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Prepublished online as a Blood First Edition Paper on August 22, 2002; DOI 10.1182/blood-2002-06-1732.
NEOPLASIA
From the Department of Haematology, University of Wales
College of Medicine, Cardiff, United Kingdom; and
Department of Pathology, St Jude Children's Research Hospital,
Memphis, TN.
The t(8;21) translocation, which encodes the AML1-ETO fusion
protein (now known as RUNX1-CBF2T1), is one of the most frequent translocations in acute myeloid leukemia, although its role in leukemogenesis is unclear. Here, we report that exogenous expression of
AML1-ETO in human CD34+ cells severely disrupts normal
erythropoiesis, resulting in virtual abrogation of erythroid colony
formation. In contrast, in bulk liquid culture of purified erythroid
cells, we found that while AML1-ETO initially inhibited proliferation
during early (erythropoietin [EPO]-independent) erythropoiesis,
growth inhibition gave way to a sustained EPO-independent expansion of
early erythroid cells that continued for more than 60 days, whereas
control cultures became growth arrested after 10 to 13 days (at the
EPO-dependent stage of development). Phenotypic analysis showed that
although these cells were CD13 The t(8;21)(q22;q22) translocation is one of the
most frequent chromosomal abnormalities associated with acute myeloid
leukemia (AML), occurring in approximately 40% of AML cases that have
M2 morphology in the French-American-British
classification.1-4 One of the targets of this
translocation is the AML1 gene (also referred to as
core-binding factor CBF The mechanisms of how AML1-ETO expression may contribute to
leukemogenesis are not well understood (for reviews, see Downing et
al16 and Downing17). The AML1-ETO chimeric
protein retains the ability to dimerize with the CBF In this study, we have introduced the AML1-ETO fusion gene
into human CD34+ hematopoietic cells by using a retroviral
vector in which the AML1-ETO fusion gene is coexpressed with
green fluorescent protein (GFP). We report here that expression of this
gene profoundly affected the growth of normal human erythroid
progenitors. Although dramatically reducing the capacity of these cells
to generate erythroid colonies, in bulk liquid culture we show that
AML1-ETO was able to inhibit early erythroid development and, at the
same time, allow greatly extended proliferation of these cells. This study demonstrates the capacity of this leukemia-associated gene to
promote self-renewal of normal human cells with a progenitor phenotype.
Generation of retrovirus
Isolation of human CD34+ erythroid cells
expressing AML1-ETO
Cell culture and colony assay Initially, liquid cultures were maintained in growth medium containing 5 ng/mL IL-3, IL-6, and SCF but without erythropoietin (EPO). On day 10, a portion of each culture was supplemented with EPO (R & D Systems) at 1 U/mL and cultured for a further 10 days, whereas the remainder were cultured for a further 3 days in the absence of EPO. To calculate the growth of erythroid cells at each time point indicated, a portion of each culture was analyzed by flow cytometry (see "Data analysis") to determine the proportion of CD13 cells present in the culture. Colony assays were
performed on day 3 by limiting dilution in 96-U plates (0.3 cells/well)
in the same liquid medium containing IL-3, IL-6, SCF, and EPO and incubated at 37°C with 5% CO2. Individual colonies
(> 50 cells) and clusters (> 5 cells) were harvested 14 days
following plating and were scored and analyzed for differentiation (see
"Data analysis").
Analysis of AML1-ETO expression To confirm the expression of the AML1-ETO-transduced gene in human cord blood progenitor cells, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNAs from Kasumi-1-, control-, and AML1-ETO-transduced cells. Total RNA was isolated following guanidinium thiocyanate lysis and RT using an ETO-outer primer (5'-GGTGTAAATGAACTGGTTCTTGGAG-3') was performed according to manufacturer's instructions (Applied Biosystems, Warrington, United Kingdom). cDNA was added to the first-round PCR containing AML1-outer primer (5'-TGACCTCAGGTTTGTCGGTCGAAG-3') and ETO-outer primer. Water was used as a negative control. The PCR was run at 95°C for 4 minutes followed by 30 cycles of 94°C (1 minute); 63°C (1 minute) with a final extension time of 10 minutes at 72°C. First-round product was used as a template in the second round containing AML1-inner primer (5'-AAGCTTCACTCTGACCATCACTGTC-3'), and ETO-inner primer (5'-CATT GTTGGAGGAGTCAGCCTAGA-3'). The second-round product from samples containing the AML-ETO transcript was 181 bp in length. The quality of RNA was assessed by amplifying the ABL housekeeping gene in a single-round RT-PCR using random hexamers. The ABL product was observed at 183 bp. Each PCR product was run on a 2% agarose gel containing ethidium bromide and observed under UV light. AML1-ETO RNA was identified by comparison with molecular weight markers and to the positive control (Kasumi-1).Cell surface phenotype and differentiation analysis At the time points indicated, liquid cultures were analyzed by 4-color immunophenotypic analysis. In addition, cells were centrifuged onto slides for staining with Wright-Giemsa for morphologic examination. Cells were stained with CD13 allophycocyanin (APC; Leinco Technologies, St Louis, MO) in combination with CD36-biotin (Ancell, Bayport, MN) and one of the following PE-labeled antibodies (BD): CD34, CD33, HLA-DR, or glycophorin A (gly A) (Dako, Ely, United Kingdom). Biotinylated CD36 was subsequently labeled with streptavidin-peridinin chlorophyll protein (PerCP). For analysis of individual colonies, 2 × 103 latex beads (BD) were added to each colony-containing well to control for recovery in estimation of the number of cells/colony. Colonies were then carefully aspirated and stained with gly A-PE (BD) and CD11b-QR (Sigma, Poole, Dorset, United Kingdom), as described. All reactions were controlled with the appropriate isotype-matched irrelevant antibody. Incubations were carried out at 4°C for 30 minutes in the presence of 0.5% human gamma globulin (Sigma). Reagent concentrations were as recommended by the manufacturer. Data acquisition and analysis are described in "Data analysis."Cell cycle analysis For the analysis of DNA content, day 6 control and AML1-ETO-transduced cultures were washed in PBS and fixed in 70% ethanol for 30 minutes on ice followed by overnight incubation at 20°C. Fixed cells were washed free of alcohol, resuspended in PBS
containing 40 µg/mL propidium iodide (Molecular Probes, PoortGebouw,
The Netherlands) and 100 µg/mL RNAse type I-AS (Sigma), and
incubated at 37°C for 30 minutes. Cell cycle distributions were
immediately evaluated by flow cytometry (see "Data analysis") and
assessed using Cylchred (T.H., Department of Haematology, University of Wales College of Medicine, Cardiff, United Kingdom).
PKH26 staining On day 6, 5 × 104 cells from each culture were stained with the fluorescent PKH26 dye, according to the manufacturer's instructions (Sigma). Subsequent to PKH26 labeling (on days 6, 8, 10, and 13), the cells were labeled with CD13-APC (see "Cell surface phenotype and differentiation analysis") and analyzed by flow cytometry. The cell division history of CD13 cells was based on the fact that with each cell
division the fluorescence intensity of PKH26 is reduced by one
half.32
Data analysis Flow cytometric data were acquired using a FACSCalibur cytometer (BD). Acquired flow cytometric data were analyzed using WinMDI (Joe Trotter, Pharmingen, San Diego, CA). The threshold for GFP positivity was determined from the autofluorescence of identically treated mock-transduced cultures; for antibody-labeled cells, this was determined from control stained cells (in each case, set to 95% of controls). In the case of colony analysis, background staining/autofluorescence was taken as the maximum observed in 6 randomly selected mock-transduced control colonies. Debris and ejected nuclei were excluded from all analyses on the basis of light scatter. Committed myeloid cells were excluded from all analyses based on CD13 positivity. In the analysis of DNA content, doublets were excluded on the basis of pulse width, as were particles with a DNA content of less than 10% of 2N.33 Significance of difference was tested using the Student t test. Minitab software version 12.0 (Minitab, State College, PA) was used for all analyses.
Expression of GFP and AML1-ETO in human CD34+ cells To investigate the effects of the AML1-ETO fusion protein in human primary erythroid development, we retrovirally infected human CD34+ progenitor cells with a recombinant retrovirus expressing the AML1-ETO fusion gene in combination with GFP. Following infection (day 3), 4-color analysis demonstrated that these cultures consisted of predominantly CD34+ cells of which approximately 30% were of erythroid phenotype (CD13CD36+/) and of these cells
approximately 14% were GFP+ (Figure
1A). To facilitate the analysis of the
retrovirally transduced erythroid population, we enriched the day 3 cultures for GFP+CD13 cells by FACS.
Subsequent analysis showed that this resulted in cultures that were
more than 90% GFP+ and predominantly of erythroid
phenotype (Figure 1B). As others have previously
reported,30 we did not detect any effect of GFP expression
on erythroid development compared with mock-transduced cells.
To confirm the expression of AML1-ETO in transduced human
cord blood progenitor cells, we performed RT-PCR analysis. As shown in
Figure 2, the AML1-ETO fragment was
clearly detected in cells transduced with this fusion gene while absent
in cells transduced with GFP alone. ABL amplification was used as an
internal control and RNA from the Kasumi-1 cell line was used as a
positive control for AML1-ETO detection.34
AML1-ETO suppresses erythroid colony formation Having generated a purified population of retrovirally transduced primitive erythroid cells (predominantly GFP+CD34+CD13) equivalent to a
late erythroid burst-forming unit (BFU-E) population,35 we
set out to determine the effects of AML1-ETO expression on erythropoiesis. First, we assessed the erythroid colony-forming capacity of these cells in the presence of IL-3, IL-6, SCF, and EPO. To
facilitate subsequent analysis of these colonies, the assays were
performed by limiting dilution in liquid medium in 96-well plates.
After 14 days, individual colonies were harvested and double-labeled
with antibodies to gly A and CD11b to distinguish any myeloid colonies.
Under these conditions, expression of AML1-ETO significantly suppressed
erythroid colony formation by 83% when compared with the control
(Table 1); in each case more than 90% of
colonies analyzed were gly A+CD11b,
confirming the identity of the sorted population. As part of the
analysis we also estimated the number of cells constituting each colony
(see "Materials and methods"). We found that expression of AML1-ETO
resulted in a significant decrease in the average number of cells
constituting each colony (Table 1). These data show that expression of
the AML1-ETO gene grossly impairs erythroid colony
formation.
Expression of AML1-ETO promotes amplification of early human erythroblasts To establish in more detail the effect of AML1-ETO on human erythropoietic development, we introduced GFP+CD34+CD13 cells into bulk
liquid culture. Erythropoiesis can be considered to occur in 2 distinct
stages: early development, which occurs independently of the
requirement of EPO,36 and late development, which depends
on the presence of this cytokine. We therefore initially cultured these
cells in the presence of IL-3, IL-6, and SCF but in the absence of EPO,
which allowed us to study the effect of AML1-ETO on early
erythropoiesis independently of its effect on late (EPO-dependent)
development.35 Under these conditions, control cultures
proliferated rapidly for 8 to 10 days, following which the cells became
growth arrested at the EPO-dependent boundary. In contrast, we found
that the pattern of growth of cells expressing AML1-ETO was profoundly
altered. During the first few days of culture, the effect of AML1-ETO
was to inhibit erythroid growth (Figure
3A). The average population doubling time
of control-transduced cells (GFP) over the first 3 days following
retroviral transduction was approximately 18 hours, whereas the
expression of AML1-ETO prolonged the doubling time to about 32 hours.
To determine the basis of this increased doubling time, we analyzed the
cell cycle distribution of these cultures. AML1-ETO-transduced
cultures exhibited almost 30% reduction in the proportion of cells in
cycle (S + G2/M) compared with controls, indicating an
inhibition of the G1-to-S phase transition (Figure 3B). At
this point, AML1-ETO expression had no significant effect on the
proportion of cells with a sub-G1 DNA content, indicating
that inhibition of proliferation did not arise as a consequence of an
increase in the frequency of apoptosis. This analysis was supported by
morphologic scoring where the number of cells expressing AML1-ETO
undergoing apoptosis (2.4% ± 1.3%) was not significantly different
from control cultures (3.6% ± 1.3%).
In control cultures, later stages of EPO-independent growth were
characterized by a reduction in proliferative rate as the cells
approached the EPO-dependent stage of development. By day 13, no
further growth of erythroid cells was observed. In contrast, cells
expressing AML1-ETO did not become growth arrested at day 13 (Figure
3A). We observed that AML1-ETO promoted expansion of erythroid
progenitors for many weeks, giving rise to a 1000-fold increase in the
total number of erythroid cells compared with control cultures (Figure
3A). Despite this continued expansion, cells expressing AML1-ETO
retained the characteristic of poor erythroid colony-forming
efficiency, which showed no change from when assayed on day 3 (Table 2). As expected, control
cells showed a marked reduction in colony-forming capacity at day 10. The presence of EPO itself was not inhibitory to the growth of cells
expressing AML1-ETO under colony-forming conditions because in the
absence of this cytokine no colonies were formed. These cells also
remained cytokine dependent both for survival and proliferation, a
characteristic also observed by Okuda et al,21 in
hematopoietic progenitor cells from fetal livers of AML1-ETO knock-in
mice. Morphologically, these cells retained a basophilic cytoplasm with
an area of increased translucence that is characteristic of erythroid
precursors; however, the nuclei of these cells were abnormal,
demonstrating marked lobulation (Figure
4). Such disturbances of erythroblast
nuclear morphology are, however, common in both acute leukemia and
myelodysplasia.37 Phenotypically these cells were
predominantly CD13
Having made these observations, we wished to establish whether this
greatly extended proliferative capacity arose from a subpopulation of
AML1-ETO-expressing cells that escaped the growth inhibitory effects
of this oncogene, or whether all AML1-ETO-expressing cells responded
in a similar manner. To distinguish these possibilities, we labeled day
6 cultures with the red fluorescent membrane dye, PKH26, which
partitions in daughter cells after cell division. Differential
proliferation of a subpopulation of cells would give rise to
heterogeneity in the distribution of fluorescence intensity of the
labeled population. Following labeling with this aliphatic dye, the
cells became brightly fluorescent (Figure
5) with no loss of viability or
proliferative capability when compared with unlabeled cells (data not
shown). During subsequent culture, cell aliquots were removed at the
time intervals indicated and examined for PKH26 staining. During this
period of culture, the fluorescence intensity of both control and
AML1-ETO-transduced populations showed a relative decline, indicating
that these cells were actively dividing. Because beyond day 6 the
inhibition of cell cycle progression by AML1-ETO was greatly
diminished, there was little difference in the relative decrease in
fluorescence intensity between the 2 populations. At the end of a
typical experiment (day 13), the majority of cells were
PKH26dim, with no evidence of a population that were
PKHbright, which would have indicated heterogeneity of
proliferative potential within the culture (Figure 5). These data
provide evidence that the continued proliferative expansion of
AML1-ETO-expressing cells was the property of the majority of cells
expressing this oncogene and not of a subpopulation that escaped the
growth inhibition.
AML1-ETO expression disturbs early erythroid differentiation The above data indicated that AML1-ETO expression was able to promote the expansion of erythroid cells during the EPOindependent stage of development. We next examined whether expression of AML1-ETO also affected the early differentiation of these cells. By following changes in cell surface marker expression in these cultures by 4-color cytometric analysis, it is possible to monitor the differentiated status of these cells throughout early development. Normal EPO-independent development is characterized by loss of the cell surface markers CD34, HLA-DR, and subsequently CD33 and by concomitant up-regulation of CD36 (thrombospondin receptor).38-42 We found that in both control and AML1-ETO-transduced cultures, the expression of CD34 and HLA-DR declined as they underwent differentiation (Figure 6A-B). The loss of expression of these markers was slightly delayed in AML1-ETO-transduced cultures although this could be attributed to the reduced rate of proliferation as noted. Abnormalities in the pattern of CD33 and CD36 expression were more marked. In contrast to control cells, AML1-ETO-expressing cells retained CD33 expression throughout early development (Figure 6C). In the case of CD36, control cells showed a progressive up-regulation of this antigen, whereas cells expressing AML1-ETO showed no significant increase in expression (Figure 6D). Overall these data are consistent with a suppression of early differentiation or an increase in self-renewal imposed by AML1-ETO during early erythroid development.
Addition of EPO partly relieves the differentiation block imposed by AML1-ETO To establish whether these cells retained the capacity to complete their differentiation program we added EPO to day 10 cultures. Over the following 10 days in the presence of EPO, control cultures underwent a further 400-fold expansion after which no net increase in cell number was observed (Figure 7). Cultures expressing AML1-ETO responded to EPO in a similar manner with no evidence of a significant population of cells, which exhibited an extended proliferative capacity. Thus, despite the failure of these cells to proliferate under colony-forming conditions (Table 2), these cells displayed a normal proliferative response in bulk liquid culture. We then examined the differentiated phenotype of these cells over the course of the culture in terms of the up-regulation of gly A and reduction in cell size that accompanies erythroid maturation. In AML1-ETO-expressing cultures there was a similar reduction in cell size when compared with control cells (Figure 8A). In addition, these cells demonstrated up-regulation of CD36 and gly A and rapid down-regulation of CD33 and HLA-DR in a similar manner to control cells (Figure 8B-E, respectively). However, morphologic analysis (Figure 8F) showed a significantly higher percentage of immature erythroblasts in day 17 AML1-ETO-expressing cultures. These results suggest that AML1-ETO-expressing cells underwent a differentiation response to EPO, although their full maturation may have been impaired.
The capacity of AML1-ETO-expressing cells to differentiate is reduced following extended culture in the absence of EPO Because the addition of EPO to day 10 cultures was able to promote the differentiation of cells expressing AML1-ETO, we next addressed the question whether cells cultured for extended periods of time in the absence of EPO were also able to differentiate. We added EPO to AML1-ETO-expressing cells that had been cultured in the absence of this cytokine for 29 days and followed their proliferation and development over the following 3 weeks. EPO had no effect on the proliferation or morphology of the cells compared with cells expressing AML1-ETO that were cultured in the absence of this cytokine. Immunophenotypic analysis showed a variable but generally weak modulation of gly A and CD33 (Figure 9A-E). These data suggest extended culture of AML1-ETO-transduced cells gave rise to cells that were refractory to the differentiating influence of EPO.
To investigate the effect of AML1-ETO expression on normal hematopoietic development, we have devised a model system based on the expression of this fusion gene in normal human cells. Here, we describe the consequences of AML1-ETO expression on erythroid development and demonstrate the capacity of this fusion gene to promote extensive self-renewal. One of the most striking effects of AML1-ETO we observed was a gross inhibition of erythroid colony formation; the few colonies that did form were significantly smaller than control colonies, consistent with a negative effect of AML1-ETO on cell growth under these conditions. This represents the first report of the effect of AML1-ETO on erythroid colony formation of human cells and draws a contrast with transgenic models. Although AML1-ETO disrupts primitive hematopoiesis,21 there have been no reports of inhibition of colony formation when expressed in adult animals.27 These data indicate that human cells may respond differently to mouse cells in this respect, although it has been shown that it is possible to block the differentiation of mouse erythroleukemia (MEL) cells by expression of AML1-ETO.43 Interestingly, our data also show that these cells performed differently when grown in bulk liquid culture; although an antiproliferative effect was also observed in early (EPO-independent) development, the inhibitory effect imposed by AML1-ETO was overcome by day 8 of culture, at which point the proliferation of these cells continued at a similar rate to controls. In light of this, it was surprising that the same cells did not eventually form colonies in vitro (at day 14). Although a matter for speculation, it is conceivable that the harsher conditions of clonal growth may have resulted in a block in proliferation or loss of viability thereby explaining the dramatic reduction in colony-forming efficiency. We examined the basis for the inhibition of growth from a number of perspectives. One possibility was that AML1-ETO expression promoted the apoptotic death of these cells during their development. However, we found no evidence to support this in terms of the frequency of morphologically apoptotic cells or the proportion of cells with a sub G1-DNA content. A second possibility was that AML1-ETO imposed a temporary inhibition of cell cycle progression. This was confirmed by analysis of cell cycle distribution, which indicated an inhibition of cell cycle progression at the G1 phase. These data are supported by a number of studies describing the effects of AML1-ETO on cell cycle progression. For example, in a tetracycline-inducible model, expression of AML1-ETO induced a block of proliferation in U937 cells.44 In addition, Amann et al45 demonstrated that expression of AML1-ETO resulted in an accumulation of MEL and 32D cells in the G1 phase of the cell cycle. Finally, in studies using a dominant-negative inducible AML1 protein, KRAB-AML1-ER, Lou et al46 observed an inhibition of cell cycle with a rapid reduction in endogenous CDK4 mRNA levels. In this latter study, overexpression of exogenous CDK4 overcame the inhibition of G1 progression. Despite the inhibitory effect on proliferation observed in early cultures, AML1-ETO expression promoted a massive amplification of early erythroblasts compared with control cells. Labeling of these cells with the "tracker dye" PKH26 suggested that the extended proliferative potential of these cells did not arise due to outgrowth of a subpopulation of cells. From our data, it would appear that expression of AML1-ETO was able to confer this property to the bulk of the transduced population. As might be expected, the antiproliferative effect seen in the EPO-independent stage of growth was combined with developmental changes. This was observed both in the context of morphology and immunophenotype. During EPO-independent development, phenotypic analysis of AML1-ETO-expressing cells suggested that it stalled differentiation following loss of CD34 and HLA-DR but preceding loss of CD33 and up-regulation of CD36. Evidence indicates that transcription of the CD36 gene is dependent on binding of AML1 to its promoter,47 suggesting that the failure to up-regulate this antigen may be partly due to the dominant-negative effect of AML1-ETO on the transcriptional activity of AML1. Morphologic analysis demonstrated that even though these cells appeared to have primitive erythroid morphology, there were some nuclear abnormalities, which became more apparent as the culture progressed. These data suggest that as well as perturbing cell cycle progression AML1-ETO was able to interrupt normal developmental progress and thereby allow a massive amplification of this population. Interestingly, addition of EPO to these cultures was able to partially overcome this developmental suppression and allow near-normal maturation of these cells. However, AML1-ETO-expressing cells cultured for prolonged periods in the absence of this cytokine did not respond to EPO in terms of proliferation or differentiation, suggesting a developmental block had arisen. Our provisional data also indicate that this effect is specific to erythroid progenitors, because we have observed no comparable effects on granulocytic or monocytic development or on myeloid colony formation (A.T., unpublished data, February 2002). Because it is known that erythroid cells from patients with AML M2 express AML1-ETO transcripts,48,49 it is pertinent to consider the possible consequences for such developmental disruption in vivo. In the bone marrow, it is thought that early erythroid progenitors form a reservoir of red cell potential and the availability of EPO (which is the major regulator of red cell production) controls the subsequent development of these cells.50 Therefore, it is conceivable that early erythroblasts expressing AML1-ETO may undergo extensive EPO-independent proliferation and contribute to bone marrow hyperplasia. We also observed that erythroblasts expressing AML1-ETO were markedly dysplastic. Interestingly, t(8;21)+ AML M2 is unusual in the high degree of dysplastic maturation observed and this includes the erythroid lineage.51,52 This feature is also characteristic of fetal liver cells cultured from AML1-ETO "knocked-in" mice21 and of preleukemia with t(8;21), which probably represents an early presentation of AML-M2.53 We should add that given the nature of the nuclear abnormalities we have observed, it is possible that erythroid-lineage dysplasia has to some extent been misclassified as granulocyte dysplasia. Together, the above observations strongly suggest that disrupted erythroid differentiation is associated with the pathogenesis of t(8;21) disease. The proliferation of early progenitor cells without detectable changes in their differentiated status implies a capacity of AML1-ETO to promote self-renewal. This view supports Okuda et al,21 in which fetal liver cells from AML1-ETO knocked-in mice demonstrated dysplastic maturation and could be passed indefinitely in cultures, suggesting that AML1-ETO altered the balance of self-renewal and maturation of hematopoietic stem cells. Further evidence comes from Mulloy et al28 who, using a similar system to that described here, demonstrated that AML1-ETO expression in human CD34+ cells enhanced the self-renewal of stem cells in a cobblestone area-forming cell assay. Despite the capacity to promote self-renewal, expression of AML1-ETO alone appears to be insufficient for leukemic transformation. Induction of AML1-ETO expression in adult mice throughout their normal life span failed to give rise to leukemia,27 though these animals did display abnormal maturation and proliferation of progenitor cells. In a separate study, Yuan et al29 demonstrated that mice expressing AML1-ETO under the control of a myeloid-specific promoter from the human MRP8 gene did not develop leukemia even when AML1-ETO-expressing cells were transplanted into syngeneic recipients. However, after treatment of these newborn mice with a strong DNA-alkylating mutagen, N-ethyl-N-nitrosourea, the majority of these mice developed leukemia. More recently, Higuchi et al26 demonstrated in a mouse model that activation of a conditional AML1-ETO knock-in allele by cre-mediated recombination resulted in an enhanced replating efficiency of myeloid progenitors and that expression of AML1-ETO was not sufficient to induce leukemia. However, many features of AML were mimicked when cooperating mutations were induced in these mice. The mechanism by which AML1-ETO may promote self-renewal may arise from
deregulation of transcription in hematopoietic cells. Data increasingly
suggest that AML1-ETO disturbs one or more of the transcription factors
or partner proteins involved in hematopoietic development. AML1
interacts with a number of proteins such as p300/CBP, ets, and
C/EBP In summary, despite the ability of AML1-ETO to inhibit colony growth, in bulk liquid culture it was able to promote extensive self-renewal of erythroid progenitors. During the early stages of this amplification of progenitors, an almost normal differentiation response could be induced by the addition of EPO; however, after prolonged culture, these cells became refractory to this cytokine, indicating that either directly or indirectly, expression of AML1-ETO may also gave rise to a population that had lost the capacity to differentiate. These data provide a clear demonstration of the capacity of this fusion gene to promote self-renewal of progenitor cells and may provide a tractable model for determining the mechanism by which AML1-ETO influences self-renewal in normal human cells.
Submitted June 12, 2002; accepted August 12, 2002.
Prepublished online as Blood First Edition Paper, August 22, 2002; DOI 10.1182/blood-2002-06-1732.
Supported by the Leukaemia Research Fund of Great Britain.
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: Alex Tonks, Department of Haematology, University of Wales College of Medicine, Cardiff, CF14 4XN, United Kingdom; e-mail: tonksa{at}cf.ac.uk.
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Top
Abstract
Introduction
, CBFA2,
PEBP2
. The
AML1 gene contains a conserved region that is highly homologous with the product of the Drosophila melanogaster
segmentation gene, runt. This region is termed the Runt
domain and is required for DNA binding and dimerization with
CBF
P < .01, and 
P < .001. Error bars
represent 1 SD.
