of Tokyo, Tokyo, Japan.
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ABSTRACT |
The E2A-HLF fusion gene, generated by t(17;19)(q22;p13) in
acute lymphoblastic leukemia (ALL), encodes a chimeric transcription factor in which the trans-activating domains of E2A are
fused to the DNA-binding and dimerization domains of hepatic leukemic factor (HLF). To investigate its biological role, we
generated transgenic mice expressing E2A-HLF using Ig enhancer
and promoter, which direct transgene expression in cells committed to
the lymphoid lineage. The transgenic mice exhibited abnormal
development in the thymus and spleen and were susceptible to infection.
The thymus contained small numbers of thymocytes, and TUNEL staining
showed that higher population of thymocytes were undergoing apoptosis. The spleen exhibited a marked reduction in splenic lymphocytes and the
flow cytometric analyses and the in vitro colony formation assays
showed that the B-cell maturation was blocked at a very early
developmental stage. These findings indicated that the expression of
E2A-HLF induced T-cell apoptosis and B-cell maturation arrest in vivo and that the susceptibility of the transgenic mice to infection
was due to immunodeficiency. Moreover, several transgenic mice
developed acute leukemia, classified as T-ALL based on the surface
marker analysis and DNA rearrangements, suggesting that an additional
event is required for malignant transformation of lymphoid cells
expressing E2A-HLF. Our findings provide insight into the
biological function of E2A-HLF in lymphoid development and also
its role in leukemogenesis.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
CHROMOSOMAL TRANSLOCATIONS are recurrent
features of human hematopoietic malignancies.1 Consistently
observed chromosomal abnormalities in specific tumors indicate that the
underlying genetic events are essentially implicated in the causation
of the diseases. In acute leukemias, several characteristic chromosomal translocations have been noted, and studies on the chromosomal breakpoints have identified candidates of genes whose dysregulated or
aberrant expression would be responsible for the tumor
development.1
Transcription factors are frequent target of chromosomal translocations
observed in acute lymphoblastic leukemia (ALL).2 Among
them, E2A gene, encoding a basic helix-loop-helix (bHLH) transcription factor on chromosome 19, has been known to be involved in
two chromosomal translocations, t(1;19)(q23;p13)3,4 and t(17;19)(p22;q13)5,6 that are observed in human B-lineage leukemias. As a result of t(1;19)(q23;p13), the C-terminal region of
E2A gene, including the bHLH DNA-binding and dimerization
domains, is replaced with the DNA-binding domain of PBX1
homeobox gene on chromosome 1.3,4 On the other hand,
following the t(17;19)(p22;q13), the same region of the E2A
gene is fused to the DNA-binding and dimerization domains of hepatic
leukemic factor (HLF) gene belonging to the basic
region/leucine zipper (bZIP) family on chromosome 17.5,6
These events create novel fusion gene products, E2A-PBX1 and
E2A-HLF, respectively, and the expression of these chimeric transcription factors with altered structural and functional features would play a substantial role in the leukemogenic process(es).
One of the E2A-involving chimeric molecules, E2A-HLF,
was cloned in t(17;19)(p22;q13)-bearing childhood ALL with pro-B
immuophenotype.5,6 Structurally, E2A-HLF is
composed of the trans-activation domains of E2A and the
DNA-binding and dimerization domains of HLF.5,6 E2A was originally identified as an Ig  chain
enhancer-binding protein7 and has been shown to be
expressed in many different types of cells.8 On the other
hand, HLF is normally expressed in the liver, kidney, and lung,
but not in lymphoid cells.5,6 Although both E2A-HLF
and HLF have been shown to bind to the same consensus sequence,
5'-GTTACGTAAT-3', a core dyad-symmetric motif characteristic of bZIP
family DNA-binding proteins,9 studies have shown that
E2A-HLF and HLF possess different transcriptional and
transforming properties.10 E2A-HLF has an ability
to transactivate a reporter gene in various types of cells, such as
HepG2 hepatocellular carcinoma cells, NIH 3T3 fibroblasts, and 293 fetal kidney cells, whereas transactivating ability of wild-type
HLF was detected only in 293 cells but was silent in HepG2 and
NIH 3T3 cells.10 In addition, expression of E2A-HLF
in NIH 3T3 cells induced anchorage-independent cell growth in soft agar
and rendered these cells tumorigenic in nude mice, while overexpression
of HLF has not been reported to be oncogenic.10 A
subsequent study showed that E2A-HLF has an anti-apoptotic
effect in hematopoietic cell lines.11 Introduction of a
dominant-negative form of E2A-HLF, which lacks the
trans-activation domains of E2A and contains a
defective HLF basic domain, in E2A-HLF-expressing human leukemic cells rapidly induced apoptosis.11 In
addition, the expression of E2A-HLF in mouse factor-dependent
Ba/F3 cells blocked factor-depleted- and radiation-induced
apoptosis.11 These findings suggest that E2A-HLF
would activate genes that are normally quiescent in lymphoid
development and that the aberrant gene expression would contribute to
the development of ALL.12
Although these studies have provided us with insights in the biological
properties of E2A-HLF, the in vivo role of E2A-HLF in
lymphoid development and leukemogenesis has not been fully understood.
To address this issue, we generated and analyzed mice expressing
E2A-HLF in cells committed to the lymphoid lineage.
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MATERIALS AND METHODS |
Construction of the transgene and generation of the transgenic mice.
The E2A-HLF cDNA5 was subcloned into an expression
vector which contains Ig enhancer and promoter as regulatory elements. A fragment encompassing the Ig enhancer/promoter, the E2A-HLF cDNA, and the SV40 early splicing and polyadenylation signals was
microinjected into pronuclei of eggs from C57BL/6XDBA/2 F2 mice as
described earlier.13 The transgenic mice were identified by
hybridizing tail DNA with the injected fragment13 and the transgenic progeny were generated by successive mating of the founder
mice or transgenic descendants with C57BL/6XDBA/2 F1 mice. The mice
were kept under a conventional maintaining condition or in a separated
clean isolator.
Pathological examination.
After gross examination, all the tissues were fixed in
10% phosphate-buffered saline (PBS)-buffered formaldehyde, embedded in
paraffin, and stained with hematoxylin-eosin (HE). Smears of peripheral
blood cells were stained with Wright-Giemsa (WG).
Protein extraction, immunoprecipitation, and Western blotting.
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, 1% sodium deoxycholate) with 50 U/mL aprotinin. For detecting
E2A-HLF protein, 1 mg of protein aliquots were incubated with
1:1,000 diluted anti-E2A antibody5 and
immunoprecipitated proteins were probed with 1:1,000 diluted
anti-HLF antibody.5 The positive signals were
visualized using the ProtoBlot Western AP system (Promega, Madison, WI).
Immunofluorescent staining.
Transgenic spleens were fixed in 10% PBS-buffered formalin and
embedded in paraffin. Specimens were mounted on saline-coated slides,
dewaxed in xylin, rehydrated through a graded series of alcohols, and
treated with 20 µg/mL proteinase K in 10 mmol/L Tris-Cl (pH 7.4) at
37°C for 15 minutes. After washing two times with PBS, specimens were
stained with 1:100 diluted fluorescein isothiocyanate (FITC)-conjugated
anti-B220 monoclonal antibody (MoAb) (Pharmingen, Osaka, Japan) and
1:50 diluted anti-HLF polyclonal antibody5 coupled
with 1:50 diluted Texas-red-conjugated anti-rabbit antibody (Jackson
ImmunoResearch Laboratories Inc, West Grove, PA). After washing three
times with PBS, specimens were observed under a confocal microscopic
system, MRC1024 (BioRad, Hercules, CA).
Detection of apoptotic cells with TdT-mediated dUTP nick end
labeling (TUNEL).
Transgenic and nontransgenic thymuses were fixed in 10% PBS-buffered
formalin and embedded in paraffin. Specimens mounted on saline-coated
slides were dewaxed as described above. After washing two times with
PBS, specimens were subjected to TUNEL reaction using in situ cell
death detection kit (Boehringer, Mannheim, Germany) according to the
manufacturer's instructions and were observed under a confocal
microscopic system, MRC1024 (BioRad).
Flow cytometric analysis.
Cells were stained with FITC- or phycoerythrin-conjugated commercial
MoAbs including anti-Thy-1.2, anti-B220, anti-CD4, anti-CD8, and
anti-CD43 (Pharmingen) according to the manufacturer's instructions. The stained cells were washed three times with PBS and analyzed on a
FACScan (Becton Dickinson, Sunnyvale, CA).
Colony assays.
Bone marrow cells of transgenic mice and nontransgenic littermates were
subjected to the in vitro colony formation assays as described
elsewhere.14
RNA extraction and reverse transcription-polymerase chain reaction
(RT-PCR).
Total RNA was extracted using the acid guanidine/phenol-chloroform
method.15 The RT and PCR amplification reactions were performed as previously detailed.13
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RESULTS |
Expression of the E2A-HLF transgene product in T and B
lymphocytes in the transgenic mice.
We planned to generate transgenic mice expressing E2A-HLF in
hematopoietic cells committed to the lymphoid lineage. With this aim,
Ig enhancer and promoter were chosen as regulatory elements. The
schematic model of the transgene is shown in Fig
1A. Microinjection resulted in generation
of three founder mice containing approximately 10 copies of the
transgene in a head-to-tail manner (data not shown) and two of these
three founders (named 1-17 and 2-17) transmitted the transgene stably
to their progeny.
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| Fig 1.
(A) Schematic model of the injection fragment for
generating E2A-HLF transgenic mice. The Ig enhancer/promoter,
the E2A-HLF cDNA, and the SV40 early splicing and poly(A)
signals are shown as white, black, and shaded boxes, respectively. The
positions of primers used for RT-PCR (P1-P4) are also indicated. The
bar shows 1 kb. (B) Western blot analysis for the expression of the
E2A-HLF transgene product in the thymus and spleen of a control
mouse (C) and a transgenic mouse from two independent lines (1-17 and
2-17). The positions of E2A-HLF protein and Ig are indicated by
arrows, and the positions of the molecular marker are shown on the
left. (C) Immunofluorescent staining of the transgenic
spleen with anti-B220 and anti-HLF antibodies. The
B220-expressing and HLF-expressing cells are visualized by
green and red signals, respectively.
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To examine the expression of the transgene product in the lymphoid
tissues, proteins extracted from the thymus and the spleen of a
transgenic mouse and a nontransgenic littermate were immunoprecipitated with anti-E2A antibody and immunoprecipitated proteins were
blotted with anti-HLF antibody. As shown in Fig 1B, the
expression of E2A-HLF protein was detected in the thymus but
was undetectable in the spleen in both lines, raising a question of
whether B lymphocytes expressed the E2A-HLF transgene product.
To address this issue, we performed immunofluorescent staining of the
transgenic spleen using FITC-conjugated anti-B220 antibody and
anti-HLF antibody coupled with Texas-red-conjugated
anti-rabbit antibody. The results showed that cells expressing B220
were positive for HLF (Fig
1C). Because
HLF has not been shown to be expressed in the hematopoietic cells,5 HLF+ lymphocytes are
considered to express E2A-HLF protein. These results showed
that both T and B lymphocytes expressed the E2A-HLF transgene
product in the transgenic mice.
E2A-HLF transgenic mice were susceptible to infection.
The transgenic mice were initially kept under a conventional
maintaining condition. Under this condition, although the transgenic mice grew normally until weaning (4 weeks of age), they gradually exhibited emaciation and almost all the transgenic mice died within several months after birth, in contrast to the fact that no
nontransgenic littermates died within the same period. Autopsies of the
dead animals showed abscesses, strongly suggesting that the transgenic mice were susceptible to infection. To address this issue, newly weaned
transgenic and nontransgenic littermates (4 weeks of age) were divided
into two groups and mice in one group were kept under the same
conventional condition, whereas mice in the other group were maintained
in a separated clean isolator. As a result of about 3 months of
observation, we found that the transgenic mice that grew under the
conventional condition also showed a high mortality rate (5 of 17 transgenic mice were alive more than 4 months of age), whereas the
transgenic mice that grew in a clean isolator exhibited much better
surviving rate (14 of 16 transgenic mice are surviving over 4 months of
age) (Fig 2, right). During this period, no
nontransgenic mice died under either condition (Fig 2, left). To
investigate the cause of the death of the transgenic mice, tissues of
the dead or moribund mice were pathologically examined. In about half
of the mice examined, macroscopic abscesses were found in several major
organs such as the liver, spleen, and intestine. Microabscesses were
detected in all the cases and were accompanied by tissue necrosis and
infiltration of granulocytes. Staining of stamp specimens of the
abscesses showed proliferation of bacteria. These results indicated
that the transgenic mice were susceptible to infection and that the
major cause of early death of the transgenic mice was bacterial
infection.
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| Fig 2.
Survival curves of transgenic mice (Tg, right) and
nontransgenic controls (C, left) that are kept under a conventional
condition (solid lines) or in a clean isolator (dotted lines). The life
span is presented by the Kaplan-Meier method.
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Decrease in thymocytes and marked reduction in splenic lymphocytes in
E2A-HLF transgenic mice.
To examine whether the expression of E2A-HLF affected the
development of the lymphoid tissues, transgenic mice and nontransgenic littermates at 4 weeks of age were autopsied and macroscopically examined. Consistent and remarkable changes were detected in the thymus
and in the spleen. The thymuses of the transgenic mice were apparently
smaller than those of the nontransgenic littermates. The spleens of the
transgenic mice were consistently larger than those of the
nontransgenic littermates. The representative macroscopic appearances
of a transgenic mouse and a nontransgenic littermate are shown in Fig
3.
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| Fig 3.
Macroscopic appearances of a transgenic mouse (Tg) and a
nontransgenic control (C) (4 weeks of age). The thymuses and spleens
are indicated by ( ) and ( ), respectively. The transgenic mouse
exhibits regressed thymus and enlarged spleen.
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To investigate the histological changes underlying the phenotypic
abnormalities, the thymuses and spleens of transgenic mice and
nontransgenic littermates at 4 weeks of age were subjected to
microscopic examination. Significant changes were observed in the
medulla of the thymus and in the white pulps of the spleen. In
comparison with the nontransgenic thymus, the transgenic thymus showed
enlargement of the medulla, and obscurity and irregularity of the
medulla/cortex junction (Fig 4A, top panels). The medulla of the
transgenic thymus contained small numbers of lymphoid cells and
consisted mainly of epithelial cells and interdigitating cells. Infiltration of neutrophils, multinucleated giant cells, and
tingeble-body macrophages were frequently seen (Fig 4A, bottom panels).
As for the spleen, the fundamental structure of the spleen (integrity of white and red pulps) was disorganized in the transgenic mice in
comparison with the nontransgenic littermates (Fig 4B, top panels). The
periarteriolar lymphoid sheaths in the transgenic spleen were
irregularly shaped where lymphoid cells were markedly depleted and
neutrophils and megakaryocytes infiltrated (Fig 4B, bottom panels).
These findings showed that the thymic regression was due to decrease in
thymocyte number and that the splenic enlargement was associated with
marked depletion of splenic lymphocytes and diffuse proliferation of
other hematopoietic cells.
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| Fig 4.
HE-stained sections of the thymus (A) and spleen (B) of a
transgenic mouse (Tg) and a nontransgenic control (C) (4 weeks of age).
The boxed areas in the top panels are magnified in the bottom panels.
(A) Thymus. In the lower magnification (top panels), transgenic thymus
shows enlargement of the medulla and obscurity and irregularity of the
medulla/cortex junction. In the higher magnification (bottom panels),
the medulla of the transgenic thymus contains fewer thymocytes and
consists of epithelial and interdigitating cells. Infiltrated
neutrophils and multi-nucleated giant cells are indicated by black and
white triangles, respectively, and the tingeble-body macrophages are
indicated by an arrow. (B) Spleen. In the lower magnification (top
panels), transgenic spleen shows marked disorganization in the
fundamental structures. In the higher magnification (bottom panels), in
a white pulp of the transgenic spleen, lymphocytes are markedly reduced
and proliferation of neutrophils and megakaryocytes is observed. The
central splenic artery, which is located in the middle of the white
pulp, is indicated by an arrow.
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Increased thymocyte apoptosis in E2A-HLF transgenic mice.
The decrease in thymocyte number is suggestive of either reduction of
thymocyte production or alteration in thymocyte survival. The
pathological findings that the thymocyte decrease was associated with
macrophage infiltration is in agreement with the latter possibility. To
confirm this, the thymuses of transgenic mice and nontransgenic littermates were subjected to TUNEL staining, which detects apoptotic cells by in situ labeling using TdT and dUTP.16 As shown in Fig 5 (see page
2785),
the number of positively stained cells was significantly increased in
the transgenic thymus in comparison with the nontransgenic thymus,
showing that the decrease in thymocyte number was caused by increased
apoptosis. To investigate whether the thymocyte apoptosis was due to a
differentiation block at a specific developmental stage, the transgenic
thymocytes were subjected to flow cytometric analysis using anti-CD4
and anti-CD8 antibodies. Repeated experiments showed that the
percentage of each CD4/CD8 subset
(CD4 /CD8,
CD4+/CD8+, CD4+/CD8,
and CD4/CD8+) was not significantly
altered between the transgenic and nontransgenic thymuses (data not
shown). These results indicated that the expression of E2A-HLF
induced thymocyte apoptosis with no obvious interference in thymocyte
differentiation.
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| Fig 5.
TUNEL staining of the thymus of a transgenic mouse (Tg)
and a nontransgenic control (C) (4 weeks of age). Apoptotic thymocytes
are detected as yellow signals and background cells are seen as green
signals. The transgenic thymus exhibits increased number of thymocytes
undergoing apoptosis.
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Maturation block of B-cell progenitors in E2A-HLF transgenic
mice.
The pathological analysis of the transgenic spleen showed a marked
decrease in splenic lymphocytes (Fig 4B). These findings strongly
suggest that the expression of E2A-HLF impaired B-cell development as well as induced T-cell apoptosis. To address this possibility and to identify the stages in which the B-cell development was impaired, splenic and bone marrow cells of the transgenic mice and
nontransgenic littermates were subjected to flow cytometric analysis
with anti-B220 and anti-CD43 antibodies. The B220+
CD43+ population contains mainly early B-cell progenitors,
whereas B220+ CD43 population consists of
pre-B and mature B lymphocytes.17 As shown in Fig
6A and B, significant changes were observed
in the B220+ CD43+ as well as the
B220+ CD43 population both in the spleen and
in the bone marrow. The nontransgenic spleen and bone marrow contained
approximately 6% to 10% of B220+ CD43+ cells,
whereas only 2% to 3% cells were positive for both B220 and CD43 in
the transgenic spleen and bone marrow (Fig 6A and B). More remarkable
is the reduction in B220+ CD43 late
progenitor population. In the transgenic spleen and bone marrow, this
population was reduced to 5% to 8%, as compared with 30% to 40% in
the nontransgenic spleen and bone marrow (Fig 6A and B). In addition,
no B cells expressing high levels of B220 were detected in the
transgenic spleen or bone marrow (Fig 6A and B). These results showed
that both early and late B-cell progenitors were severely reduced in
the transgenic mice.
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| Fig 6.
Flow cytometric analysis of the spleen (A) and bone
marrow (B) of a transgenic mouse (Tg) and a nontransgenic control (C)
with B220 and anti-CD43 antibodies (4 weeks of age). The transgenic
spleen and bone marrow show marked reduction of both
B220+ CD43+ and B220+
CD43 populations.
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We then investigated whether hematopoietic progenitor cells having a
potential to differentiate into B220+ cells might exist in
the transgenic bone marrow. The bone marrow cells from transgenic mice
and nontransgenic littermates were cultured with interleukin-7 (IL-7)
and stem cell factor (SCF), which can recapitulate B-cell
differentiation in vitro.14 Interestingly, although the
colony numbers varied among experiments, we found that the transgenic
bone marrow could develop B-cell colonies as well as the nontransgenic
bone marrow (Table 1). To confirm that the
colonies generated with IL-7 and SCF were committed to the B-cell
lineage, cells from the colonies were subjected to flow cytometric
analysis with anti-B220 and anti-CD43 antibodies. As shown in Fig
7A, both colonies were positive for B220
and showed the same broad pattern for CD43, demonstrating that they
both consisted of early and late B-cell progenitors. To further confirm that the B-cell colonies developed from transgenic bone marrow expressed E2A-HLF transgene product, mRNA extracted from the
colonies were subjected to RT-PCR analysis. By use of the primers
encompassing the splicing signal (P1-P4, see Fig 1), the mRNA-derived
PCR product (160 bp) can be distinguished from any contaminating
genome-derived PCR product (230 bp), because 70 bp should be removed by
the splicing. As shown in Fig 7B, we detected the 160-bp band in
addition to the 230-bp band in the PCR products generated from the
transgenic colonies, indicating that the B-cell colonies developed from
transgenic bone marrow expressed transgene-derived mRNA. The overall
results indicated that although hematopoietic progenitor cells having a
potential to differentiate into the B-cell progenitors did exist in the
transgenic bone marrow, their commitment to the B-cell lineage was
blocked at a very early developmental stage.
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| Fig 7.
(A) Flow cytometric analysis of the B-cell colonies
generated from bone marrow of a transgenic mouse (Tg) and a
nontransgenic control (C) with anti-B220 and anti-CD43 antibodies (4 weeks of age). Colonies established from transgenic mice and
nontransgenic controls are similarly positive for B220 and show the
same broad pattern for CD43. (B) Expression of transgene-derived mRNA
in the transgenic B-cell colonies. The RT-PCR products of the
transgenic colonies (Tg) and nontransgenic colonies (C) were
electrophoresed in an agarose gel, stained with ethidium bromide, and
photographed. In the top panel (E2A-HLF), products of genomic
and mRNA amplification are indicated by arrows and the position of the
DNA size markers are shown on the left. In the bottom panel
(K-ras), K-ras RT-PCR products are shown as an internal
control.
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Acute lymphoblastic leukemia in E2A-HLF transgenic mice.
We found that several transgenic mice developed acute leukemia. Among
26 transgenic mice that survived more than 4 months, 5 have developed
acute leukemia. All the leukemic mice exhibited massive thymic
enlargement, splenomegaly, and lymph node swelling (Fig 8A [see page
2785]). The peripheral blood smears of these mice showed massive
proliferation of blast cells with no granules, having the appearance of
lymphoblasts (Fig 8B). Pathological analyses showed infiltration of
leukemic cells into almost all tissues examined, especially in the
thymus, spleen, liver, and kidney (Fig 8C through F). The
characteristics of the leukemic mice are summarized in Table
2. Surface marker analysis of the leukemic cells showed that all the leukemic cells were positive for Thy1.2 but
negative for B220, indicating that the leukemias were of a T-cell
phenotype. Staining of the leukemic cells with anti-CD4 and anti-CD8
antibodies showed that one tumor was double positive (CD4+
CD8+) but three cases were single positive for CD4
(CD4+ CD8) and the other one case was
single positive for CD8 (CD4 CD8+). The
clonality of the leukemic cells was shown by Southern blot analysis
using T-cell receptor (TCR)- as a probe. These results indicated that the leukemias developed in the transgenic mice were
clonal T-cell tumors.
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| Fig 8.
Pathological analysis of acute leukemia developed in
E2A-HLF transgenic mice. (A) Macroscopic appearance of a
leukemic mouse. The enlarged thymus and spleen are indicated by black
and white triangles, respectively, and subcutaneous lymph node
swellings are indicated by arrows. (B) WG-stained peripheral blood
smear of a leukemic mouse. Massive proliferation of lymphoblasts is
apparent. (C through F) HE-stained thymus (C), spleen (D), liver (E),
and kidney (F) of a leukemic mouse. The fundamental structures of the
thymus and the spleen are destructed due to massive proliferation of
leukemic cells (C and D, respectively) and infiltration of leukemic
cells is detected around the blood vessels in the liver and the kidney
(E and F, respectively).
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DISCUSSION |
Generation of transgenic mice has successfully been used for
investigating gene functions in vivo.18 Transgenic mice
have provided significant insights into the biological properties of the chimeric gene product, especially for genes that are activated by
chromosomal translocations, such as Bcr/Abl and
PML/RAR .13,19-22 Thus, we applied
this technique to investigate the biological function of
E2A-HLF. The use of Ig enhancer/promoter allowed us to express
the E2A-HLF transgene product in both T and B lymphocytes (Fig
1B and C), as shown in previous reports.23-25 Failure in
detecting the E2A-HLF protein in the transgenic spleen by
immunoprecipitation/Western blot (Fig 1B) was probably due to the
reduction in B lymphocytes (Figs 4B and 6A).
Histological analysis showed remarkable changes in the thymus and in
the spleen (Fig 4A and B). The overall decrease in thymocytes and
marked reduction in splenic lymphocytes indicated that the expression
of E2A-HLF impaired normal lymphocyte development in both T and
B lineages. These results enabled us to explain the cause of the high
mortality rate observed in the transgenic mice. Reduction in the
lymphocyte number would render the transgenic mice immunodeficient and
highly susceptible to fatal infections. The finding that transgenic
mice kept in a clean isolator survived much longer than those
maintained under a conventional condition supports this idea. The
reason why the phenotypic changes (emaciation and death) were not
observed before weaning could be explained by the notion that the
maternal Ig would be transmitted through the milk and rescue the
immunodeficiency of the transgenic mice, as suggested in a previous
report.26
The TUNEL staining showed that the decrease in thymocyte number was
caused by increased apoptosis (Fig 5). In addition, the flow cytometric
analysis demonstrated that the marked reduction in splenic lymphocytes
was caused by the maturation block of B lymphocytes at a very early
developmental stage (Fig 6A and B). Thus, the expression of
E2A-HLF in lymphoid cells induced T-cell apoptosis and B-cell
maturation arrest in vivo. Although the precise mechanism is not clear,
it could be supposed that E2A-HLF induced expression of genes
regulating cell survival and cell death and led to impairment of normal
lymphoid development. Thus, we compared the expression levels of
several candidate genes, including bcl-2,27 bcl-x,28 and Fas,29 in the transgenic and
nontransgenic thymuses. Because we did not detect any differences in
their expression levels (data not shown), the abnormal lymphocyte
development would be caused by other mechanism(s) than we examined.
Interestingly, although B-cell maturation was blocked at a very early
stage in vivo, transgenic bone marrow cells restored the ability of
developing B-cell progenitors in vitro (Fig 7 and Table 1), suggesting
that some in vivo factor(s) might contribute to the abnormal lymphoid development in cooperation with E2A-HLF.
Increased T-cell apoptosis observed in E2A-HLF transgenic mice
seems to be inconsistent with the results of experiments using in vitro
cell systems, which demonstrated an anti-apoptotic function of
E2A-HLF.11,30 One possible explanation is the
difference in the cell lineages. We observed that UOC-B1 cells, which
was established from human pro-B leukemia expressing E2A-HLF,
underwent apoptosis by the induction of a dominant-negative form of
E2A-HLF.11 In addition, two murine IL-3-dependent
pro-B cell lines, Ba/F3 and FL5.12, became resistant to apoptosis
caused by IL-3 depletion when E2A-HLF was expressed in these
cells.11,30 However, E2A-HLF failed to reverse
apoptosis in cytokine-derived 32D cells, a murine IL-3-dependent
myeloid cell line (our unpublished results). These results
suggest that the anti-apoptotic function of E2A-HLF might be
confined to B-lineage lymphocytes. Maturation arrest of B lymphocytes, on the other hand, may agree with our recent result obtained using an
in vitro experimental system that CD20, a well-known cell-surface marker for B-cell maturation, is induced by the expression of a
dominant-negative form of E2A-HLF in UOC-B1
cells.31
It is to be noted that several transgenic mice developed acute
leukemia. The leukemic mice exhibited thymic enlargement, splenomegaly, and lymph node swelling (Fig 8A). The leukemic cells, having the appearance of lymphoblasts (Fig 8B), were highly malignant, showing massive infiltration into almost all the tissues examined (Fig 8C
through F). The leukemic cells were positive for Thy1.2 and carried
rearrangements in TCR loci (Table 2), indicating that they were clonal
T-cell tumors. However, the expression patterns of CD4 and CD8 in each
leukemia were heterogeneous; one tumor was double-positive for CD4 and
CD8, three tumors were single-positive for CD4, and the other one case
was single-positive for CD8. Thus, E2A-HLF rendered an
oncogenic activity to T cells that are in transition from
double-positive to single-positive stage of development. These results
indicated that E2A-HLF possesses an oncogenic potential in
addition to inducing T-cell apoptosis and B-cell maturation block. The
finding that 5 of 26 mice that survived more than 4 months developed
ALL may suggest that an additional genetic event might be required for
disease development. This idea is in agreement with the findings that
E2A-HLF exhibited an oncogenic ability in NIH 3T3
cells10 and showed an anti-apoptotic activity in Ba/F3
cells,11 since the transfected cells are immortalized and
would carry a genetic abnormality(ies) for immortalization. Therefore,
it is likely that E2A-HLF contributes to leukemogenesis in
cooperation with another or more genetic event(s).
Interestingly, the phenotypic aspects observed in the E2A-HLF
transgenic mice (T-cell apoptosis, B-cell maturation block, and T-cell
leukemia) closely resemble those reported in the transgenic mice
expressing another E2A-involving chimeric protein,
E2A-PBX1, under the control of the same Ig
promoter/enhancer.32 If aberrant gene transcription induced
by E2A-HLF or E2A-PBX1 is responsible for the
phenotypes, the phenotypic coincidence between both types of transgenic
mice seems to be anomalous, because the DNA-binding activities of
E2A-HLF and E2A-PBX1 are dependent on HLF- and
PBX1-derived regions, respectively, and their experimentally
defined binding motifs are completely different (5'-GTTACGTAAT-3' for
HLF and 5'-ATCAATCAA-3' for PBX1).9,33 One
possible explanation for this is that a gene(s) that is a common target
of both E2A-HLF and E2A-PBX1 exists, and its aberrant
expression in lymphoid cells impaired normal lymphoid development and
led to phenotypic abnormalities. Another possibility, which seems to be
more likely, is that the phenotypes commonly observed in both
transgenic mice were caused by a dominant-negative inhibition of
E2A activity. This idea could be supported by the finding that
mice lacking E2A also exhibited very similar phenotypes.
E2A-deficient mice failed to generate B220+ cells
because of maturation arrest of B cells at an early developmental stage.34,35 In addition, they contained a low number of
thymocytes and eventually developed T-cell lymphomas.36
Thus, it would be possible to consider that the biological properties
of E2A-HLF and E2A-PBX1 are caused, at least in part,
by interfering E2A function.
The results of functional studies of E2A-HLF and
E2A-PBX1 using cell lines support this idea. The
E2A-derived regions of E2A-HLF and E2A-PBX1
have been shown to be required for the transactivating and transforming
abilities in both chimeric proteins10,37-39 and also for
the anti-apoptotic function of E2A-HLF in IL-3-dependent murine lymphocytes.30 In contrast, the bZIP domain of
HLF and the homeodomain of PBX1, which mediate
site-specific DNA-binding capacities of E2A-HLF and
E2A-PBX1, respectively, have been shown to be unnecessary for
the transforming ability of E2A-PBX138,39 or for
anti-apoptotic function of E2A-HLF.30 Tal-1
(also called as SCL), which is a transcription factor
belonging the bHLH family and is involved in human T-ALL, is shown to
directly bind to E2A40,41 and is considered to
contribute to leukemogenesis by interfering normal E2A function
through forming a heterodimer with E2A.42 However,
it is not the case of E2A-HLF and E2A-PBX1, because the dimerization domain of E2A is deleted in both
proteins.3-6 Thus, although it is likely that the abnormal
lymphocyte development observed in the transgenic mice would be induced
by a dominant-negative effect on E2A function, the underlying
mechanisms have remained to be established. One possibility to be
investigated is that E2A-HLF and E2A-PBX1 might
competitively bind to a specific interaction partner of the activation
domain of E2A that is expressed in lymphoid cells and is
required for E2A activity, through which they inhibit E2A function and induce an E2A-deficient phenotype.
There remains a question concerning the difference in the
immunophenotypes of the leukemias between humans and mice. In humans, E2A-PBX1 is detected in pre-B cell leukemias3,4 and
E2A-HLF is associated with pro-B type leukemias,5,6
whereas the leukemias developed in the transgenic mice have so far been
diagnosed as T-ALL32 (and this report). Although the reason
is not clear, one possibility is that Ig enhancer/promoter allowed
transgene expression in both in T and B lymphocytes, and T cells might
be more susceptible to the oncogenic properties of the chimeric
proteins. The generation of knock-in mice expressing E2A-HLF
and E2A-PBX1 under the control of native E2A promoter
might clarify the problem concerning the phenotypic disparity between
humans and mice.
| |
ACKNOWLEDGMENT |
We thank Dr T.W. Mak for providing the mouse TCR- probe. We thank
Yoshikazu Oh-hira for preparing pathological specimens, and we also
thank Haruki Kume for photographs.
| |
FOOTNOTES |
Submitted October 12, 1998; accepted February 10, 1999.
Supported in part by Grants-in-Aids from the Ministry of Education,
Science and Culture of 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.
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; e-mail: hhirai-tky{at}umin.ac.jp.
| |
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ABSTRACT
INTRODUCTION