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From the Laboratory of Environmental Carcinogenesis and
Mutagenesis*
and the Laboratory of Experimental
Pathology,
National Institute of Environmental
Health Sciences, and the Chemical Industrial Institute of
Toxicology,
Research Triangle Park,
North Carolina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-globin
promoter develops an array of spontaneous epithelial and mesenchymal
neoplasms. In this report we describe the morphological,
immunophenotypic, and molecular features of a unique
hematopoietic neoplasm in these mice. The cardinal lesion of this
disease is marked hepatomegaly due to leukemic proliferation and
infiltration. In the peripheral blood, there is a marked
increase in the number of metarubricytes and other less differentiated
erythroid progenitor cells. Leukemic cells stain positively with an
erythroid-associated nuclear transcription factor (GATA-1). Using a
reverse transcription polymerase chain reaction assay,
co-expression of GATA-1 and endogenous -globin genes is detected in
hematopoietic tissues of nonleukemic transgenic and nontransgenic mice.
ras transgene expression is, however,
detected only in normal bone marrow and leukemic tissues of transgenic
mice, and 5' mapping experiments using S1 protection analysis
of total RNA from leukemic tissue indicates that transcription of the
transgene mRNA is initiated from the natural -globin promoter start
site, supporting the belief that the -globin promoter
directs v-Ha-ras expression in erythroid progenitor
cells, ultimately leading to leukemic
transformation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The advent of transgenic technology has allowed the introduction of specific oncogenes into mice and targeted tumorigenesis in various tissues, including the hemolymphatic system.5,6 Transgenic models of lymphoid neoplasia have been developed, but transgenic lines expressing specific disorders of nonlymphoid hematopoietic cells have not been reported.
The Tg.AC transgenic mouse line, carrying a v-Ha-ras
oncogene linked to the embryonic -globin promoter, was developed by
Leder and colleagues to study the development of the embryonic
hematopoietic system.7
Interestingly, the Tg.AC strain
develops an array of spontaneous epithelial and mesenchymal
tumors8,9
and has been used in mechanistic studies of
chemically induced skin carcinogenesis due to its sensitivity to
induction of skin papillomas.10,11
However, hematopoietic
abnormalities have not been reported other than descriptions of
hepatosplenomegaly associated with infiltrations of lymphoid or
myelomonocytic cells and typically found in animals bearing skin
tumors.7,8
In our laboratory, cases of hepatosplenomegaly in Tg.AC mice have been
observed due to a distinct neoplastic disease of erythroid cells
associated with expression of the transgene. We propose that the
transcription factors required for the constitutive expression of the
endogenous -globin promoter in adult erythroid stem cells are
functioning to stimulate expression of the -globin promoted
ras transgene and that accumulation of the oncogenic
ras protein results in aberrant proliferation of
erythropoietic cells. Therefore, in this report we present an initial
morphological characterization of this disease as well as an
investigation of the roles of the ras transgene and the
erythroid-specific transcription factor, GATA-1, in its development.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The animals described in this report were identified from a colony of homozygous Tg.AC transgenic mice and parental strain FVB/N mice obtained from Taconic Farms (Germantown, NY) at approximately 4 to 5 weeks of age, held for up to 10 weeks, and then placed on skin tumor induction protocols as described elsewhere.10,11 Standard treatment was twice-weekly applications of test chemical or acetone vehicle to the shaved dorsal skin. Two affected animals in this data set, as well as all randomly selected unaffected Tg.AC and FVB/N mice used as controls, were not on any chemical treatment protocol and had no chemical exposure history. Mice were multiply housed (five per cage), kept on a 12-hour light/dark cycle, and fed either NIH-07 pellet ration or Purina Pico Chow 5058 ad libitum.
Pathological Evaluations
Observation of marked abdominal distention due to hepatosplenomegaly provided antemortem identification of affected mice. Blood collections for hematological analyses were made from the retro-orbital venous sinus under anesthesia with 70% CO2/30% O2. Mice were killed by CO2 asphyxiation, and body and organ weights were recorded. Tissues were collected, fixed in 10% neutral buffered formalin, paraffin embedded, sectioned at 6 µm, and stained with hematoxylin and eosin for light microscopic evaluation. Tissues routinely collected at necropsy were liver, spleen, kidney, heart, lung, femur, sternum, and all gross lesions.
Automated complete blood counts were made with a Technicon H*1 hematology analyzer (Miles, Tarrytown, NY) outfitted with specialized rodent software. Hematological measurements included the following parameters: white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and platelet count. Nucleated cell differentials were made visually from blood smears stained with Wright-Giemsa solution, and reticulocyte counts were obtained from smears stained with New Methylene Blue (EK Industries, Joliet, IL).
Immunohistochemical Analysis of GATA-1
Four-micron serial sections of formalin-fixed, paraffin-embedded tissues were deparaffinized and rehydrated to 1x Automation buffer (Biomeda Corp., Foster City, CA). Endogenous peroxides were blocked in 3% aqueous H2O2 for tissue sections and 0.3% for air-dried cytocentrifuge preparations. Tissues were subjected to antigen retrieval in the microwave (twice for 5 minutes each at 700 W in 0.1 mol/L citrate buffer, pH 6.0).12,13 After cooling to room temperature, all slides were blocked with 1% normal goat serum (Vector Laboratories, Burlingame, CA) and then incubated with rat anti-mouse GATA-1 (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution for 1 hour at room temperature. Detection was accomplished with StrAviGen supersensitive detection kit (BioGenex Laboratories, San Ramon, CA), followed by incubation with diaminobenzidine as the substrate and counterstaining with Harris or Mayer's hematoxylin (Sigma Chemical Co.).
Preparation of Total RNA from Leukemic and Nonleukemic Tissue
Liver, spleen, and bone marrow were collected and flash frozen in liquid nitrogen. Total RNA was extracted from frozen tissue using TriReagent (Molecular Research Center, Cincinnati, OH) and following the manufacturer's protocol. Organs were homogenized in TriReagent using a Brinkmann polytron (Brinkmann Instrument Co., Westbury, NY), extracted once with chloroform, and then precipitated with isopropanol. The final RNA pellet was washed once in 75% ethanol, resuspended in sterile 0.1% diethylpyrocarbonate-treated water, and then heated for 15 minutes at 55°C to facilitate dissolution of the pellet. RNA was quantified by spectrophotometric measurements at A260.
Reverse Transcription PCR
Reverse transcription (RT)-PCR was performed as described
previously.9,13
RNA-specific primers were directed to
either the SV40 polyadenylation/splice region of the
transgene,9,13
GATA-1,14
mouse
ß2-microglobulin,13
or endogenous mouse
-globin. Primer sequences for -globin were selected from genomic
DNA of mouse -globin15
with the sense primer located in
exon 1 from bases 435 to 462 and the antisense primer in exon 2
spanning bases 1509 to 1536. Sequences for the -globin primers are
as follows: sense 5'-GCC AGT CTT GAG TGC ACT CAA CTC CAG C-3' and
antisense 5'-CAC TAG AGA GGT TGT CGA TGC TCT TAA C-3'. All primers were
synthesized by BioServe Biotechnologies (Laurel, MD). RT-PCR products
were 214-bp RNA-derived product and a 279-bp DNA-derived product for
the v-Ha-ras transgene, 578 bp for GATA-1, 300 bp for
-globin, and 212 bp for mouse ß2-microglobulin.
Amplification of the cDNA was carried out for 30 cycles using annealing
temperatures of 50°C for transgene and mouse
ß2-microglobulin primers, 55°C for GATA-1 primers, and
60°C for -globin primers. Denaturing, annealing, and extension
were all done for 30 seconds. RT-PCR products were electrophoresed on
2% agarose gels prepared with 1x TAE (0.04 mol/L Tris acetate, 0.001
mol/L EDTA) and containing 0.5 µg/ml ethidium bromide.
5' Transcript Mapping via S1 Nuclease Protection Assay
32P-labeled DNA used for the S1 nuclease protection
assay was prepared from a pGEM plasmid containing the mouse -globin
promoter (gift from A. Leder, Harvard Medical School). A 4-kb
BamH1 fragment was excised, gel purified, and end-labeled
with [
32P]ATP using polynucleotide kinase (Promega,
Madison, WI). The labeled fragment was then digested with
EcoR1, generating a 1000-bp 32P-end-labeled
fragment, which served as the S1 probe, and 0.1 pmol of probe was
hybridized to 25 µg of total RNA overnight at 42°C in hybridization
buffer (40 mmol/L PIPES, 1 mmol/L EDTA, 0.4 mmol/L NaCl, and 80%
formamide). After incubation, the hybridized samples were digested to
completion with S1 nuclease (Promega) containing salmon testes DNA
(Sigma Chemical Co., St. Louis, MO) as carrier for 1 hour at 37°C,
and then the reaction was terminated with the addition of 1 mol/L
ammonium acetate, 0.5 mol/L EDTA, and 50 mg/ml tRNA (Sigma). The
S1-protected samples were ethanol precipitated, resuspended in
formamide loading buffer, and electrophoresed on an 8% polyacrylamide
sequencing gel containing 7.7 mol/L urea.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six affected mice with complete histopathological and
hematological evaluations were selected to constitute the data set of
this report. These cases occurred over a 16-month time span. Because
the animal colony is used for various studies and not maintained for
tumor incidence statistics, the exact incidence of erythroleukemia in
Tg.AC mice has not been documented, but it is estimated to be <5%.
Erythroleukemia incidence in control heterozygous females used in
chemical testing experiments is approximately 1% (William Eastin,
personal communication). Case histories and necropsy data of the six
diseased mice as well as age- and sex-matched controls are presented in
Table 1
. Although the six cases evaluated
in this study were females, a true gender predisposition could not be
determined due to a preponderance of female Tg.AC mice in the animal
colony due to study design. The age of onset of hepatomegaly and
necropsy ranged from 4 to 9 months. Affected mice were on skin tumor
studies and had been treated topically with various agents, including
TPA, benzene, and acetone or untreated. To date, no association has
been made between occurrence of erythroleukemia and any particular
chemical treatment.
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. Splenomegaly of variable severity was also a
consistent gross finding. The only other gross observation noted with
regularity was one or more enlarged lymph nodes, usually the
submandibular and/or abdominal nodes (Table 1)
.
Abnormalities of the peripheral blood were found in all cases evaluated
(Table 2)
. Automated counts of total
nucleated cells in the peripheral blood were markedly elevated.
Examination of blood smears revealed numerous metarubricytes (average
of 40% of total nucleated cells), erythroblasts, and smudge cells
(Figure 1A)
. In addition to the large
numbers of metarubricytes, anisocytosis and polychromasia of the
non-nucleated red cells were also apparent, and reticulocyte counts
were elevated. Other abnormalities of red blood cell parameters were
decreased RBC, Hgb, and Hct and increased MCV, indicating an anemia.
Numerous mitotic blasts were present in the peripheral blood of some
cases (Figure 1A)
. WBCs corrected for nucleated erythroid and smudge
cells ranged from 0.4 x 103
to 18 x
103/ml. A small subpopulation in the peripheral blood
consisted of cells with horseshoe-shaped nuclei and light blue-gray
cytoplasm but without granules, interpreted to be possibly of the
myelomonocytic lineage.
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. The predominant cell type
was poorly differentiated with a round to folded nucleus containing
coarsely clumped chromatin and a moderate amount of basophilic
cytoplasm. Erythroid differentiation was evidenced by intermingled
metarubricytes and erythrocytes, often forming sinusoidal blood lakes
with more undifferentiated large cells around the periphery. Many of
the infiltrating cells stained positive for the cell proliferation
marker Proliferating Cell Nuclear Antigen (data not shown). Mitoses
were abundant, as well as focal areas of necrosis of both infiltrating
and parenchymal cells.
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In cases with gross involvement of nodes or other tissues (Table 1)
,
microscopic examination revealed leukemic infiltration by sheets of
poorly differentiated cells with focal areas of erythroid
differentiation. An increased number of nucleated cells within blood
vessels was apparent in multiple tissues.
Expression of GATA-1 in Erythroleukemic Tissues and Cytological Preparations
GATA-1 has been shown to be a key regulator of erythroid
differentiation, may be a central regulator of erythroid-specific gene
expression, and has also been shown to modulate all globin gene
expression.16,17
Targeted disruption of mouse GATA-1 as
well as differentiation assays show that an absence of GATA-1 results
in an arrest of erythroid precursors at the proerythroblast
stage.18-20
Given the important role of GATA-1 in
erythropoiesis, peripheral blood smears and paraffin-embedded liver
sections were immunostained for protein expression. Intense reactivity
was present within neoplastic cells in the peripheral blood (Figure 1B)
and infiltrating the hepatic sinusoids (Figure 2B)
. At both sites,
GATA-1 immunolocalization was limited to the nuclei of the more poorly
differentiated cells.
The ras Transgene, GATA-1, and Endogenous -Globin Are
Co-Expressed in Leukemic Tissues
Liver, spleen, and bone marrow from normal
(n = 3) and leukemic (n =
5) animals were examined by RT-PCR for the expression of the
ras transgene. Results of representative cases are shown in
Figure 3A
. Primers directed to the SV40
polyadenylation/splice region of the transgene construct encompass a
65-bp intervening sequence that gives rise to a 279-bp product derived
from contaminating DNA and to a 214-bp product derived from processed
messenger RNA. As can be seen in Figure 3A
, the 279-bp product was
detected in all Tg.AC tissues (lanes 2, 3, and 6 to 11). However, the
RNA-specific 214-bp product was found only in normal Tg.AC bone marrow
(lanes 2 and 3), leukemic spleen (lanes 8 and 10), and leukemic liver
(lanes 9 and 11). Although the normal Tg.AC spleen sample of this study
(lane 6) did not show evidence of transgene expression, other studies
have demonstrated expression in approximately 50% of normal Tg.AC
spleens8
(and unpublished observations). Background strain
FVB/N tissues showed no evidence of either the 279- or 214-bp transgene
product (lanes 4, 5, 12, and 13).
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-globin gene was expressed in
leukemic tissue in view of the fact that the ras transgene
construct contains the -globin promoter sequence. Expression of the
endogenous embryonic -globin gene would indicate the presence of
transcription factors common to both. RT-PCR results of GATA-1 and
endogenous -globin are shown in Figure 3, B and C
, respectively.
Nonleukemic bone marrow and spleen from Tg.AC (lanes 2, 3, and 6) and
parental strain FVB/N (lanes 4, 5, and 12) mice co-expressed GATA-1 and
endogenous -globin. In leukemic liver and spleen from Tg.AC mice
(lanes 8 to 11), both GATA-1 and endogenous -globin are expressed in
addition to the transgene. Normal liver from both Tg.AC (lane 7) and
FVB/N (lane 13) mice show no evidence of transgene (Figure 3A)
, GATA-1
(Figure 3B)
, or -globin (Figure 3C)
.
5' Mapping Assay Demonstrates That the Transgene -Globin
Promoter Is Utilized in Erythroleukemia in the Tg.AC Mouse
Expression of the transgene in cutaneous tumors has been shown to
be dependent on the -globin promoter of the ras transgene
(R.E. Cannon, R.S. Faircloth, J.W. Spalding, C.S. Trempus, K.M. Virgil,
M.C. Humble, G.D. Lacks, J.-L. Klein, R.W. Tennant, submitted for
publication). To determine whether the transgene initiates
transcription from its -globin promoter in erythroleukemia, RNA from
leukemic spleen and liver was analyzed by S1 protection experiments.
The assay, illustrated at the top of Figure 4
, predicts that, if transcription
initiates from the known natural start site of the mouse -globin
promoter of the transgene, a 120-bp 32P-labeled fragment
will be protected from S1 nuclease digestion. Figure 4
shows, as
predicted, that a 120-bp S1-resistant product was produced from cell
lines derived from squamous cell carcinomas taken from Tg.AC mice
(lanes 4 and 5) but not in normal, non-tumor-bearing FVB/N skin (lane
2) or Tg.AC skin (lane 2). An S1-resistant product of identical size
was also found in RNA from the spleen (lane 6) and liver (lane 7) of an
animal diagnosed with erythroleukemia. The leukemic tissues and cell
lines were also found to be expressing the ras transgene by
RT-PCR (data not shown). These data indicate that transgene expression
in erythroleukemias utilizes the -globin promoter sequences for the
start of transcription. The start of transcription identified in these
experiments is identical to that observed in cell lines derived from
cutaneous, transgene-expressing malignancies taken from Tg.AC mice
(Figure 4)
. This suggests that factors necessary for the expression of
the transgene are common to cells within both the cutaneous reservoir
and progenitor blood cells of erythrocytic origin.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several lines of evidence make it clear that the
-globin/v-Ha-ras transgene is involved in leukemogenesis
in Tg.AC mice. First, a similar disease has never been seen in the
parental FVB/N strain of mouse.23
Second, a high level of
transgene expression is invariably present in leukemic tissues, whereas
expression is generally undetectable or low in normal
tissues.9
Furthermore, in situ hybridization
studies with a transgene-specific probe produce a signal localized to
the leukemic infiltrating cells in the liver9
(and data not
shown). No viral particles have been seen in electron microscopic
examination of leukemic cells (data not shown).
Our current working hypothesis is that transgene activation targets accumulation of the activated ras protein to erythroid progenitor cells resulting in abnormal clonal proliferation, as evidenced by the known ability of ras-containing oncogenic viruses to induce a malignant phenotype in erythroid cells, both in vivo and in vitro.24 Most of the erythroleukemias observed to date in the Tg.AC mouse have a mixture of cells at different stages of differentiation. Attempts to culture cells from erythroleukemic tissues were largely unsuccessful because the cells rapidly terminally differentiated in culture, even in the presence of supplemental interleukin-3 (data not shown). These observations are in agreement with our findings of large numbers of metarubricytes in leukemic mice as well as with other studies where it has been shown that Ha-ras infection of hematopoietic cells confers a proliferative advantage to cells of erythroid lineage although not blocking their capacity to differentiate.25,26 Therefore, we conclude that overexpression of the v-Ha-ras transgene results in neoplastic transformation of erythroid progenitor cells and confers a proliferative advantage but not maturation arrest.
It has been shown that the start of transcription in cell lines derived
from cutaneous malignancies in Tg.AC mice is dependent upon the
-globin promoter of the transgene construct (Cannon et al, submitted
for publication). To determine whether transgene expression was also
dependent upon the -globin promoter in erythroleukemia, we undertook
S1 protection experiments of RNA from leukemic spleen and liver tissue.
Because we detected a protected product that is identical to the
endogenous -globin gene promoter, we can conclude that activation of
transgene expression is dependent upon the -globin promoter of the
transgene construct and that initiation of transcription is not due to
the activity of the transcriptional domain of another gene flanking the
transgene integration site in chromosome 11.
In view of the fact that the ras transgene expression is
driven by the -globin promoter, it was of interest to examine both
normal adult hematopoietic organs as well as tissues from animals with
erythroleukemia for the presence of erythroid-specific transcription
factors that function to regulate globin gene expression and that could
thereby act as a transcriptional activator of the transgene. GATA-1, a
member of the GATA family of nuclear DNA-binding transcription factors,
has emerged as an important regulator of normal
erythropoiesis,17,20,22
including erythroid differentiation
and globin gene regulation. Particularly noteworthy in regards to Tg.AC
mice, the transgene -globin promoter contains GATA consensus binding
sites. Therefore, in addition to immunostaining for GATA-1 as a nuclear
marker of erythroid lineage, the expression of this gene was also
examined by RT-PCR as a potential regulator of transgene expression.
Those results demonstrated co-expression of GATA-1 and endogenous
murine -globin in nonleukemic hematopoietic tissues of both Tg.AC
and FVB/N mice. In addition, analogous to its expression in normal
erythropoiesis, GATA-1 was also expressed in erythroleukemic
infiltrates of the liver and spleen, consistent with previous findings
in erythroleukemia cells.27-29
There is also emerging
evidence that suggests that GATA-1 functions as an anti-apoptotic
factor to protect developing erythroid cells.30
The
possibility exists, then, that overexpression of GATA-1 in neoplastic
erythroid cells acts as a survival mechanism in conjunction with
oncogenic ras-driven proliferation in the development of
erythroleukemia in these mice.
The tissue of origin of erythroleukemia in Tg.AC mice is unclear. Liver
origin is suggested by the fact that this organ is the most
consistently involved and severely affected in all cases examined.
However, our selection of cases based on hepatomegaly obviously biased
the phenotype observed toward those mice with marked liver involvement.
Bone marrow or spleen origin is suggested by the RT-PCR detection in
these tissues of all the proposed necessary molecular ingredients for
leukemogenesis in these mice, ie, transgene, GATA-1, and -globin
gene. Elucidation of the histogenesis of erythroleukemia in Tg.AC mice
awaits development of a reproducible model to induce the disease such
that mechanistic studies of its initiation and progression can be
performed. Attempts thus far have been unsuccessful. Induction studies
using blood loss and erythropoietin treatments have been attempted with
the rationale that physiological stimulation of benign erythropoiesis
may ultimately lead to transgene expression and aberrant proliferation
of erythroid cells. Apparently these methods did not inflict all the
genetic or epigenetic events necessary for malignant transformation or
did not target the susceptible progenitor cell population, perhaps one
in which -globin rather than adult hemoglobin is being expressed.
Despite its sporadic spontaneous occurrence, however, erythroleukemia
in the Tg.AC transgenic mouse offers potential as a novel murine model
for the study of normal and abnormal erythropoiesis as a mouse model of
myeloid neoplasia rarely seen in other strains.
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Acknowledgements |
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Footnotes |
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G. Farris's current address: Los Alamos National Laboratory, Los Alamos, NM 87545.
D. Malarkey's current address: College of Veterinary Medicine, Department of Microbiology, Pathology, and Parasitology, North Carolina State University, Raleigh, NC 27695.
Accepted for publication April 24, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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