1. Art and Diseño

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BCR/ABL P210 and P190 Cause Distinct Leukemia in Transgenic Mice
By Jan Willem Voncken, Vesa Kaartinen, Paul K. Pattengale, Wilfred T.V. Germeraad, John Groffen,
and Nora Heisterkamp
gin, with a relatively short period of latency. The observed
DNA constructs encoding BCR/ABL P210 have been introdissimilarities are most likely due to intrinsically different
duced into the mouse germ line using microinjectionof oneproperties of the P190 and P210 oncoproteins and
may also
cell fertilized eggs. Kinetics of BCR/ABL P210 expression in
involve sequences that control transgene expression. The
transgenic micewere very similarto those ofBCR/ABL P190
delayed progressionof BCR/ABL P210-associated diseasein
constructs in transgenic mice. mRNA transcripts were dethe transgenic mice is consistent with the apparent indotectable early in embryonic development and alsoin hemalence ofhuman chronicmyeloid leukemia duringthe chronic
topoietic tissue of adult animals. Expressionof BCR/ABL in
phase. We conclude that, in transgenic models, comparable
peripheral bloodprecededdevelopment of overtdisease.
expression of SCR/ASL P210 and BCR/ABL P190 results in
P210 founder and progeny transgenic animals,
when becomclinically distinct conditions.
ing ill, developedleukemiaof B, T-lymphoid, or myeloid
0 1995 by The American Society of Hematology.
In contrast, P190origin after a relatively long latency period.
transgenic mice exclusively developed leukemia of B-cell ori-
recipients developed a CML-like myeloproliferative disease,
RECIPROCAL TRANSLOCATION between chromolymphoblastic leukemia, or macrophage cell type tumors
somes 9 and 22 results in the formation of the Philadelwithin 5 months.” Other investigators reported BCWABLphia (Ph) chromo~ome.”~
Although invariably associated
mediated hematopoietic disease involving macrophage,
with chronic myeloid leukemia (CML), the Ph-chromosome
lymphoid, erythroid, and mast cell lineages. The incidence
also occurs in a percentage of adult and childhood acute
(69% to 100%) of the exact type of disease was strainlymphoblastic leukemias (ALL)4*5and in acute nonlymphodependent.” Myelomonocytic leukemias, granulocytic leucytic leukemia (ANLL). The [t(9;22)(q34;ql)] translocation
kemias, and lymphocytic leukemiallymphomas were found
fuses BCR to the ABL proto-oncogene. Activation of the Ab1
both with P210 and P190.23*24
It appears that the kinetics and
tyrosine kinase in hybrid Bcr/Abl oncoproteins is considered
the nature of the induced disease in this experimental setting,
essential for the development of leukemia.6 The two major
besides depending on the choice of internal promoter, depend
forms of Bcr/Abl proteins, P190 and P210, differ only in the
on culturing conditions, infection procedure, and genetic
amount of Bcr amino acid residues included. P190 contains
background of the grafted mi~e.’~.’~
It was further concluded
only residues encoded by BCR gene exon l, whereas P210
that retroviral regulatory sequences may have influenced the
contains residues encoded by BCR exons 1 to 13 or 14,
outcome of the experiments. A major obstacle in retroviral
depending on the exact location of the breakpoint on chromotechnology is targeting the infection to stem cells. This is
some 22. P190 appears to be associated mainlywith Ph+
essential, because CML is thought to be a clonal disease
ALL, whereas P210 is found both in Ph+ ALL and in
originating in the hematopoietic stem cell.”
CML.’”
A different model system is created through the use of
The 160-kD BCR gene product contains several distinct
transgenic mice because the gene of interest will be present
domains. The segment encoded by exon 1 harbors a serine/
in every cell. In previous studies, we have generated mice
threonine kinase activity in vitro and is held responsible for
transgenic
for a BCWABL P190 construct under the tranactivating ABL in the chimeric BCWABL gene
scriptional regulation of a mouse metallothionein (MT-l)
BCR exons 3 through 10, which are present in BCWABL
promoter; most transgenic founder animals and progenydied
P210 but not in BCWABL P190, share sequence homology
rapidly of an acute leukemia, which was clinically classified
with a guanine nucleotide-exchange factor for human
as
A L L . ~ ~ - ~ O Initial attempts to generate a transgenic mouse
CDC42, the DBL proto-onc~gene.’~”~
The C-terminal end
model for human CML with a BCWABL P210 transgene
of p16OBCr
has GTPase-activating protein (GAP) activity toward the small GTP-binding proteins Racl/2 and CDC42
in
Examination of bcr null-mutant mice recently
Fromthe Department of Pathology Research and Laboratory
connected ~160’“ GAP in vivo to regulation of superoxide
Medicine, Divisions of Molecular Carcinogenesis and Haematopaproduction by the NADPH-oxidase system in myeloid and
thology, Childrens Hospital of Los Angeles, Los Angeles, CA.
B-lymphoid cells”.
Submitted June 2, 1995; accepted August S, 1995.
Involvement of the ~ 1 4 5in~cytoplasmic
~ ’
as well as nuSupported by Grant No. CA50248 from the Public Health Service
clear processes has been suggested based on in vitro experito N.H. and by a Childrens Hospital Los Angeles Research Award
ments.I8 In vivo, abl null-mutant mice are runted and die
to J. W.V.
within 2 weeks after birth. In addition, many show B- and TAddress reprint requests to Nora Heisterkamp, PhD, Department
cell lymphopenia, the cause of which remains ~ n k n o w n . ’ ~ . ~of~Parhology Research and Laboratory Medicine, Division of Molecular Carcinogenesis, Childrens Hospital of Los Angeles, 4650 Sunset
However, the exact function of Ab1 during development and
Blvd, Los Angeles, CA 90027.
cellular differentiation in mammals is still unclear.
The publication costs of this article were defrayed in part by page
To investigate the role of the chimeric BCWABL gene
charge payment. This article must therefore be hereby marked
in leukemia, BCWABL P210-encoding retroviral constructs
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
have been introduced into mouse bone marrow and transindicate this fact.
planted into lethally irradiated recipients. Different types of
0 1995 by The American Society of Hematology.
hematopoietic disease were found. In one study, 43% of the
0006-4971/95/8612-0029$3.00/0
A
Blood, Vol 86, No 12 (December 15). 1995: pp 4603-4611
4603
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4604
VONCKEN ET AL
AMT-BCR/ABL P190
Transgenic SMT-BCWABL P190 animals have been described in
detail elsewhere.’x~’”Mice transgenic for the MT-BCWAHL P210
constructs were generated as described.’x Founder animals were the
offspring of matings between C57BLICBA F, animals. Transgenic
progeny was the result of matings between transgenics and
CS7BLxCBA F, mice. Progeny are identified by eight-digit numbers;
the first four digits are those of the founder of that specific line,
Histology. Pathologic analysis was performed, as described previously,” on autopsy material obtained at killing from mice that
were generally terminally ill. Routine histology examinations included bone marrow, lymph nodes, liver, kidney, spleen, and thymus.
Tissue sections were fixed in 10% formalin, 90% BS. All MT-BCW
ABL P21 0 males displayed fertility problems; severely reduced testis
size and aspermia were frequently encountered. For this reason. only
a limited amount of offspring was obtained from MT-BCWABL
founders. The SMT-BCWABL P210 and P190 animals apparently
had normal fertility.
Periodic monitoring of peripherul blood for signs .f‘ diseuse.
White blood cell (WBC) counts and BCWABL expression assays
were performed regularly on peripheral blood from transgenic and
control mice. Briefly, approximately 70 yL of blood was obtained
via retro-orbital sinus bleeding. WBC counts were performed manually, as were differential WBC counts. Peripheral blood films were
stained with Wright-Giemsa and evaluated histologically. Evidence
of myeloid involvement (differential counts) in the period that preceded the development of overt leukemia in
BCWABL
P210
transgenic animals was not found. Fifty microliters of heparinized
blood was used for RNA isolation. Redblood cells were lysed in
two successive treatments with 20 vol of 0.144 mol/L NH,CI and 5
volof 0.01 mol/L NH4HC03interchanged with washes in normal
phosphate-buffered saline (PBS). WBCs were pelleted and lysed in
SO0 yL of solution D; RNA was isolated essentially as described.”
The RNA pellet was suspended in 40 yL dH,Oand reverse transcriptase-polymerase chain reactions (RT-PCR) were performed on
8 yL, essentially as described:
using combinations of ALL-E and
ALL-F or CML-A and CML-H oligo-nucleotides as amplimers and
ALL-G or CML-B as hybridization probe, respectively’5; in every
experiment, RNA from the human CML cell line K562 or from a
P190-expressing mouse lymphoma was used as a positive methodologic control.
In previous experiments, the level of sensitivity of the RT-PCR
method for BCWABL P190 expression was established at 1 in
100,000 nucleated cells.‘” To verify that a certain RNA sample was
suitable for RT-PCR reactions, it was tested for mouse actin expression using a 3’ amplimer (S’-CGGTTGGCCTT AG GGTTCAGGG3 ’ ) and a S’ amplimer (S’-GTGGGCCGCTC-TAGGCACCAA-3‘).’”
In some experiments, an oligonucleotide probe (S’-ACTCCTATGTGGG-TG ACGAGG-3’) contained within the amplified region was used for hybridization probe.
Composition of hematopoietic tumors und peripheral blood .sumples. Composition of hematopoietic tissues was determined by
double-color flowcytometry on a FACScan (Becton Dickinson, Lincoln Park, NJ). Briefly, tissues were disaggregated and washed twice
in cold PBS. Approximately 1 X 10‘ WBCs were stained with phycoerythrin (PE)-and/or fluorescein isothiocyanate (F1TC)-conjugated
antibodies for 20 to 30 minutes in 50 to 100 yL RPM1 containing
3% fetal bovine serum and 0.1% sodium azide on ice in the dark.
Cells were then washed in PBS, fixed in I % paraformaldehyde, and
analyzed. Monoclonal antibodies used include anti-CD45R (RA36B2). anti-CD45 (30F1 I . l ) , antiLCD36 (145-2C1 l), anti-Thy1.2 (532.1). anti-GR-l (RB6-8CS; PharMingen, San Diego, CA), and antiC D l l b (M1/70.15; Caltag, San Francisco, CA).
Southern analysis. Tissue DNAs were isolated as described.’x
DNAs digested with EcoRI, HindIII, and BamHl were run on 0.7%
gels, blotted to nitrocellulose, and hybridized to a 1.2-kb Msp I/
I
MT-BCRIABL P210
A MT-BCR/ABL P210
Fig 1. Schematicrepresentation of human BCR/ABL transgenic
constructs. Expression of the MT-BCR/ABL P210 transgene (middle
line) is regulated by the completemouse metallothionein (MT-1)
gene promoter, whereas the SMT-BCR/ABL P210 transgena (lower
line) is controlledby a part of the MT promoter (seethe Materials and
Methods). Promoter sequencesin the SMT-BCR/ABL P190 construct
(upper line) are identicalto those of thesMT-BCR/ABL P210 construct
(lower line). As described in the Materials and Methods, different
combinations of BCR and ABL genomic and cDNA sequenceswere
used to generatetheseconstructs.Thinlinesindicate
intron sequences. Hatched boxes represent
BCR gene exons. Black boxesr e p
resent ABLgeneexons. The smallblackboxes 5’ to BCR exon 1
represent murine MT-1 promoter sequences.
controlled by the human BCR promoter failed because of
deleterious effects of the construct on embryogenesis; no live
offspring were obtained.3’We present here the generation of
mouse lines transgenic for MT-BCWABL P210 constructs
and describe the development of hematopoietic neoplasia
caused by the P210 transgenes.
MATERIALS AND METHODS
Transgenes and mice. The MT promoter in the bMT-BCWABL
P210 construct (Fig 1) lacks the glucocorticoid-responsive element
and is identical to that used before to generate GMT-BCRLABL P190
transgenic animals.**The GMT-BCWABL P210 construct was generated by ligating a 5’ Clu I-Sal I fragment containing the truncated
MT-I promoter (a 200-bp Sst I-Bgl I1 fragment’*) and BCR gene
exon 1 until the Sal I site to a large Sal I-Cia I fragment encompassing the remainder of BCWABL. The latter fragment was derived
from the previously described cosmid cl(8)der, a BCWABL DNA
construct containing the human BCR gene promoter.” The Sal ICia I fragments were ligated into vector pHEP and packaged in
vitro. The 32.5-kb cosmid insert was separated from the vector by
pulsed-field gel electrophoresis. In this construct, the entire BCR
coding region up to Mbcr exon 3 (BCR exon 14) and ABL exons 2
through 8 are contained within one large cDNA fragment. The rest
of ABL includes introns and is a genomic sequence. The MT-BCW
ABL P210 was constructed from three segments: (1) the complete
murine MT-1 promoter fused to BCR exon 1 plus a segment of 3’
intron; (2) the intron immediately preceding BCR gene exon 2 and
additional BCR gene exons up to and including Mbcr exon 2 and
Mbcr exon 3 with flanking introns; and ( 3 ) ABL exon 2 with 5’ and
3’ introns and ABL exons 3 through I 1 as cDNA.
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BCRIABL P210 TRANSGENIC MICE
EcoRI
JH, a 0.7-kb Pst I T-cell receptor-P (TCR-P), or
a 2.8-kb
BamHI Ck fragment respectively. Posthybridization washings were
at 0.1 X SSC at 65°C.
RNA isolation and expression in other tissues. RNAswereisolated as described34 usingguanidine-isothiocyanate. To test for early
expression,timedmatingsbetween
transgenic males and nontransgenic C57BlxCBAF1 females were setup and embryos isolated
between days 13 and 15 of gestation. Three fetuses were pooledand
homogenized immediately in solution D and stored at -80°C. Bone
marrow, spleen, or tumor RNA was isolated after homogenizing part
ofthetissue in slnD. One to three micrograms of total RNA was
used per RT-PCR reaction. RT-PCR products were separated on 1%
agarose gels, blotted to Nytran (Schleicherand Schuell, Keene, NH)
and hybridized as indicated above.
Immunoblotting. Tissueswereminced
and homogenized in a
Potterhomogenizer in 2Xsodiumdodecylsulfate
(SDS) sample
buffer and analyzed by Western blotting, as described.” The chimeric Bcr/Abl P190 and P210 oncoproteins were detected with the
mouse monoclonal antibody Ab-3 (anti-c-Abl; Oncogene Science,
Inc, Uniondale, NY).
RESULTS
Transgenes and transgenicanimals. To circumvent the
problem of embryonic lethality observed with the human
BCR pr~rnoter,~’
the mouse metallothionein-l promoter was
chosen as an alternative regulatory sequence for BCWABL
P210 transgenes. Analysis of a mouse model for human
ALL in which this MT promoter was used to control P190
expression (the GMT-BCWABL P190 c o n s t r u ~ t ~ *Fig
. ~1)
~;
showed that transgenic animals developed pre-B-cell leukemia without prior exposure to heavy metals. BCWABL
mRNA could be detected, by RT-PCR, as early as the 1 cell
stage embryo and was found in all tissues examined at later
stages.30This low level of transgene expression is apparently
enough to predispose for cancer development. Subsequently,
two BCWABL P210 DNA constructs were made (Fig l),
both controlled by the murine MT-1 promoter. They differ
in the length of the MT promoter segment included and
in their intrordexon structure; the GMT-BCWABL P210 and
GMT-BCWABL P190 constructs share identical MT promoter sequences (Fig 1).In transfected mouse NIH-3T3
cells, expression of both BCWABL P210 constructs resulted
in the production of 210-kD proteins, which were active in
autophosphorylation assays in vitro (data not shown).
Transgenic founder and progeny animals were generated
with both BCWABL P210 constructs (Table 1) and hematologic tumors were obtained. Animals containing a double
GMT-BCWABL P210 transgene were also generated by
breeding (ie, P210/P210). However, the transgene copy number had no influence on the kinetics and/or type of disease
that developed (data not shown).
Disease. Unlike mice transgenic for BCWABL P190,
which developed pre-B-cell acute lymphoblastic leukemia,
BCWABL P210 animals developed B- as well as T-cell leukemias with approximately the same frequency; no difference was found in type of disease between the two BCW
ABL P210 constructs (Table 1). Tumor phenotype was confirmed by flow cytometric analysis (Fig 2) and Ig heavy and
light chain and TCR-0 rearrangement (data not shown). The
incidence of hematologic disease inGMT-BCWABL P210
mice was 58% and in MT-BCWABL P210 was 53%; in
4605
comparison, GMT-BCWABL P190 had a disease incidence
of nearly 100% (Fig 3; see also Groffen et a13’). The experimental end point was arbitrarily chosen at 44 weeks; some
€910-transgenic animals died ofBCWABL related disease
after more than 1 year.
In contrast to studies by others that reported the development of T-cell neoplasias upon injection of either BCWABL
P190 or P210 retroviral vectors directly into the thymus,39
we have found no evidence that T-lymphoid cells are targets
for BCWABL P190. Subsequent to the first reported study,28
among 14 newly generated founders andmorethan
200
transgenic offspring, none has shownany leukemia other
than of the B-lineage.
Neoplasias inBCWABL P210 animals were of both Band T-cell type and, less frequently, of myeloid origin. Blymphoid leukemias or lymphomas were invariably accompanied by splenomegaly and involvement of lymph nodes,
but never involvement of the thymus. The majority of T-cell
lymphoid leukemias involved the thymus, frequently lymph
nodes and spleen; WBC counts were often extremely high.
Mature lymphocytic leukemia (B or T) was further characterized by absence of mitotic figures in the peripheral blood.
In both B-and T-lymphoid tumors, a coexisting but separate
and presumably non-neoplastic prominent hematopoiesis
(erythropoiesis and megakaryocytosis) was frequently observed in the spleen (data not shown). Because clonality (Ig
heavy or light chain and/or TCR rearrangements) and/or Bcr/
Ab1 protein levels of isolated megakaryocyte populations
were not evaluated, we cannot exclude the possibility that
these phenomena related to direct oncogenic action of BCW
ABL. Macrophage tumors were not found in either BCW
ABL P190 or BCWABL P210 animals; primary granulocytic
disease was not very prevalent. All leukemias were rapid in
onset; no evidence for chronic disease was found in the
peripheral blood.
In a few cases, the myeloid compartment was involved
at terminal stages of disease. Circulating mature neutrophil
counts were increased in 1 case (1994/2663), and a relatively
high percentage of cells in the bone marrow compartment
of another terminally sick animal (1994/2685) stained positive for both B220 and GR-l (Fig 2B). Myeloblastic leukemia with a sarcomatous component in animal 2663 involved
the red pulp in the spleen, lymph nodes, liver, and kidney
(Table 1). In contrast to neoplastic lymphoblasts, the myeloblasts in animal 2663 showed a conspicuous rim of pinkish
cytoplasm and appeared cohesive, particularly in the nonlymphoid red-pulp area of the s ~ l e e n . ~Because
~ , ~ ’ mouse
2685 showed a mild lymphoid proliferative disorder at an
earlier stage, the myeloid involvement could have been be
secondary to lymphoid tumorigenesis (ie, a deregulated cytokine production), as has been suggested before.22,2s
Alternatively, BCWABL P210 expression could disturb differentiation or proliferation of relatively rare myeloid-lymphoid
precursors. Taking these findings together, we conclude from
our data that P190 and P210 cause clinically distinct disorders in a transgenic setting.
Kinetics of transgene expression. To investigate whether
differences in type of disease were associated with different
levels of BCWABL expression, total protein was isolated
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4606
VONCKEN ET AL
Table 1. Leukemogenesis in BCR/ABL P210-Transgenic Animals
Tissue lnvolvernentt
Age*
Animal
MT-BCR/ABL P210
1063
1063-1164
1102-1684
1102-1691
2160-2302
2179
6MT-BCR/ABL P210
1994-2619
1994-2661
1994-2663
1994-2683
1994-2685
1994-2710
1994-2720
1994-2725
1994-2744
1994-2748
1994-2762
1994-2777
1994-2779
1994-2781
1994-2784
(wkl
BM
SPL
11
13
17
15
20
15
+
+
18
44
54
22
21
14
19
11
18
25
12
9
47
28
28
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
-
THY
LN
WBC*
(xWirnLJ
+
110
53
376
+
219
- lymphoma
lymphocytic
+B-lineage ND
leukemia/lymphoma
T-lineage
+
ND
-
+
+
+
+
Diagnosis§
B-lineage lymphoblastic leukemia
Small B-lymphocytic leukemia
Small T-lymphocytic leukemia/lymphoma
Small T-lymphocytic leukemiallymphorna
+
+
+
-
+
+
+
-
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
ND
126
98
1
1
2
128
84
ND
19
29
56
32
196
ND
T-lymphoblastic leukemia
T-lymphocytic leukemia/lymphoma
Myeloblastic leukemia//(
B-lineage lymphoblastic lymphoma
B-lymphocytic leukemia/lymphoma/l
B-lymphoblastic lymphoma
Small T-lymphoblastic leukemia/lymphoma
T-lymphoblastic leukemia/lymphoma
T-lineage leukemia/lymphoma
T-lymphoblastic leukemia
Small B-lymphocytic leukemia/lymphoma
T-lymphocytic leukemia/lymphoma
T-lymphoblastic lymphoma
B-lineage lymphoblastic leukemia
T-lymphoblastic lymphoma
Abbreviation: ND, not done.
* A g e at death; autopsies were performed on terminally ill animals or animals were followed up until they died.
t Tissue involvement was established by gross pathologic and histologic examination.
WBC count was either determined at or a few days before
death.
§ Phenotype of lymphoid tumors was assessed by flow cytometry and lg and
TCR gene rearrangement (see the Materials and Methods).
11 These animals displayed granulocytic involvement in the peripheral blood at terminal stages ofdisease.
1Diagnosis of myeloblastic leukemia in animal 2663 was based on pattern, general morphology, and anatomic sites involved (see also the
Results).
*
from different end-stage tumors. However, lymphomas of
all three transgenic animal lines expressed comparably high
levels of the chimeric Bcr/Abl oncoproteins (Fig 4).
We examined transgene expression using RT-PCR early
in development and in bone marrow. Both BCWABL P210
transgenes were expressed early in utero in nonhematopoietic tissues (testis and brain; data not shown) and in the
bone marrow and spleen of different transgenic animal lines
(Table 2) without prior exposure to zinc or cadmium. As
was the case in the 6MT-BCWABL P190 animal model,
the appearance ofBCWABL P210 expressing cells in the
peripheral blood preceded the development of overt leukemia.”’ Transgene expression in three transgenic mouse lines
(founders animals 1102, 1989, and 1994) was investigated
in more detail. Transgene expression in the bone marrow of
nonleukemic animals was low (on average, 50 to > l O O X
lower than in K562; data not shown); there was no apparent
correlation between relative transgene expression levels and
the development of disease among the different lines. Not
all transgenic animals within the lines developed detectable
peripheral transgene expression (Table 2). Moreover, appearance of transgene expression in circulating blood cells
did not necessarily precede immediate development of disease. Some animals for which transgene expression in circulating nucleated cells had been confirmed between 10 and
16 weeks of age remained disease free for a long period,
with some remaining disease free past 16 months of age.
These observations were valid for both BCWABL P210 constructs. In P190 animals, a close correlation exists between
transgene expression and development of disease.29The difference between P190 and P210 transgenic lines is also reflected in the different mortality curves (Fig 3). The large
differences in latency time among transgenic animals indicates that BCWABL P210 expression is not the sole cause
of leukemia but rather predisposes for the cancer. In
agreement with current concepts, additional (epilgenetic mutations, directly or indirectly resulting from BCWABL expression, most likely contribute to malignant tumor evolution.
DISCUSSION
Transgenic mouse models allow directcomparison oj
P210- and PI90-associateddisease.
In recent years, attempts to generate a reliable animal model for the study of
CML have been thwarted by lack of reliability and reproducibilit~.~’
The outcome of bone marrowtransplantation experiments using retroviral BCWABL P210 constructs was shown
to depend largely on infection conditions, the retroviral and
internal regulatory sequences used, andalso on genetic background of the irradiated recipient mice; in essence, the out-
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4607
BCRIABL P210 TRANSGENIC MICE
lo4
P198
10.
2762
I
I
lo2
"""_
10'
loo
loo
10'
lo2 lo3
10.
loo
lo'
lo2
lo3
10.
Thylf
0
U
U
m
t
Gr-l
Fig 2. Flow cytometric analysis of BCR/ABL-positive tumors. (A) An example of a pre-B-cell leukemiellymphoma of a SMT-SCR/ABL P190
mouse, a B-lymphocytic Ieukemiallymphoma of a GMT-BCR/ASL P210 mouse (2762). and a small T-lymphocytic leukemia of a SMT-BCR/ABL
P210 animal (2720). (B) Two examples of granulocytic involvement in P210-relatedleukemia (see Results). Animal 2685 showed B-lymphocytic
leukemia with cells staining double positivefor B220and GR-l; mouse 2663 showed acute myeloblasticleukemia. All flow cytometric analyses
were performed on peripheral blood samples. Animal identification numbers are printed in the top right corner of the respective graphs and
correspond to those in Table 1.
struct were designed to resemble the genomic BCWABL gene
come of the disease was thought to depend on which hematopoietic progenitor cell was targeted by retroviral v e ~ t o r s . ~ ~ structures,
.~~
with the exception of the MT promoter, which
Myeloproliferative disorders could be obtained by manipuwas used to circumvent embryonic lethality observed when
the BCR promoter was used." With the exception of addilating these experimental conditions. In this fashion, BCW
ABL P190 and P210 were shown to cause similar disease.24 tional BCR coding sequences in the BCWABL P210 conEvenwith v-abl, which, under natural conditions, exclustructs, the ABL and promoter sequences were identical in
sively induces pre-B-cell leukemia in mice, myeloproliferathese transgenes. One additional difference should be noted:
introns are positioned differently among the constructs.
tive illness could be produced.2'
However, both BCWABL P210 constructs gave a similar
In BCWABL transgenic animals, every cell, including heclinical spectrum of disease, despite different positioning of
matopoietic stem cells, will contain a BCWABL transgene.
Although the MT promoter used here does not target
intron sequences.
transgene expression exclusively to hematopoietic cells, it
The present study shows that BCWABL P190-and P210mediated leukemogenesis clearly differ, despite the similaridoes allow expression in these cell types. However, leukemogenesis in our BCWABL transgenic mice is independent
ties in transgene design. An independent study using an MTof influences of retroviral sequences or other experimental
BCWABL P210 transgenic construct supports the distinct
factors, such as infection and bone marrow culture condioncogenicity of BCWABL P190 and
However, in
tions. Hence, a transgenic mouse model is expected to more
this study, mice exclusively developed T-cell leukemias,
accurately reflect the leukemogenic properties of BCWABL.
which may be related to transgene design including lack of
Influence of Bcr protein moiely and BCR gene promoter
introns and integration effects in the single line studied.
on leukemogenesis. The P210 constructs and P190 conThe longer latency time of P210-associated leukemogene-
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VONCKEN ET AL
4608
100
Mortality
(%l
Table 2. Transgene Expression in Bone Marrow of Different
BCR/ABL P210 Lines
Animal of Line"
60
BCRlABL P210
40
20
0
0
4
20
8 18 12
24
28
32
38
40
44
48
Age (weeks)
Fig 3. Mortality curve of BCR/ABL P190- versus BCR/ABL P210transgenic mice. The data shown are a compilation of data from
different transgenic lines. Both GMT-BCR/ABL P210 and MT-BCR/ABL
P210 groups included F, (founder; n = 10 of both P210 transgenes
combined) and F, and F?progeny animals (An = 36). A comparable
selection of animals (including F. [n = 141, F,, and Fz animals of both
sexes) is represented in the GMT-BCR/ABL P190 graph (0n = 42).
sis, independent of whether a B- or T-lineage or myeloid
leukemia develops, would seemto indicate intrinsic variation
in biochemical properties between the two distinct oncoproteins. It has been reported that P190 Bcr/Abl has a higher
autophosphorylation activity than P210 in vitro, a property
that correlated with its higher transforming ability in cultured
fibroblast cell lines."" Such differences in tyrosine kinase
activity might, for example, have a differential effect on the
differentiation of certain hematopoietic lineages andmay
help explain the altered disease-spectrum associated with
P2 IO as compared with P190.
The combined findings in our transgenic mice show that,
as in human leukemia, the additional Bcr domains included
1
2
3
4
4
4
P210
P190
Fig 4. BcrlAbl expression in primary tumors of transgenic aniof kD is indicated
mals. A high molecular weight protein marker200
at theleft Isquare). Lanes 1 and 2, P190 expression in lymphomas of
two different GMT-BCR/ABL P190 animals; lane 3, p210 expression
in a T-cell lymphoma in a fiMT-BCR/ABL P210 mouse of the
1994 line;
lane 4, transgene expression in a B-cell lymphoma of an MT-BCR/
ABL P210 animal.
6MT-BCR/ABL P210
168
170
171
1989
1994
2039
2043
MT-BCR/ABL P210
1063
1102
1105
2148
2160
2179
TGt xpr
Disease in Mouse Line:
Founder or Offspring
ND
NO
+
ND
+
+
-
+
NO
ND
+
+
+
+
ND
NO
+
+
+
-
+
+
+
+
Of animal line170, the founder(170) and at least two of his offspring
were evaluated for transgene expression; founder and 2 offspring of
the 1989 line were found positive for low transgene expression in
bone marrow; of line 1994, at least 20 F1 and F2 animals have been
monitored periodically fortransgene expression and development of
disease (see the Materials and Methods). Founders and at least two
offspring oflines 2039 and 2043 were evaluated for transgene expression in bone marrow; only the2043 line had detectable BCWABL P210
mRNA. Animal lines 1063,1102,2148,2160,
and 2179 all showed
equal levels of transgene expression in bothspleen and bone marrow
on average somewhat higher than the bMT-P210 lines. Offspring of
lines 1063 (n = l ) , 1102 (n = 8). and 2148 (n = 3) were evaluated for
transgene expression and found to bepositive. Of line 1102, at least
7 offspring were monitored periodically for
transgene expression and
development of disease (see the Materials and Methods). Transgene
integrity in the different transgenic lines was assessed by restriction
endonuclease digestion and Southern analysis.
Abbreviations: ND, not determined; NO, no offspring sired by this
founder.
Animal lines are represented by founder identificationnumbers.
t Transgene expression in bone marrow was evaluated in at least
one transgenic animal per line by RT-PCR.
in the P2 IO oncoprotein modulate the outcome of the disease.
However, the mechanisms that govern these distinct leukemic processes are ngt understood. As indicated above, the
Bcr domains modify the tyrosine kinase activity of the Ab1
moiety6 and also F-actin binding activities of c-Abl." In
addition, the Dbl-homology'3 and pleckstrin-homology dom a i n only
~ ~ ~present in P210 may allow protein-protein interactions with other cellular factors that cannot occur with
P190.
Apart from its structural role in the Bcr/Abl oncoprotein,
there appears to be a second discrete mechanism through
which BCR may contribute to leukemogenesis. We have
recently shown that the effect of bcr gene ablation specifically affects hematopoietic cells in mice affected by BCW
ABL in humans and mice." It seems reasonable to conclude
from our data that a correlation exists between the cell type
affected in Ph+ leukemia and Bcr function. The BCR promoter controls expression of both the remaining nonrearranged BCR allele and the BCWABL oncogene in human
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
ECRIAEL P210 TRANSGENIC MICE
leukemia. It is very well possible that regulatory sequences
that normally govern BCR expression have substantial influence in directing BCWABL-mediated leukemogenesis.
The prominent myeloid development in BCWABL P210mediated human CML may be related to the presence of
factors in myeloid lineages that regulate BCR expression
through specific elements in the BCR promoter. These factors
may be less prevalent in human T- and B-cell lineages and
preferentially direct BCR-promoted malignant transformation in granulocytes.
All experimentally derived BCWABL lymphoid tumors
were generated with regulatory sequences other than the
BCR promoter. To date, all animals models for human Ph+
leukemia, whether generated through
or
transgenic technologies (Heisterkamp et al?' Honda et ai?
Hariharan et al?' and this study), develop predominantly
lymphoid malignancy of the B and T type. Although hematologic tumors in Ph+ ALL are principally of the pre-B phenotype, B-lymphoid involvement occurs in about 25% of cases
of human CML in blast crisis and T-cell involvement is
rarely seen in humans. It is tempting to speculate that regulatory sequences within the BCR transcriptional unit are important for the type of disease found in humans and mice.
This concept contrasts with the postulate that (intransplantation experiments using BCWABL-expressing, retrovirus-infected bone marrow) neither BCR sequences nor myeloid cell
promoter elements are essential for myeloid leukemogenic
tran~formation.~~
Similarly, in a comparable experimental
setting, the mere introduction of an activated ab1 gene into
a certain target cell was proposed to control the selection of
particular hematopoietic cell types andnot the BCR sequences that distinguish BCWABL P190 and BCWABL
P210.24The findings presented here do not support these
conclusions.
Ph+ myeloidleukemia in human and mouse.BCWABL
expression in human chronic myeloid leukemia results in
myeloid expansion, which may increase the chance for additional mutagenic events, that ultimately accompany malignant transition to fatal blast crisis. We found evidence of
myeloid disease at terminal stages of several BCWABL
P210-transgenic animals, although the frequency was relatively low. Because no obvious evidence was found for the
development of myeloid proliferative disorders secondary to
other primary malignancies, we assume thatthe observed
myeloid involvement was neoplastic. The mixed lymphoidmyeloid leukemia, which was found in 1 case, seems to
indicate that BCWABL P210 expression, in contrast to BCW
ABL P190, can disturb differentiation or proliferation of relatively rare myeloid-lymphoid precursors in the mouse. Such
rare stem cell origins in Ph+ leukemia are occasionally en~ o u n t e r e d ~ ' .also,
~ ~ ; switching within myeloid and pre-B
lymphoid tumor lineages has been rep~rted,~'showing a
close developmental link between these hematopoietic lineages.
The absence of a chronic phase and the low frequency of
myeloid disease in the present study could be a direct result
of the restricted size of the cell population subject to secondary mutational events in a small animal like the mouse. A
similar scenario was suggested to explain the absence of
4609
retinoblastoma in animals heterozygous for the tumor-suppressor retinoblastoma susceptibility gene (Rb)-mutant anim a l ~ . Alternatively,
~'
the mouse may be more prone to develop lymphoid neoplasias than spontaneous myeloid
disorders." This finding, combined with the observation that
BCWABL P210 targets cells of both myeloid and lymphoid
origin in a transgenic setting, may explain the natural predilection for BCWABL-mediated lymphoid leukemia.
In humans, about 50% of cases ofPh' ALL carry the
BCWABL P190 translocation, with the other 50% carrying
the P210 translocation. Consequently, the issue of whether
Ph+ ALL is actually CML in blastic phase has been controversial for some time. However, clinical and molecular studies have recently established that Ph' ALL and acute-phase
CML are indeed two distinct diseases.53All results of the
present study support this concept. In transgenic mice, BCW
ABL P210 clearly causes a distinct disease from BCWABL
P190. This is caused by inherently distinct properties of
P190 and P210, possibly including the intrinsically different
kinase activities of the ABL moiety in the hybrid oncoproteins and/or cellular interactions of the Dbl and pleckstrin
homology domains or as of yet to be identified domains in
Bcr. In addition, regulatory sequences upstream from or
within the BCR gene are likely to contribute to the occurrence of myeloid leukemia in humans and presumably in
mice. These ideas will be the subject of future investigations
in our laboratory.
REFERENCES
1. Nowell PC, Hungerford DA: A minute chromosome in human
chronic granulocytic leukemia. Science 132:1497, 1960
2. De Klein A, Geurtsvan Kessel A, Grosveld G, Bartram CR,
Hagemeijer A, Bootsma D, Spurr NK, Heisterkamp N. Groffen J,
Stephenson JR: A cellular oncogene is translocated to the Philadelphia chromosome in chronic myelocytic leukemia. Nature 300:765,
1982
3. Rowley JD: A new consistent chromosomalabnormalityin
chronic myelogenous leukemia identifiedby quinacrine fluorescence
and giemsa staining. Nature 243:290, 1973
4. Sandberg AA: Chromosomeinstabilitysyndromes,inThe
Chromosomes in Human Cancer and Leukemia (ed 2). New York,
NY, Elsevier, 1990, p 185
5. Priest JR, Robinson LL, McKenna RW, Lindquist LL, Warkentin PI, LeBien W, Woods WC, Kersey JH, Coccia PF, Nesbit ME
Jr: Philadelphia chromosomepositive childhood acute lymphoblastic
leukemia. Blood 56:15, 1980
6. Van Etten RA: The molecular pathogenesisof the Philadelphiapositive leukemias: Implications for diagnosis and therapy, in Freireich EJ,Kantarjian H (eds): Leukemia:AdvancesResearchand
Treatment.Deventer,TheNetherlands,Kluwer
Academic, 1993, p
295
7. Kurzrock R, Gutterman JU, Talpaz M The molecular genetics
of Philadelphia chromosome-positive leukemias. N Engl J Med
319:990, 1988
8. Sawyers CL, Denny CT, Witte ON: Leukemia and the disruption of normal hematopoiesis. Cell 64:337, 1991
9. Heisterkamp N, Groffen J: Molecular insights into the Philadelphia translocation. Hematol Pathol 5:1, 1991
10. M m Y, Witte ON: TheBCR gene encodes a novel serine/
threonine kinase activity within a single exon. Cell 67:459, 1991
1 1 . McWhirter JR, WangJYJ:Activation
of tyrosinasekinase
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
4610
and microfilament-binding functions ofc-ab1by bcr sequences in
bcrlabl fusion proteins. Mol Cell Biol 11:1553, 1991
12. Muller AJ,Young JC, Pendergast AM, Pondel M, Landau
NR, Littman DR, Witte ON: BCR first exon sequences specifically
activate theBCWABL tyrosine kinase oncogene of Philadelphia
chromosome-positive human leukemias. Mol Cell Bid 1 1 : 1785,
1991
13. EvaA, Aaronson SA: Isolation of a new human oncogene
from a diffuse B-cell lymphoma. Nature 316:273, 1985
14. Hart MJ, Eva A, Evans T, Aaronson SA, Cerione RA: Catalysis of guanine nucleotide exchange on the CDC42Hs protein by the
dbl oncogene product. Nature 354:311, 1991
15. Diekmann D, Brill S, Garrett MD, Totty N, Hsuan J, Monfries
C, Hall C, Lim L, Hall A: Bcr encodes a GTPase-activating protein
for p21". Nature 351:400, 1991
16. Hart MJ, Maru Y, Leonard D, Witte ON, Evans T, Cerione
A: A GDP dissociation inhibitor that serves as a GTPase inhibitor
for the Ras-like protein CDC42. Science 258:812, 1992
17. Voncken JW, Van Schaick H, Kaartinen V, Landing B, Pattengale PK, Deemer K, Coates T, Dorseuil 0, Bokoch GM, Groffen
J, Heisterkamp N: Increased neutrophil respiratory burst in bcr null
mutants. Cell 80:719, 1995
18. Wang JYJ: Ab1 tyrosine kinase in signal transduction and
cell-cycle regulation. Cum Opin Gen Dev 3:35, 1993
19. Tybulewicz VLJ, Crawford CE, Jackson PK, Bronson RT,
Mulligan RC: Neonatal lethality and lymphopenia in mice with a
homozygous disruption of the c-ab1 proto-oncogene. Cell 65: I 1 53,
I991
20. Schwartzberg PL, Stall AM, Hardin JD, Bowdish KS,Humaran T, Boast S, Harbinson ML, Robertson EJ, Goff S P Mice
homozygous for the abl'"' mutation show poor viability and depletion
of selected B and T cell populations. Cell 65: 1 165, 1991
21. Daley GQ, Van Etten R, Baltimore D: Induction of chronic
myelogenous leukemia in mice by the P210 BCWABL gene of the
Philadelphia chromosome. Science 247:824, 1990
22. Elefanty AG, Hariharan IK, Cory S: BCWABL, the hall mark
of chronic myelogenous leukemia in man, induces multiple hemopoietic neoplasms in mice. EMBO J 9:1069, 1990
23. Kelliher MA, McLaughlin J, Witte ON, Rosenberg N: Induction of a chronic myelogenous leukemia-like syndrome in mice with
v-ab1 and BCWABL. Proc Natl Acad Sci USA 87:6649, 1990
24. Kelliher MA, Knott A, McLaughlin J, Witte ON, Rosenberg
N: Differences in oncogenic potency but not target cell specificity
distinguish the two forms of the BCWABL oncogene. Mol Cell Biol
11:4710, 1991
25. Elefanty AG, Cory S: Hematologic disease induced in BALBl
c mice by a BCWABL retrovirus is influenced by the infection conditions. Mol Cell Biol 12:1755, 1992
26. Kelliher MA, Weckstein DJ, Knott AG, Wortis HH, Rosenberg N: ABL oncogenes directly stimulate two distinct target cells
in bone marrow from S-fluorouracil-treatedmice. Oncogene 8:1249,
1993
27. Fialkow PJ, Jacobson RJ, Papayannopoulou T, Penfold GK,
Jacobson RT, Hansen JA: Chronic myelocytic leukemia: Clonal origin in a stem cell common to the granulocyte, erythrocyte, platelet
and monocytelmacrophage. Am J Med 63:125, 1977
28. Heisterkamp N, Jenster G, Ten Hoeve J, Zovich D, Pattengale
PK, Groffen J: Acute leukaemia in bcrhbl transgenic mice. Nature
344:2S I , 1990
29. Voncken JW, Morris C, Pattengale P, Dennert G, Kikly C,
Groffen J, Heisterkamp N: Clonal development and karyotype evolution during leukemogenesis ofBCWABL transgenic mice. Blood
79: 1029, 1992
30. Voncken JW, Griffiths S, Greaves MF, Pattengale PK, Hei-
VONCKEN ET AL
sterkamp N, Groffen J: Restricted oncogenicity of BCWABL P190
in transgenic mice. Cancer Res 52:4534, 1992
3 1. Heisterkamp N. Jenster G, Kioussis D, Pattengale PK, Groffen J: Human BCWABL gene has a lethal effect on embryogenesis.
Transgenic Res1:45,1991
32. Stuart GW, Searle PF, Chen HY, Brinster RL, Palmiter RD:
A 12 base-pair DNA motif that is repeated several times in metallothionein gene promoters confers metal regulation to a heterologous
gene. Proc Natl Acad Sci USA 81:73 18, 1984
33. Pattengale PK, Stewart TA, Leder A, Sinn E, Muller W,
Tepler I, Schmidt E, Leder P: Molecular analysis of transgenic mice
carrying and expressing activated oncogenes. Am J Pathol 135:39.
1989
34. Chomczynski P, Sacchi N: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
AnalBiochem 162:156, 1987
35. Kawasaki ES, Clark SS, Coyne MY, Smith SD, Champlin R,
Witte ON, McCormick FP: Diagnosis of chronic myeloid and acute
lymphocytic leukemias by detection of leukemia specificmRNA
85:5698,
sequences amplified in vitro. Proc NatlAcadSciUSA
1988
36. Alonso S, Minty A, Bourlet Y, Buckingham M: Comparison
of three actin-coding sequences in the mouse; evolutionary relationships between the actin genes of warm-blooded vertebrates. J Mol
Evol 23: 1 1, 1986
37. Harlow E, Lane D: Antibodies: A Laboratory Manual. Cold
Spring Harbor, NY, Cold Spring Harbor Laboratory, 1988
38. Groffen J, Voncken JW, Kaartinen V, Moms C, Heisterkamp
N: Ph'-positive leukemia: A transgenic mouse model. Leuk
Lymphoma IO: l , 1993 (suppl 2)
39. Clark SS, Chen E, Fizzotti M, Witte ON, Malkovska V: BCW
ABL andv-ab1
oncogenes induce distinct patterns of thymic
lymphoma involving different lymphocyte subsets. J Virol 67:6033,
1993
40. Hanecak R, Pattengale PK, Fan H: Addition or substitution
of simianvirus 40 enhancer sequences into the Moloney murine
leukemia virus (M-MuLV) long terminal repeat yields infectuous
M-MuLV with altered biological properties. J Virol 62:2427, 1988
41, Pattengale PK: Tumours ofthe lymphohaematopoietic system, in Turusov V, Mohr U (eds): Pathology of Tumours in Laboratory Animals. Lyon, France, International Agency for Research on
Cancer (WHO), 1994, p 651
42. Daley GQ: Animal models of BCWABL-induced leukemias.
Leuk Lymphoma 11:57, 1993
43. Honda H, Fujii T, Takato M, Mano H, Witte ON, Yazalu Y,
Hirai H: Expression of p21OBCWABL
by metallothionein promoter
induced T-cell leukemia in transgenic mice. Blood 85:2853, 1995
44. Lug0 TG, Pendergast A-M, Muller AJ, Witte O N Tyrosine
kinase activity and transformation potency of bcr-ab1 oncogene
products. Science 247: 1079, 1990
45. Mayer BJ, Ren R, Clark KL, Baltimore D: A putative modular
domain present in diverse signalling proteins. Cell 73:629, 1993
46. Gishizky ML, Johnson-White J, Witte ON:Efficient transplantation of BCWABL-induced chronic myelogenous leukemia like
syndrome in mice. Proc Natl Acad Sci USA 90:3755, 1993
47. Hariharan IK, Hams AW, Crawford M,Abud H, Webb E,
Cory S, Adams JM: A bcr-v-ab1 oncogene induces lymphomas in
transgenic mice. Mol Cell Biol 9:2798, 1989
48. Akashi K, Taniguchi S, Nagafuji K, Harada M, Shibuya T,
Hayashi S, Gondo H, Niho Y: B-lymphoidmyeloid stem cell origin
in Ph-positive acute leukemia with myeloid markers. LeukRes
17:549, 1993
49. Akashi K, Mizuno S, Harada M, Kimura N, Kinjyo M, Shibuya T, Shimoda K, Takeshita M, Okamura S, Matsumoto I, Kikuchi
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
BCRIABL P210 TRANSGENICMICE
M, Niho Y: T-lymphoidmyeloid bilineal crisis in chronic myelogenous leukemia. Exp Hematol21:743, 1993
50. Martin M, Strasser A, Baumgarth N. Cicuttini FM, Welch K,
Salvaris E, Boyd AW: A novel cellular model (SPGM 1) of switching between pre-B cell and myelomonocytic lineages. J Immunol
150:4395, 1993
5 1. Jacks T, Fazeli A M , Schmitt EM, Bronson RT, Goodell MA,
461 1
Weinberg RA: Effects of an Rb mutation in the mouse. Nature
359:295, 1992
52. Frith CH, Wiley LD: Classification an incidence of hyperplastic and neoplastic hematopoietic lesions in mice. J Geront 36534,
1981
53. Berger R: Differences between blastic chronic myeloid leukemia and Ph-positive acute leukemia. Leuk Lymphoma 11 :235, 1993
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 86: 4603-4611
BCR/ABL P210 and P190 cause distinct leukemia in transgenic mice
JW Voncken, V Kaartinen, PK Pattengale, WT Germeraad, J Groffen and N Heisterkamp
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