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PLENARY PAPER
From the Departments of Ematologia and
Oncologia-Patologia Sperimentale, University of Florence; Azienda
Ospedaliera Careggi, Florence; Department of Biomorfologia, University
G. D'Annunzio, Chieti; and Servizio Qualità Sicurezza Animale,
Biologia Cellulare, and Biochimica Clinica, Istituto Superiore
Sanità, Rome, Italy.
The phenotype induced by the GATA-1low (neo Idiopathic myelofibrosis (IM), also known as
agnogenic myeloid metaplasia, is a myeloproliferative disorder of
clonal origin of unknown etiopathology characterized by fibrotic
degeneration of the marrow and extensive extramedullary hemopoiesis in
the spleen and liver.1-3 The fact that human IM is often
associated with alterations of megakaryocytopoiesis, such as
thrombocytopenia and accumulation of megakaryocytes (Mk) in the
tissues,1-3 has prompted the hypothesis that the disease
may be the consequence of chronic activation of bone marrow stromal
cells by growth factors, such as transforming growth factor- GATA-1 is a transcription factor that exerts a well-established role in
erythroid and megakaryocytic cell differentiation.13 Definitive proof for its importance in hemopoiesis came from genetic manipulation of the expression of GATA-1 in mice. In fact, mice expressing ectopic levels of the gene show an increased rate of erythroid differentiation,14 whereas those lacking the
gene by targeted mutation die in utero as the consequence of severe anemia.15,16 Instrumental to determining the role of
GATA-1 in megakaryocytopoiesis has been the generation of mutant mice with reduced GATA-1 expression.17,18 These mice are anemic but, unlike the deletion mutants, survive for longer periods during gestation, up to the stage when alterations in megakaryocytopoiesis are
detectable. One of these mutants, the mutant in which the first
enhancer (DNA hypersensitive site 1) has been replaced with a
neo-cassette and which lacks the distal GATA-1 promoter (neo To further confirm the hypothesis that the development of IM may result
from an accumulation of Mk in the hemopoietic tissues, it was
investigated whether the GATA-1low mice would develop the
disease with age. Indeed, early IM traits (presence of reticulotic
fibers in the marrow and spleen) could be detected since 10 months of
age. However, a frank IM picture (anemia, occurrence of tear-drop
poikilocytes and progenitor cells in the blood, fibrotic degeneration
of the marrow and spleen, and active hemopoiesis in the liver)
developed only after 15 months. These results indicate that Mk
accumulation in the marrow, as a consequence of impaired GATA-1
expression, may contribute to the pathogenesis of IM.
Mice
Hematologic blood parameters
Immunohistochemical analysis Samples of liver, spleen, and bone marrow were routinely fixed in 10% (vol/vol) phosphate-buffered formalin; bones were decalcified using standard techniques with acidified EDTA. Fixed tissues were paraffin embedded, and 2.5- to 3-µM sections were prepared for hematoxylin-eosin staining or Gomori reticulum staining.22 For the search of extramedullary foci of hematopoiesis, liver, lung, and kidney samples were serially cut at 100-µM intervals, and at least 25 sections per organ per mouse were examined and their sequences numbered. Immunohistochemical staining with a rat monoclonal antibody directed against murine CD45 (Ly-5; PharMingen, San Diego, CA) was performed on numbered liver sections immediately preceding or following those stained with hematoxylin-eosin in which foci of extramedullary hematopoiesis had been discovered. CD45+ cells were identified with the avidin-biotinylated horseradish peroxidase system (ABC Staining system; Santa Cruz Biotechnology, Santa Cruz, CA) using diaminobenzidine as a substrate.Progenitor cell counts The frequency of progenitor cells (erythroid colony-forming unit [CFU-E]; erythroid burst-forming unit [BFU-E]; granulocyte macrophage-colony-forming unit [CFU-GM]; megakaryocytic pure (p) or mixed (m) colony-forming unit [CFU-Mkp, CFU-Mkm]) was analyzed by culturing low-density mononuclear cells (0.25-1.0 × 105 cells/plate) of selected organs (marrow, spleen, liver) from wild-type and mutant littermates under specific culture conditions. In the case of peripheral blood, 10 µL whole blood was directly plated per milliliter culture, as described.23 Briefly, cultures were established in standard methylcellulose culture (0.9% wt/vol) in the presence of fetal bovine serum (FBS, 30% vol/vol; Hyclone, Logan, UT) and of a combination of recombinant growth factors including rat stem cell factor (SCF, 100 ng/mL; Amgen, Thousand Oaks, CA), mouse interleukin 3 (IL-3, 10 ng/mL; Genetic Institute, Cambridge, MA), and human erythropoietin (EPO, 2 U/mL; Boehringer Mannheim, Mannheim, Germany) for BFU-E growth or human granulocyte-colony stimulating factor (G-CSF, 50 ng/mL; Neupogen; Dompè Biotech, Milan, Italy) and murine granulocyte-macrophage colony stimulating factor (GM-CSF, 50 ng/mL; a gift from Dr K. Kaushansky, University of Washington, Seattle) for CFU-GM growth.24 The growth of CFU-E-derived colonies was stimulated with EPO alone (2 U/mL).25 The growth of CFU-Mk-derived colonies was obtained in collagen-supplemented cultures (MegaCult; Stem Cell Technologies, Vancouver, British Columbia, Canada) stimulated with the combination of recombinant cytokines including murine IL-3 (25 ng/mL), IL-6 (100 ng/mL; Sigma), IL-11 (50 ng/mL; Sigma), and human TPO (50 ng/mL; Amgen). Cultures were incubated at 37°C in a humidified incubator containing 5% CO2 in air and scored after 3 days (for CFU-E-derived colonies) or 7 days (for CFU-GM-, and BFU-E-derived colonies) of culture under an inverted microscope according to standard morphologic criteria. Megakaryocytic colonies and isolated megakaryocytes were counted at day 7 on dehydrated and acetone-fixed collagen cultures stained for acetylcholinesterase (AChE) for 6 hours, followed by methyl-green nuclear counter-staining, as described.26 Megakaryocytic colonies (5 or more cells) were considered pure or mixed when containing only AChE+ cells or both AChE+ and AChEneg cells, respectively.Flow cytometry analysis and megakaryocyte purification Bone marrow cells were flushed from femurs in Ca++- and Mg++-free phosphate-buffered saline (PBS) supplemented with 1% (vol/vol) bovine serum albumin, 2 mM EDTA, and 0.01% NaN3 and were labeled on ice with the erythroid-specific phycoerythrin (PE)-conjugated Ter-119 (Ly-76) (PharMingen) monoclonal antibody and the megakaryocyte-specific fluorescein isothiocyanate (FITC)-conjugated 4A5 antibody as described.27 Cells incubated with the corresponding irrelevant isotype-matched antibodies were used for gating nonspecific fluorescence, and dead cells were excluded by propidium iodide staining (5 µg/mL; Sigma, St Louis, MO). Megakaryocytes were purified from the spleen as described.27 Briefly, monocellular spleen suspensions were prepared by cutting the spleens into small fragments in 5 mL Ca++- and Mg++-free PBS containing 10% (vol/vol) FBS, 5 mM EDTA, and 2 mM theophylline and by passing the fragments through progressively smaller needles. Spleen light-density mononuclear cells were isolated by centrifugation over Lympholyte-M ( = 1.0875 g/mL; Cedarlane Lab, Hornby, British Columbia, Canada)
at 800g for 20 minutes at room temperature and washed twice
in Ca++- and Mg++-free PBS, and adherent cells
were first removed by 2 cycles of adherence to plastic at 37°C for 1 hour. The nonadherent fraction was then enriched for megakaryocytic
cells by immunomagnetic selection: briefly, cells were incubated with
FITC-conjugated 4A5 rat monoclonal antibody (approximately equal to 1 µg/106 cells) for 30 minutes on ice, washed twice with
PBS containing 0.5% (wt/vol) bovine serum albumin and 2 mM EDTA, and
suspended in 80 µL buffer and 20 µL MACS microbeads conjugated with
a monoclonal mouse antifluorescein antibody (Miltenyi Biotech GmbH,
Bergisch Gladbach, Germany). The cell suspension was washed twice with labeling medium and loaded on a MS+/RS+
MiniMacs column placed in a MACS separator; 4A5-negative cells were
recovered in the effluent fraction, whereas 4A5-positive cells were
flushed out of the column. The purity of the sorted fractions was
evaluated by reanalysis of the fluorescent cells with the cytometer.
Cell aliquots concurrently incubated with irrelevant isotype-matched
antibodies were included in each analysis as a negative control.
RNA isolation and semiquantitative RT-PCR analysis Total RNA from femurs was prepared by freezing the bones, carefully cleaned from the soft tissues, in liquid nitrogen and then grinding them with mortar and pestle directly into a commercial guanidine thiocyanate-phenol solution (Trizol; Gibco BRL, Paisley, United Kingdom). Total RNA (1 µg) was reverse transcribed at 42°C for 30 minutes in 20 µL 10 mM Tris-HCl, pH 8.3, containing 5 mM MgCl2, 1 U RNAse inhibitor, 2.5 U Moloney murine leukemia virus reverse-transcriptase, and 2.5 µM random hexamers (all from Perkin-Elmer, NJ). Gene expression was analyzed by amplifying reverse-transcribed cDNA (2.5 µL) in the presence of the specific sense and antisense primers (100 nM each) described in Table 1. The reaction was performed in 100 µL of 10 mM Tris-HCl, pH 8.3, containing 2 mM MgCl2, 200 µM each dNTP, 0.1 µCi of [ 32P]dCTP (specific
activity, 3000 Ci/mmol [110 TBq/mmol]; Amersham Italia,
Cologno Monzese, Italy), and 2 U AmpliTaq DNA polymerase. Primers
specific for  2-microglobulin (50 nM each)
were added to each amplification after the first 10 cycles as a control
for the amount of cDNA used in the reaction.33 PCR
conditions were as follows: 60 seconds at 95°C, 60 seconds at 60°C,
and 60 seconds at 72°C. All reactions were performed using a GeneAmp
9700 Perkin-Elmer thermocycler and were analyzed in the linear range of
amplification defined by preliminary experiments to be between 20 and
35 cycles for osteocalcin, TGF-1,
PDGF, FGF-A, and VEGF and 20 to 30 cycles for 2-microglobulin. Positive (RNA
from adult marrow) and negative (mock cDNA) controls were included in
each experiment. Aliquots (20 µL) were removed from the PCR mixture
after amplification at different cycle numbers, and the amplified bands
were separated by electrophoresis on 4% polyacrylamide gel. Gels were
dried using a Bio-Rad apparatus (Hercules, CA) and were exposed to
Hyperfilm-MP (Amersham) for 2 hours at  70°C. All procedures were
performed according to standard protocols.34
Statistical analysis Statistical analysis of the data was performed by analysis of variance using Origin 3.5 software for Windows (Microcal Software, Northampton, MA). The probability of mice to survive over time was calculated by the Kaplan-Meier method with censored data using the SigmaStat 2.0 program for Windows (SPSS, Erkrath, Germany).23 Animals were censored from the survival curve when killed for experimental purposes. Survival probability curves obtained for the GATA-1low and the W/Wv mice were compared using the Mann-Whitney rank sum test.
Changes in the hematologic parameters of GATA-1low mice with age Blood values in progressively older GATA-1low and wild-type littermates are summarized in Table 2. Mice were divided into 3 age groups (4-8, 8-12, and 15-20 months of age) for convenience. In the normal littermates, small but not statistically significant variations in the blood cell counts occurred during the aging of the animals (Table 2). The Hct (50.1 ± 3.9 vs 45.3 ± 3.1 at 4-8 vs 15-20 months of age, respectively) and the white cell counts (8.1 ± 4.3 vs 6.7 ± 2.3) slightly decreased with age, whereas the platelet counts slightly increased (728 ± 135 vs 1060 ± 189). In contrast, age-matched GATA-1low animals underwent significant changes of hematologic parameters with age. In particular, the number of circulating red cells decreased significantly at 15 to 20 months of age (Hct 47.2 ± 2.3 vs 30.0 ± 3.9; P < .05) (Table 2) when tear-drop poikilocytes (Figure 1) and erythrocytes with Howell-Jolly bodies and polychromatophilia were detectable in the blood. The frequency of Howell-Jolly body-positive erythrocytes increased from less than 0.1% at 12 months to 1% to 2% at 15 months of age. Although the total number of circulating white cells did not significantly change with age, differential counts revealed changes in relative cell distributions. There was a progressive increase in the percentages of neutrophils, which became statistically significant at 15 to 20 months, and a corresponding decrease in the percentages of lymphocytes. Furthermore, immature myeloid cells were released in the circulation of the mutant mice starting from 8 months of age (Table 2). No variations were observed during aging of the mutant mice in terms of platelet counts that remained significantly lower than normal all through the lifespan (Table 2). The morphology of the circulating platelets remained abnormally large and dysplastic (Figure 1).
In the blood of the wild-type animals, the number of circulating
progenitor cells did not significantly change with age but remained at
the lower limit of detection in all the experimental groups (fewer than
10 CFC/10 µL; Table 3). In contrast,
the number of progenitor cells (of all types) circulating in the blood
from the GATA-1low mice increased by 2- to 10-fold at 8 to
12 months and remained higher than normal in older mice (15-20 months).
Larger changes were observed for CFU-E, detected at all ages in the
blood from the mutants, and reached a peak concentration as high as
114 ± 13 CFU-E per 10 µL blood at 8 to 12 months of age (Table 3).
Development of myelofibrosis in the GATA-1low mice with age Myelofibrotic degeneration of the marrow and spleen of the GATA-1low mice with age is documented in Figures 2 and 3, respectively. Hematoxylin and eosin examination of bone marrow sections from 1-month-old mutants shows the great prevalence of Mk in the marrow without any sign of alterations detectable in the intracellular space by Gomori staining (Figure 2A). As shown by FACS analysis (Figure 2D), the marrow of 1-month-old GATA-1low animals contains approximately 7 times more Mk cells than the marrow from their normal littermates (8.6% vs 1.3%, respectively).
Starting from 12 months of age, there was a progressive reduction of the space available in the marrow for hemopoietic cells because of the simultaneous increase of bone trabeculae and of intercellular matrix. The massive presence of newly formed bone trabeculae in the femoral cavity led, by 18 months, to a decrease in the space available for hemopoietic cells (see the hematoxylin-eosin staining in the left panels of Figure 2A). In the oldest mice, increased osteogenesis eventually occluded the femoral cavity, as evident in the cross-sections of the femur diaphysis that show the thickness of the cortical region with the interconnecting bone trabeculae occluding the lumen. Progressive accumulation of fibers in the intercellular space of the marrow from the GATA-1low mutants was also documented by Gomori silver staining (Figure 2A, right panels). The fibers changed from fine and diffuse reticulin fibers at 6 months of age to gross collagen fibers from 12 months on and ultimately almost completely filled the femoral cavity. The reduction of the space available in the marrow for hemopoietic cells was associated with a reduction in total marrow cellularity (Figure 2C). Although the cellularity of the marrow from the wild-type mice increased with age, that of the mutant animals, which was slightly but significantly lower than normal already in the 4- to 8-month group, was further reduced by 2-fold in the 15- to 20-month-old group (Figure 2C). Changes were specifically observed in the frequency of progenitor cells in the marrow from the GATA-1low mice with age. In fact, though no consistent change was found to be associated with age in normal mice, a significant (2- to 3-fold) decrease was observed in the frequency of all types of progenitor cells in the GATA-1low mice with age (Table 3), which, if adjusted for the reduction in total marrow cellularity, corresponds to a 4-fold reduction in total number of progenitor cells per femur (Figure 2D). The accumulation of reticulin fibers with age was also documented by
Gomori staining in the GATA-1low spleen (Figure 3A).
Additional proof for the fibrotic degeneration of this organ came from
the fact that the size of the already massive spleen of the mutant mice
further increased with age (Figure 3B), but the total spleen
cellularity No changes were observed in the frequency of progenitor cells in the spleen of the wild-type littermates with age (Table 3), but, in the spleen of the GATA-1low mice, only the frequency of the myeloid progenitors (CFU-GM) remained constant. Those of the erythroid progenitors (BFU-E, CFU-E), though higher than in normal animals, decreased 2- to 3-fold over time. On the other hand, the frequency of megakaryocytic progenitors (CFU-Mkp and CFU-Mkm), which had already been higher than normal in the 4- to 8-month group,21 further increased to 62 ± 6 per 105 cells to 80 ± 15 per 105 cells at 15 to 20 months. However, because of the overall decrease in spleen cellularity (Figure 3B), the total number of progenitor cells of all types in the spleen actually decreased with age. Liver as site of extramedullary hematopoiesis in the old Gata-1low mice As expected, the hematoxylin-eosin staining of fetal liver shows a hemopoietic organ with abundant Mk in its parenchyma (Figure 4A), but no sign of active hemopoiesis was detectable in the liver of the GATA-1low mice after birth (1-12 months of age) (Figure 4A and results not shown). However, by 18 months, foci of hematopoiesis were recognized again within the liver parenchyma of the mutant animals, as confirmed by CD45
immunostaining of the consecutive liver section (Figure 4A, right
panel, inset). These foci were never found in liver sections obtained
from age-matched wild-type mice (results not shown).
Progressive involvement of the liver as a site of active hemopoiesis in the old GATA-1low mice was further confirmed by the analysis of progenitor cells in this organ (Figure 4B). Until 4 to 8 months of age, progenitor cells were detectable in the liver of the mutant mice at low frequencies and were similar to those found in the liver of normal mice of all ages (Kurata et al35 and results not shown). However, starting from 8 to 12 months, significant numbers of progenitor cells were detected in the liver of the mutants (0.35% of all the cells; Figure 4B). The frequency of these cells further increased (0.8% of all the liver cells) in the 15- to 20-month-old mice. CFU-E were among the most frequent types of progenitor cells in the liver of old animals. Foci of hematopoiesis were not detectable in sections of lungs and kidneys of the mutant or normal littermates at any age (results not shown). RT-PCR of the expression of TGF- , PDGF, and VEGF were amplified at
detectable levels only at the highest PCR cycles (Figure
5A). On the other lane, cDNA fragments
for osteocalcin were never amplified, even when the reaction was
extended up to 40 cycles (Figure 5A and results not shown). Our results
confirm that these genes are expressed at low levels by preparations of unfractionated bone and marrow cells from adult wild-type mice. In
contrast, with RNA prepared from GATA-1low femurs, cDNA
fragments for osteocalcin were readily amplified, and amplification of
cDNA fragments for TGF-, PDGF, and VEGF was detectable even after a
few cycles (Figure 5A). These results indicate that all these genes are
expressed at higher levels in the femur of the mutant mice than in that
of the normal ones. Because fragments for FGF-A were barely amplified
at the highest PCR cycles using RNA from normal and
GATA-1low femurs, it is not possible to draw any conclusion
about its relative levels of expression in the GATA-1low
versus the wild-type femur (Figure 5).
To clarify whether the high expression of growth factors known to be
produced by Mk in the GATA-1low femur resulted from its
increased Mk content (Shivdasani et al18, Vannucchi et al21, and Figure 2A) or from
altered gene expression by the mutated cells, Mk were purified (greater
than 90%; see the FACS dot plots in Figure 5B) with standard
immuno-affinity techniques from the spleens of normal and mutant
littermates, and the RNA extracted from them was used to analyze the
levels of expression of TGF- Survival of the GATA-1low mice with age Kaplan-Meier analysis of the probability of survival of the GATA-1low mice with age is shown in Figure 6. Mice killed for experimental purposes were censored from the analysis. It is difficult to calculate the percentage of survival of the GATA-1low mice at birth because only 30% of the pregnancies induced (as established by the presence of the vaginal plug 24-48 hours after mating) resulted in viable pups. Once the pups were born, however, they were highly vital. Only 9 of 178 GATA-1low mutants born alive died within the first 15 days of life (Figure 6). Few mice died from 15 days to 13 months. Most of the deaths (10 of 25 natural deaths recorded) occurred between 14 and 20 months of age, and only 9 animals were alive after 20 months. Because normal littermates in numbers sufficient for survival studies were unavailable, the probability for survival of the mutant mice was compared with that of mice harboring the W/Wv mutation, a mild genetic defect that, as the GATA-1low mutation, causes anemia and mast cell deficiency (Nocka et al36 and manuscript in preparation). GATA-1low mice had a probability of survival over time significantly higher than that of the W/Wv mice (50% of mice dead at 20 vs 14 months of age, respectively; P < .001 by rank sum test) (Figure 6).
This paper describes that mice harboring the GATA-1low mutation, unlike their normal littermates, develop at 15 months of age an IM-like disease characterized by the presence of (1) anemia (Table 2), tear-drop poikilocytes (Figure 1), and progenitor cells (Table 3) in the blood; (2) collagen fibers in the marrow (Figure 2) and in the spleen (Figure 3); and (3) hemopoietic foci in the liver (Figure 4). The phenotype was expressed by hemizygous males and homozygous females, with an apparent penetrance of 100%. These results suggest that development of the disease in these mice may be the direct consequence of the GATA-1low mutation. Although the GATA-1 gene has been identified since 1989 as a pivotal gene in erythroid differentiation,37 genetic GATA-1 alterations have not been found associated with human abnormalities for a long time. In fact, the extreme severity of the phenotype expressed by GATA-1 mutations experimentally induced in mice had suggested that alterations of this gene, possibly occurring in humans, would go undetected because of their lethality. Most recently, GATA-1 mutations altering either the FOG-binding or the DNA-binding domain of the protein have been described to be associated with human inherited diseases characterized by alterations in the megakaryocytic and the erythroid differentiation pathways, such as dyserythropoietic anemia,38 and X-linked thrombocytopenia,39,40 and thalassemia.41 IM is a clonal disease that can be experimentally transmitted in mice10 and cured in mice9 and humans42 by bone marrow transplantation. It is associated with Mk accumulation in the marrow and with thrombocytopenia and anemia in the blood. These facts suggest that IM may be caused by gene defects that impair the stem cell ability to differentiate along the Mk and the erythroid pathways. TPO and its receptor, Mpl, the 2 genes most likely to cause defective Mk differentiation,6 have not been found altered in 14 IM patients carefully analyzed for this purpose.12 Therefore, the search for the genetic defect involved in the development of IM is still open. We suggest that, at least in some patients, human IM may result from altered regulation of GATA-1 gene expression or from other mutations in the GATA-1 functional pathway because IM, as do the human genetic diseases associated with GATA-1 mutations described so far,38-41 presents defective differentiation of the megakaryocytic and the erythroid pathways, because the slightly higher incidence of IM in males than in females (0.73 and 0.40 new diagnoses every year per 100 000 males and females, respectively43) indicates that its cause may be partially X-linked, and because of the phenotype of the GATA-1low mice as described in this paper. Current experiments are under way to verify this hypothesis. Several animal models have been developed in recent years for the study of IM. The major difference found between the IM traits expressed by the GATA-1low mice and those expressed by other models for the disease is represented by the rate of the progression toward its final exitus that was slow in the GATA-1low mice but rapid in the other models. In fact, wild-type mice that received transplants of bone marrow cells infected with a TPO-containing retrovirus developed a fatal IM syndrome within 2.5 to 3 months, and the final exitus was recorded approximately 7 months from transplantation.9,10 Furthermore, mice transgenic for the TPO gene survived only shortly after birth.7 In contrast, the GATA-1low mice manifested their first IM symptoms (reticulin fibers in the marrow and in the spleen [Figures 2, 3] and alterations in the frequencies of progenitor cells in the various organs [Table 3; Figure 4]) at 8 to 12 months of age, whereas a clear picture of IM was not observed until 15 months (Figures 1-3). However, few of the GATA-1low mice survived more than 2 to 4 months after the first sign that the disease had became manifested (Figure 6). The slow appearance of the first IM traits followed by a rapid final exitus has strong analogies with the human disease. In fact, human IM is usually diagnosed in the second half of life, but, when the first sign of the disease becomes manifested, the final exitus occurs within 4.5 years.3 In contrast with human IM that may evolve into acute leukemia, leukemic cells and growth factor-independent hemopoietic progenitors were never detected in tissues from GATA-1low mice (Vannucchi et al21 and data not shown). However, the transformation of an abnormally proliferating stem-progenitor cell pool represents the final step of a disease progression that may not have the time to occur within the 2-year lifespan of a mouse. Serial transplantation in congenic mice will be required to clarify whether the GATA-1low stem cell pool will undergo transformation with time. GATA-1low mice express in their marrows and spleens 10 times more Mk cells than normal tissues at 1 month of age
(McDevitt et al,17,20 Shivdasani et
al,18 and Figure 2), but they manifest the first
signs of fibrosis 11 months later. In contrast, TPO over-producing mice
develop the disease 2 to 3 months after their marrow Mk content
increases by 2-fold.6-9 To clarify this difference, we
compared by semiquantitative RT-PCR the expression levels of genes
encoding growth factors associated with the development of IM by the
femur and by Mk cells of GATA-1low mice and of their normal
littermates. Osteocalcin, TGF- In conclusion, the GATA-1low mutants represent a new animal model for human IM, and the observed association between poor capacity of the mutated Mk cells to produce growth factors with a low morbidity of the disease may provide insights into possible treatment strategies for humans.
We thank Dr Stuart Orkin for providing the GATA-1low mouse model and for continuous support and critical reading of the manuscript, and we thank Prof Giuliano D'Agnolo for encouragement. We also thank Luca Marsilli for assistance with the breeding program, Andrea Beni for help in cell cultures, and Eugenio Torre for technical assistance.
Submitted November 20, 2001; accepted April 10, 2002.
Prepublished online as Blood First Edition Paper, May 17, 2002; DOI 10.1182/blood-2001-11-0006.
Supported by Ministero per la Ricerca Scientifica e Tecnologica, Progetti di Ricerca di Interesse Nazionale 2000, 2001, and 2002; MURST (PRIN 2000, grant MM06103241); grant E1172 from the Telethon Foundation, Fondi Ricerca Corrente, Ministry of Health and institutional funds from Istituto Superiore di Sanità, Rome, Italy; Associazione Italiana per le Leucemie "30 ore," Florence and by a donation from Famiglia Cecchi. L.B. and C.C. are recipients of fellowships from Fondazione Italiana Ricerca sul Cancro, Milan, Italy.
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.
Reprints: Anna Rita Migliaccio, Clinical Biochemistry, Istituto Superiore Sanità, Viale Regina Elena 299, 00161 Rome, Italy; e-mail: migliar{at}iss.it.
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H. Chagraoui, M. Tulliez, T. Smayra, E. Komura, S. Giraudier, T. Yun, N. Lassau, W. Vainchenker, and F. Wendling Stimulation of osteoprotegerin production is responsible for osteosclerosis in mice overexpressing TPO Blood, April 15, 2003; 101(8): 2983 - 2989. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
HS)
mutation is here further characterized by analyzing the hemopoietic
system during the aging (up to 20 months) of a GATA-1low
colony (135 mutants and 40 normal littermates). Mutants expressed normal hematocrit values (Hct = 45.9 ± 4.0) until 12 months but became anemic from 15 months on (Hct = 30.9 ± 3.9;
P < .05). Anemia was associated with several markers of
myelofibrosis such as the presence of tear-drop poikilocytes and
progenitor cells in the blood, collagen fibers in the marrow and in the
spleen, and hemopoietic foci in the liver. Semiquantitative reverse
transcription-polymerase chain reaction showed that growth factor
genes implicated in the development of myelofibrosis (such as
osteocalcin, transforming growth factor-
though remaining significantly higher than that of the
spleen from the normal mice
