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Blood, Vol. 95 No. 4 (February 15), 2000:
pp. 1144-1150
PLENARY PAPER
Acquired loss of p53 induces blastic transformation in
p210bcr/abl-expressing hematopoietic cells: a
transgenic study for blast crisis of human CML
Hiroaki Honda,
Toshikazu Ushijima,
Kuniko Wakazono,
Hideaki Oda,
Yuji Tanaka,
Shin-ichi Aizawa,
Takatoshi Ishikawa,
Yoshio Yazaki, and
Hisamaru Hirai
From the Third Department of Internal Medicine, Faculty of Medicine,
University of Tokyo, Tokyo, Japan; Carcinogenesis Division, National
Cancer Center Research Institute, Tokyo, Japan; the Department of
Pathology, University of Tokyo, Japan; and the Department of
Morphogenesis, Institute of Molecular Embryology and Genetics, Kumamoto
University School of Medicine, Kumamoto, Japan.
 |
Abstract |
Chronic myelogenous leukemia (CML) begins with an indolent chronic
phase but inevitably progresses to a fatal blast crisis. Although the
Philadelphia chromosome, which generates
p210bcr/abl, is a unique chromosomal abnormality in
the chronic phase, additional chromosomal abnormalities are frequently
detected in the blast crisis, suggesting that superimposed genetic
events are responsible for disease progression. To investigate whether
loss of p53 plays a role in the evolution of CML, we crossmated
p210bcr/abl-transgenic
(BCR/ABLtg/ ) mice with p53-heterozygous
(p53+/) mice and generated
p210bcr/abl-transgenic, p53-heterozygous
(BCR/ABLtg/p53+/)
mice, in which a somatic alteration in the residual normal p53 allele directly abrogates p53 function. The
BCR/ABLtg/p53+/ mice
died in a short period compared with their wild-type
(BCR/ABL/p53+/+),
p53 heterozygous
(BCR/ABL/p53+/),
and p210bcr/abl transgenic
(BCR/ABLtg/p53+/+)
litter mates. They had rapid proliferation of blast cells, which was preceded by subclinical or clinical signs of a myeloproliferative disorder resembling human CML. The blast cells were clonal in origin and expressed p210bcr/abl with an
increased kinase activity. Interestingly, the residual normal p53
allele was frequently and preferentially lost in the tumor tissues,
implying that a certain mechanism facilitating the loss of p53 allele
exists in p210bcr/abl-expressing hematopoietic
cells. Our study presents in vivo evidence that acquired loss of p53
contributes to the blastic transformation of
p210bcr/abl-expressing hematopoietic cells and
provides insights into the molecular mechanism for blast crisis of
human CML.
(Blood. 2000;95:1144-1150)
© 2000 by The American Society of Hematology.
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Introduction |
In chronic myelogenous leukemia (CML), a clonal
disorder of multipotential hematopoietic stem cells, excessive
proliferation of immature and mature myeloid cells occurs.1
The cytogenetic hallmark of CML is the Philadelphia
chromosome,2 created by t(9;22)(q34;q11), in which the
amino-terminal bcr gene on chromosome 22 is fused to most of
the c-abl proto-oncogene on chromosome 9, thereby creating an
8.5-kilobase (kb) bcr/abl chimeric messenger RNA encoding a
210-kd hybrid protein (p210bcr/abl).3-5
The p210bcr/abl has much greater kinase activity
than the normal 145-kd c-abl gene product, and this is believed
to play a critical role in the pathogenesis of CML.6
The clinical course of CML is characterized by hematologically and
temporally distinct stages.1 In the initial stage, called chronic phase, the disease is indolent and the leukemic cells retain an
ability to differentiate into mature granulocytes. But after several
years of the chronic phase, the disease inevitably accelerates and
ultimately progresses to the terminal fatal stage, called blast crisis,
which involves aggressive proliferation of immature hematopoietic cells
arrested at an early stage of differentiation. Although the molecular
mechanisms responsible for the transition from the chronic phase to the
blast crisis are not fully understood, the appearance of additional and
nonrandom chromosomal abnormalities in the blast phase strongly
suggests that superimposed genetic events account for the disease
progression.7 One of the most frequently observed
chromosomal abnormalities is isochromosome 17q, or i(17q),7
where p53 tumor-suppressor gene is mapped.8 Studies have
shown that structural alterations (ie, rearrangement, deletion, and
mutation) of the p53 gene are extremely rare in the chronic phase but
frequently appear in the blast crisis, strongly suggesting that the
functional loss of p53 is essentially involved in the disease
evolution.9-11
To understand the complex processes involved in the pathogenesis of
human CML, it is necessary to develop animal models that recapitulate
the clinical features of the disease. Several different approaches have
been used to create a murine model for human CML. Earlier attempts
focused on bone marrow transplantation (BMT) experiments. Mice that
were lethally irradiated and given transplants of BM (BM) cells
infected with p210bcr/abl-expressing retroviruses
had CML-like granulocyte hyperplasia and other hematopoietic
malignancies, such as myelomonocytic leukemias, macrophage tumors,
pre-B- and T-cell lymphomas, reticulum-cell sarcomas, and erythroid
tumors.12-14 Another approach recently used is
transplantation of peripheral blood (PB) or BM cells from patients with
CML into sublethally irradiated nonobese diabetic (NOD) or NOD/severe
combined immunodeficient (SCID) mice. A population of the implanted CML
cells was successfully engrafted and proliferated in the recipient
mice, which eventually showed a CML-like hematologic disorder.15,16
On the other hand, generation of transgenic mice is an attractive
approach. However, transgenic models for human CML have been
unsuccessful until recently, probably owing to the lack of appropriate
promoters.17 To overcome this problem, we cloned the
promoter of mouse tec gene,18,19 which is
preferentially expressed in hematopoietic progenitor
cells,20 and generated transgenic mice expressing
p210bcr/abl under the control of the tec
promoter.21 Although the founder mice showed massive
proliferation of lymphoblasts shortly after birth and were diagnosed as
having acute lymphoblastic leukemia (ALL), the transgenic offspring,
after a long latency period, reproducibly exhibited a
myeloproliferative disorder closely resembling human CML.21
The PB smear showed remarkable granulocyte hyperplasia, and the BM was
hypercellular, with a predominance of myeloid cells at various stages
of differentiation.21 Because these pictures represent
cardinal features of human CML, our transgenic mice can be regarded as
a model for human CML.
Compared with the BMT and NOD or NOD/SCID mice approach, our transgenic
approach has several advantages. First, the disease spectrum and
disease frequency in the BMT approach are markedly influenced by the
infection conditions22,23 and the implantation efficiency
and survival of the engrafted CML cells in NOD or NOD/SCID mice vary
significantly, depending on the donor patients and recipient mice.15,16 In contrast, our transgenic mice reproducibly
exhibit CML-like granulocyte hyperplasia, with disease penetrance of
about 100% (among the 87 transgenic mice thus far generated, 84 [about 97%] developed CML and the other 3 [about 3%] developed
ALL [Honda et al, unpublished data]). Second, although the mice in
the other 2 models have p210bcr/abl only in the
hematopoietic compartment, our transgenic mice have p210bcr/abl transgene in every type of cell and can
be crossmated with other transgenic or knockout mice to investigate the
possible promoting or suppressing effect of a specific gene on
p210bcr/abl in vivo. Thus, our transgenic mice can
be regarded as a stable and inheritable model for human CML. Because of
these advantages, we used this model to examine whether loss of p53
contributes to the evolution to blast crisis of CML in vivo. With this
aim, we crossmated p210bcr/abl-transgenic mice with
p53-heterozygous mice and generated
p210bcr/abl-transgenic, p53-heterozygous mice, in
which a somatic alteration in the residual p53 gene directly abrogates
p53 function.
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Materials and methods |
Mice
Mice that were p210bcr/abl transgenic
(BCR/ABLtg/) were generated by using
the mouse tec promoter ( 1948 to +22) and the bcr/abl
(p210) complementary DNA (cDNA; b3a2 type) as previously
described.21 Because the founder mice were generated by
using eggs derived from C57Bl/6 × DBA F2 (BDF2) mice and the
transgenic progeny were generated by mating the transgenic mice with
BDF1 mice, the genetic background of the
BCR/ABLtg/ mice was a mixture of
C57Bl/6 and DBA. The p53-heterozygous (p53+/) mice
were generated by using TT2 ES cells inserted with a
neomycin-resistance cassette into the second exon of the p53 gene as
previously described.24 Because the TT2 ES cells were
established from an F1 embryo between C57Bl/6 and CBA25 and
the p53-heterozygous mice were generated by mating the chimeric mice
with C57Bl/6 mice, the genetic background of the
p53+/ mice was a mixture of C57Bl/6 and CBA.
Therefore, the background of the mice used in this experiment was a
mixture of C57Bl/6, DBA, and CBA. Identification of genotypes for
BCR/ABL and p53 was carried out by hybridizing
BamHI-digested tail DNA with bcr/abl cDNA and
SacI-digested tail DNA with the 5' flanking region of the
mouse p53 gene, as previously described.21,24
Pathological analysis
Autopsies were performed on dead or moribund animals. Smears and
stamp specimens of leukemic tissues were stained with Wright-Giemsa stain. Tissues were also fixed in 10% neutral-buffered formaldehyde and subjected to routine light microscopical examination. All the
organs were examined grossly and representative slices were prepared
for hematoxylin-eosin staining.
Western blot, immunoprecipitation, and in vitro kinase assay
For detection of p210bcr/abl-transgene product,
proteins were extracted by homogenizing leukemic tissues in RIPA lysis
buffer (150 mmol/L sodium chloride, 50 mmol/L TRIS chloride [pH 7.4],
1% Triton X-100, 0.05% sodium dodecyl sulfate, and 1% sodium
deoxycholate) with 50 U/mL of aprotinin and were probed with the
anti-Abl monoclonal antibody AB3 (Oncogene Science, Manhasset, NY) as
previously described.21 For detection of the kinase
activity of the p210bcr/abl-transgene product,
protein aliquots were incubated with AB3 and immunoprecipitated
proteins were subjected to in vitro kinase assay as previously
described.21 For detection of p53 protein, nuclear
fractions of the tissues were subjected to immunoprecipitation/Western blot analysis using anti-p53 antibodies (Oncogene Science) as previously described.24
Flow cytometric analysis
Cells were stained with fluorescein isothiocyanate-conjugated or
phycoerythrin-conjugated commercial monoclonal antibodies, including
anti-Thy-1.2, anti-B220, anti-CD4, and anti-CD8 (Pharmingen, San Diego,
CA), according to the manufacturer's instructions. The stained cells
were washed 3 times with phosphate-buffered saline and analyzed on a
FACScan (Becton Dickinson, Sunnyvale, CA).
DNA extraction and Southern blot analysis
Genomic DNAs were extracted from tumor tissues and digested with
restriction enzymes. Southern blot analysis was performed by using
phosphorus (P) 32-dCTP-labeled mouse TCR as a probe as
previously described.21
Polymerase chain reaction-single-strand conformation polymorphism
(PCR-SSCP) analysis
PCR-SSCP analysis was performed essentially as previously
described.26 In brief, genomic DNAs were amplified by using
several different sets of primers with  32P-dCTP. The PCR
products were mixed with formamide dye, electrophoresed on acrylamide
gels with or without glycerol, and autoradiographed.
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Results |
Early death and blast-cell proliferation in
p210bcr/abl-transgenic, p53-heterozygous mice
Crossmating of p210bcr/abl-transgenic
(BCR/ABLtg/) mice with
p53-heterozygous (p53+/) mice produced mice with 4 different genotypes, which were
BCR/ABL/p53+/+
(wild-type),
BCR/ ABL/p53+/
(p53 heterozygous),
BCR/ABLtg/p53+/+
(p210bcr/abl transgenic), and
BCR/ABLtg/p53+/
(p210bcr/abl transgenic, p53 heterogeneous). The
genotypes of the 58 total offspring were determined by Southern blot
assessment using tail DNAs (Figure 1A). The
numbers of the mice with each genotype were 16 for
BCR/ABL/p53+/+
(27.5%), 13 for
BCR/ABL/p53+/
(22.4%), 15 for
BCR/ABLtg/p53+/+
(25.9%), and 14 for
BCR/ABLtg/p53+/
(24.1%). These results were in good agreement with the expected ratio
based on the Mendelian rule (25% each), indicating that the
crossmating did not affect the embryonic development of the mice.
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| Fig 1.
Studies of offspring generated by crossmating
BCR/ABLtg/ mice with p53+/
mice.
(A) Genotyping of the mice. The upper lanes show the results of
Southern blot testing. The arrow indicates the position of the
bcr/abl transgene; G, the position of the germline; and T, the
targeted alleles of the p53 gene. The lower lanes show the genotypes of
the mice. (B) Survival curves for the mice, according to genotype. Thin
dotted lines indicate the percentage of surviving mice with the
BCR/ABL/p53+/+
genotype; thick dotted lines, the
BCR/ABL/p53+/
genotype; thin solid lines, the
BCR/ABLtg/p53+/+
genotype; and thick solid lines, the
BCR/ ABLtg/p53+/
genotype. (C) Changes in the white blood cell (WBC) count and mortality
of the mice. The white circle indicates the WBC counts of
BCR/ ABL/p53+/+
mice; black circle,
BCR/ABL/p53+/
mice; white square,
BCR/ ABLtg/p53+/+
mice; and black square,
BCR/ABLtg/p53+/
mice. The bar indicates SD; the arrow shows the point at which a mouse
died.
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All mice were kept under the same maintenance conditions and were
continually observed. The hematopoietic variables of the PB were
routinely examined. Sixteen
BCR/ABL/p53+/+
mice were all healthy during the observation period (Figure 1B, thin
dotted line). Among the 13 BCR/ABL/p53+/
mice, 1 mouse died with a gross subcutaneous tumor at 11 months of age
(data not shown), but the other 12 mice survived in a healthy state for
more than 1 year with no symptoms of illness (Figure B, thick dotted
line). No obvious hematologic changes were observed in these mice
(Figure 1C, upper panels). Among the 15 BCR/ABLtg/p53+/+ mice, 1 mouse died at 3 months of age from ALL and the remaining 14 gradually
exhibited CML after 6 months of age, as previously reported21 (Figure 1C, left bottom panel). About half of
the mice died within 1 year of age (Figure 1B, thin solid line). In contrast to the mice with these genotypes,
BCR/ABLtg/p53+/
mice showed high morbidity and mortality rates (Figure 1B, thick solid
line). Eleven of 14 BCR/ABLtg/p53+/
mice died within 1 year. Although detailed analysis was not possible in
2 mice because of advanced autolysis, 9 mice were subjected to
pathological, biochemical, and molecular analyses. A common macroscopic
aspect of these mice was enlargement of the thymus (Table
1). Thymoma was frequently associated with
pleural effusion, and splenomegaly was observed in some mice (Table 1).
Wright-Giemsa staining showed that blast cells with no granules and
with morphologic characteristics of lymphoblasts proliferated markedly
in the thymus (Figure 2A) and pleural
effusion (Figure 2B).
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| Fig 2.
Pathological analysis of
BCR/ABLtg/p53+/ leukemic mice.
Figures 2A to 2D show Wright-Giemsa staining of stamped or smeared
specimens of hematopoietic tissues. Massive proliferation of
lymphoblasts is observed in the thymus (A) and in the pleural effusion
(B). In mice with late onset of acute transformation, proliferation of
granulocytes is evident in the peripheral blood (C) and the bone marrow
shows marked hyperplasia of myeloid cells (D). Figures 2E to 2J show
hematoxylin-eosin staining of tissue from the following: the lung (2E
and 2F), liver (2G and 2H), and kidney (2I and 2J). Infiltration of
leukemic cells was detected around the blood vessels. The boxed areas
in Figures 2E, 2G, and 2I are magnified in Figures 2F, 2H, and 2J,
respectively.
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The age at disease onset ranged from 2 to 11 months (Figure 1B, thick
solid line, and Table 1). In mice with disease onset at a young age, no
obvious hematologic changes were observed in PB (Figure 1C, right
bottom panel), although the BM showed predominance of myeloid cells
(data not shown). On the other hand, mice with disease onset in old age
had typical symptoms of CML before thymomas developed; they showed
leukocytosis with granulocyte proliferation in the PB (Figure 1B, right
bottom panel, and Figure 2C) and marked myeloid hyperplasia in the BM
(Figure 2D). Proliferation of blast cells in the PB was observed in
some mice (data not shown). Pathological analysis showed that the blast
cells infiltrated several major organs, such as the lung, liver, and
kidney, in all mice examined (Figure 2E-2J).
Expression and enhanced kinase activity of the
p210bcr/abl-transgene product in the tumor tissues
To examine whether the blast cells in
BCR/ABLtg/p53+/ leukemic
mice expressed the p210bcr/abl-transgene product,
proteins extracted from the enlarged thymus or pleural effusion were
blotted with the anti-Abl monoclonal antibody AB3. As shown in Figure
3A (arrow), the 210-kd band was detected in
all the leukemic tissues, indicating that blast cells expressed the
p210bcr/abl-transgene product. Blotting the same
samples with antiphosphotyrosine antibody showed that the expressed
p210bcr/abl was tyrosine phosphorylated (data not
shown). To examine whether the expressed
p210bcr/abl had an enzymatically active kinase
activity, proteins extracted from the tumor tissues were subjected to
in vitro kinase assays. As shown in Figure 3B, phosphorylated 210-kd
band was observed in all the leukemic tissues (arrow), indicating that
p210bcr/abl contained an increased kinase activity.
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| Fig 3.
Expression, kinase activity, and TCR rearrangements in
p210bcr/abl in the leukemic tissues of
BCR/ABLtg/p53+/ mice.
For the expression assessment (A), 50 µg of protein aliquots
extracted from the enlarged thymus or pleural effusion of 9 BCR/ABLtg/p53+/
leukemic mice (no. 1-9 on Table 1) was separated by 6% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a
nitrocellulose membrane, and probed with the anti-Abl monoclonal
antibody AB3 (1:500). To determine kinase activity (B), 1 mg of protein
aliquot extracted from the same tissues shown in Figure 3A was
incubated with AB3 (1:200) and the immunoprecipitated proteins were
subjected to in vitro kinase assays. Phosphorylated proteins were
separated by 6% SDS-PAGE, dried, and autoradiographed. For both the
expression and kinase activity assessments, the thymus of a normal
BCR/ABL/ control mouse was
the negative control, and the thymus of a
BCR/ABLtg/ leukemic mouse21
was the positive control. The arrow indicates the position of
p210bcr/abl; the positions of the molecular markers
are shown on the left. To describe TCR rearrangements (C), 5 µg of
DNAs digested with EcoRI was separated by 0.7% agarose gel,
blotted to a nylon membrane, and probed with a phosphorus
32-dCTP-labeled TCR probe. DNA extracted from normal thymus tissue
was used as a control (denoted by C). Molecular markers are shown on
the left.
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Expression of T-cell antigen by blast cells and their clonal
origin
To determine the cell lineage of the leukemias, blast cells in the
enlarged thymus or pleural effusion from 7 BCR/ABLtg/p53+/
leukemic mice were subjected to flow cytometric analysis. Staining the
cells with anti-Thy1.2 and anti-B220 antibodies showed that all the
tumor cells were positive for Thy1.2 antigen but negative for B220
(Table 1). Staining the cells with anti-CD4 and anti-CD8 antibodies
revealed that 1 tumor was double negative (CD4
CD8), 4 tumors were double positive
(CD4+ CD8+), and the other 2 were single
positive for CD4 (CD4+ CD8) and CD8
(CD4 CD8+), respectively (Table 1). To
investigate the clonality of the blast cells, DNAs extracted from the
tumor tissues were subjected to Southern blot analysis using TCR as a
probe. The results showed that all the tumor tissues carried
rearrangements in the TCR locus, indicating that the blast cells were
clonal in origin (Figure 3C and Table 1).
Frequent and preferential loss of the wild-type p53 allele and
absence of p53 protein in the leukemic cells in
BCR/ABLtg/p53+/ mice
The rapid proliferation of blast cells in the
BCR/ABLtg/p53+/
mice strongly suggested that a somatic mutation or mutations occurred in the residual wild-type p53 gene. To address this issue, PCR-SSCP analysis was performed. This assessment can detect mutations in almost
all of the coding sequence (codon 5 to 356) and the splice donor and
acceptor sites in introns 2 through 9 of the mouse p53 gene.26 No band shifts were observed in any regions of the
p53 gene under 2 different conditions, indicating that the leukemias did not carry point mutations (data not shown). On the other hand, we
found a polymorphic site in exon 5 that allowed us to distinguish the
PCR product of the wild-type allele from that of the null allele.
Sequencing revealed that codon 134 of the wild-type allele was
G G (coding Ala), whereas that of the null allele was
G G (coding Val).
A schematic model of the region encompassing the polymorphic site and
the results of PCR-SSCP are shown in Figure
4A and Figure 4B, respectively. As shown in
the left 2 lanes in Figure 4B, the p53+/ mouse was a
heterozygote of alleles A and B, whereas the
BCR/ABLtg/ mouse was a homozygote of
allele B. Because all the tissues examined in the
BCR/ABLtg/p53+/
leukemic mice harbored allele A (numbers 1-9 in Figure 4B),
BCR/ABLtg/p53+/
mice were considered to be heterozygotes of allele A from the p53+/ mouse (null) and allele B from the
BCR/ABLtg/ mouse (wild-type).
Interestingly, among 9 tumors that developed in
BCR/ ABLtg/p53+/
mice (T lanes in Figure 4B), 6 tumors (no. 2 and no. 5-9) contained only allele A (null) and clearly lost allele B (wild-type). This allelic loss was obviously a somatic event, since the normal tissues (N
lanes of no. 2, 5, and 8 in Figure 4B) retained allele B. The absence
of the p53 protein in the tumor tissues was confirmed by
immunoprecipitation/Western blot analysis using anti-p53 antibodies (Figure 4C). These data demonstrated that the residual wild-type p53
allele was frequently and preferentially lost in the tumor tissues in
the
BCR/ABLtg/p53+/
leukemic mice and that this led to the absence of the p53 protein. The
characteristics of the leukemias that developed in
BCR/ABLtg/p53+/
mice are summarized in Table 1.
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| Fig 4.
Polymerase chain reaction-single-strand conformation
polymorphism (PCR-SSCP) analysis of the p53 gene and expression
of p53 protein in the leukemic tissues of
BCR/ABLtg/p53+/ mice.
(A) Schematic model of the region of the mouse p53 gene that
showed a polymorphic pattern between the p53 wild-type allele and the
p53 null allele. The nucleotide sequences and amino acids of the
wild-type allele and the null allele, which showed a polymorphism, are
also shown. The box denotes exon 5; black bars, adjacent introns; white
triangles, locations of the primers used for amplification (p5-1 and
p5-2); and the diamond, the polymorphic site. (B) Results of PCR-SSCP.
DNAs extracted from the tumor tissues (T) of 9 BCR/ABLtg/p53+/
leukemic mice (no. 1-9) were subjected to PCR-SSCP analysis. In mice 2, 5, and 8, DNAs extracted from the normal tissues (N) were also analyzed
to provide an internal control. DNAs extracted from the normal tissues
of a p53+/ mouse and a
BCR/ABLtg/ mouse were used as
controls for detecting the migration patterns of the parental alleles.
Black triangles denote the position of allele A (null); and white
triangles, the position of allele B (wild-type). (C) Absence of p53
protein. Proteins extracted from tumor tissues (T) and normal tissues
(N) of mice 2, 5, and 8 were subjected to immunoprecipitation/Western
blot analysis using anti-p53 antibodies. Normal tissue from a
p53+/ mouse was used as a control (denoted by C).
The arrow indicates the position of p53.
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| |
Discussion |
CML provides an appropriate model for a disease in which the gain of
an acquired genetic abnormality contributes directly to the disease
progression. The p53 is one of the genes whose alterations are
frequently detected in the blast crisis of human CML. Therefore, the
functional defect of p53 is believed to play a key role in
the evolution of the disease, but no direct evidence has been
shown.9-11 Skorski et al27 transfected
p210bcr/abl-expressing retroviruses into
p53-deficient and wild-type BM cells. They showed that the
p210bcr/abl-positive, p53-deficient BM cells had
blast-like morphologic characteristics and an increased colony-forming
ability compared with p210bcr/abl-expressing,
wild-type BM cells. In addition, when injected into SCID mice,
p210bcr/abl-positive, p53-deficient cells showed
more rapid disease progression than
p210bcr/abl-expressing, wild-type cells.
These results demonstrated that the disruption of p53 function
conferred a more aggressive leukemogenic potential on
p210bcr/abl-positive hematopoietic cells and
induced a more malignant phenotype. However, because the experimental
procedures, such as culture of BM cells and retrovirus transfection,
were performed in vitro, the synergistic effect of
p210bcr/abl and loss of p53 in vivo could not be
fully understood. We addressed this issue by generating
p210bcr/abl-transgenic, p53-heterozygous mice, in
which a somatic event in the residual p53 gene directly abrogates
p53 function.
The crossmating of p210bcr/abl-transgenic mice with
p53-heterozygous mice resulted in generation of mice with 4 different
genotypes: BCR/ABL/p53+/+,
BCR/ABL/p53+/,
BCR/ ABLtg/p53+/+, and
BCR/ABLtg/p53+/.
The
BCR/ABL/p53+/+
mice were reasonably healthy (Figure 1B and 1C). Of 13 BCR/ ABL/p53+/
mice, 1 mouse died of a skin tumor (data not shown). This result is not
consistent with those of previous p53+/ studies, in
which malignancies, including T-cell lymphoma, occurred at a higher
ratio.28,29 Although the reason for this difference is not
clear, one possibility is the genetic background of the mice, as
previously suggested.30 The genetic background of the mice
in our study was a mixture of C57Bl/6, DBA, and CBA, whereas other
p53+/ studies used mice containing the 129 strain28,29 and a study showed that an increased ratio of
129 strain accelerates disease development.30
BCR/ABLtg/p53+/+ mice
had CML-like disease and ALL (Figure 1B and 1C), as
expected.21 In contrast to these mice,
BCR/ABLtg/p53+/
mice died in a short period (Figure 1B and 1C) and with rapid proliferation of blast cells (Figure 2A and 2B). The leukemic cells
were highly malignant, as indicated by massive infiltration into
tissues (Figure 2E-2J). They expressed p210bcr/abl
with an enhanced kinase activity (Figure 3A, 3B). In addition, the
residual wild-type p53 allele was frequently and preferentially lost in
leukemic tissues (Figure 4B), which led to the absence of p53
expression (Figure 4C). These results demonstrate that the functional
loss of p53 essentially contributed to the blastic transformation of
the p210bcr/abl-expressing hematopoietic
cells in vivo.
In
BCR/ABLtg/p53+/
mice, the blastic transformation was observed in mice with a wide range
of ages (Figure 1B and 1C and Table 1). This was possibly because the
disease onset depended on when an acquired event, including loss of
p53, occurred in the p210bcr/abl-expressing
hematopoietic cells. In mice with early disease onset, the BM showed a
predominance of myeloid cells (data not shown), although no
abnormalities were observed in the PB (Figure 1B and 1C). On the other
hand, in mice with late disease onset, the PB showed excessive
proliferation of granulocytes (Figure 2C) and the BM showed marked
hyperplasia of myeloid cells (Figure 2D). Therefore, subclinical or
clinical signs of CML already existed when acute leukemias developed in
the mice. These results indicate that CML preceded the acute phase in
these mice, in recapitulation of the clinical course of human CML.
It is interesting that the loss of the wild-type p53 allele was
observed in the leukemic tissues at a high incidence but that normal
tissues retained the wild-type allele (Figure 4B). Thus, the acquired
loss of the wild-type p53 allele was not a random event; rather, there
is a certain mechanism by which genetic abnormalities preferentially
occurred in the hematopoietic cells. It could be supposed that the
expression of p210bcr/abl might contribute to the
process or processes leading to the allelic loss. The
p210bcr/abl might accelerate the mutation rate or
promote genetic instability (as reported in
p190bcr/abl transgenic mice31), which
would facilitate the loss of the wild-type p53 allele and consequently
cause the blastic transformation. Further studies will be required to
clarify the molecular mechanism or mechanisms underlying the
preferential loss of p53 in the
p210bcr/abl-expressing hematopoietic cells in the
transgenic mice.
It should be noted that all the leukemias were clonal T-cell tumors, as
demonstrated by flow cytometry and gene-rearrangement analysis (Figure
3C and Table 1). These results indicate that the loss of p53 occurred
in hematopoietic cells that had already been committed to the T-cell
lineage. This idea was further supported by the finding that
hematopoietic cells of other lineages, such as myeloid, contained the
wild-type p53 allele (data not shown). The reason for the remarkable
susceptibility of the T cells to p53 loss remains elusive. One
possibility is that gene rearrangements in the TCR loci triggered gene
alterations (possibly in cooperation with
p210bcr/abl) and led to loss of the wild-type p53
allele. The finding that p53 loss occurred preferentially in relatively
young mice (number 2 and numbers 5-9 in Figure 4C and Table 1) supports
this notion, since physiologic TCR rearrangements occur actively in the
thymus of young animals. In the 3 mice in which leukemia developed at a
relatively old age, no mutations were detected in the p53 gene (numbers
1, 3, and 4 in Figure 4C and Table 1). In these mice, the mechanism
that caused blastic transformation remains unknown. We consider it
possible that the reduction of p53 protein in p53+/
cells enhanced the oncogenicity of p210bcr/abl and
caused malignant transformation.32 Alternatively, the
decrease in p53 protein might have facilitated activation of oncogenes or inactivation of tumor-suppressor genes, with which
p210bcr/abl exerted its fully oncogenic potential.
Mouse models for clonal expansion of
p210bcr/abl-expressing hematopoietic cells have
been demonstrated in BMT experiments.33-36 Serial transplantation of murine CML cells into syngeneic recipient mice frequently caused acute leukemias. The leukemic cells contained the
same proviral integration site as the primary CML cells, indicating that they originated from the same clone. Although the molecular mechanisms responsible for the disease progression have not clearly been identified in BMT models, it may be that a secondary genetic event
or events, such as loss of p53 as observed in our study, might
contribute to the blastic transformation. One difference between our
transgenic model and the BMT experiments is the disease phenotype. Our
transgenic mice had T-cell leukemias exclusively (Table 1), whereas the
recipient mice in the BMT experiments had myeloid and B-cell acute
leukemias in addition to T-cell leukemias.33-36 This
phenotypic difference might be due to the genetic backgrounds of the
mice. The genetic background of our transgenic mice is a mixture of
C57Bl/6, DBA, and CBA, whereas the mice used in the BMT experiments
were of the BALB/C strain.33-36 An expanded study would
provide more information about the phenotypic disparity between our
transgenic mice and the mice in the BMT experiments.
There is a feature of our model that is dissimilar to human CML. A
T-cell phenotype is rarely observed in the blast crisis of human
CML.1 One possible reason is that T-cell lineage is rarely
involved in human CML, whereas every type of cell in our transgenic
mice has the transgene and the thymic cells might be more susceptible
to a somatic event or events causing blastic transformation. An animal
model for CML in which loss of p53 could be controlled in spacial and
temporal ways would clarify this issue.
In this study, we showed that
BCR/ABLtg/p53+/
mice had marked proliferation of blast cells in a short period, in
which the residual wild-type p53 allele was frequently and
preferentially lost. These results provide in vivo evidence that an
acquired loss of p53 function contributed a proliferative advantage to
the p210bcr/abl-expressing hematopoietic cells and
caused blastic transformation. Our findings provide insights into the
molecular mechanism of blast crisis of human CML. In addition, our
transgenic mice are a useful animal model for examining the biologic
effect of a specific gene on the malignant transformation of
p210bcr/abl-expressing hematopoietic cells in vivo.
| |
Acknowledgments |
We thank Yoshikazu Oh-hira for preparing pathological specimens and
Tsuyoshi Takahashi, Yoichi Imai, and Koichiro Yuji for technical
assistance. We also thank T. W. Mak for providing the mouse TCR probe.
| |
Footnotes |
Submitted May 17, 1999; accepted October 15, 1999.
Supported in part by grants-in-aid from the Ministry of Education,
Science and Culture of Japan.
Reprints: Hisamaru Hirai, Third Department of Internal
Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan; e-mail: hhirai-tky{at}umin.ac.jp.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction