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PLENARY PAPER
From the Department of Molecular Pathology, University
of California San Diego School of Medicine, La Jolla.
The molecular pathways of normal myeloid differentiation, as well
as the mechanisms by which oncogenes disrupt this process, remain
poorly understood. A major limitation in approaching this problem has
been the lack of suitable cell lines that exhibit normal, terminal, and
synchronous differentiation in the absence of endogenous oncoproteins
and in response to physiologic cytokines, and whose differentiation can
be arrested by ectopically expressed human oncoproteins. This report
describes clonal, granulocyte-macrophage colony-stimulating
factor-dependent myeloid cell lines that exhibit these properties. The
cell lines were established by conditional immortalization of primary
murine marrow progenitors with an estrogen-regulated E2a/Pbx1-estrogen
receptor fusion protein. Clones were identified that proliferated as
immortalized blasts in the presence of estrogen, and that exhibited
granulocytic, monocytic, or bipotential (granulocytic and monocytic)
differentiation on estrogen withdrawal. Differentiation was normal and
terminal as evidenced by morphology, cell surface markers, gene
expression, and functional assays. The differentiation of the cells
could be arrested by heterologous oncoproteins including AML1/ETO,
PML/RAR In normal hematopoietic progenitors, a program of
specific gene expression orchestrates commitment and differentiation to mature cells of multiple different lineages. In acute leukemias, however, oncoproteins interfere with this genetic program, resulting in
the unregulated proliferation of cells that no longer retain the
capacity to differentiate normally.1 This is particularly evident in acute myeloid leukemias (AMLs), and many known myeloid oncoproteins can block the differentiation of normal progenitors cultured in vitro in granulocyte-macrophage colony-stimulating factor
(GM-CSF) or interleukin-3 (IL-3). However, neither the genetic events
that underlie normal myeloid differentiation nor the mechanism through
which leukemic oncoproteins interfere with execution of this program
are well understood.
Primary cells and myeloid cell lines offer useful, but limited, models
to approach these questions. Although primary marrow progenitors
demonstrate normal granulocytic and monocytic differentiation in IL-3
or GM-CSF, one is limited by the scarcity of cells, the difficulty in
isolating homogenous populations, and the inability to verify
expression of nontransforming oncoproteins. Useful myeloid cell lines
that demonstrate inducible differentiation in response to changes in
cytokines include FDCPmixA4 (GM-CSF + granulocyte colony-stimulating factor [G-CSF] + macrophage colony-stimulating factor [M-CSF]),2 32Dcl3 (G-CSF),3 M1-AML
(IL-6),4 and FDB cells (GM-CSF),5 whereas
those that respond to nonphysiologic stimuli include HL60 (high levels
of retinoic acid [RA], 12-o-tetradecanoylphorbal 13-acetate [TPA],
dimethyl sulfoxide [DMSO]),6,7 EML (GM-CSF and
RA),8 MPRO (RA),9 NB4 (RA),10
and U937 cells (RA, TPA, DMSO, or vitamin
D3).11 Although these lines supply an unlimited number of clonal cells, most are limited by the fact that
they contain undefined genetic changes such that their differentiation is often incomplete, asynchronous, or accompanied by cell death. Because they are already blocked to differentiation in response to
either IL-3 or GM-CSF, it is unclear whether induction by other extrinsic factors proceeds through normal differentiation pathways. Furthermore, myeloid oncoproteins whose action it is to block differentiation induced by IL-3 or GM-CSF cannot be assayed in these
prearrested cell lines.
Therefore, an optimal myeloid cell line model would (1) lack
constitutive expression of interfering oncoproteins, (2) exhibit conditional and terminal differentiation in GM-CSF, and (3) be blocked
in differentiation by common leukemic oncoproteins. Here we describe
such a system, established by a conditional version of E2a/Pbx1, a
fusion oncoprotein that results from the t(1;19) translocation of
pediatric pre-B acute lymphocytic leukemia (ALL),12-14 that blocks myeloid differentiation in murine marrow
culture.15 E2a/Pbx1 was made conditional by inserting the
hormone-binding domain (HBD) of the estrogen receptor (ER) between the
E2a transactivation domains and the Pbx1 DNA-binding
homeodomain.16 The E2a/Pbx1-ER fusion proteins were
estrogen dependent at the level of transactivation and transformation,
and, like wild-type E2a/Pbx1, were capable of arresting the
differentiation of GM-CSF-dependent myeloid progenitors. Progenitor
clones were established which, on removal of estrogen, differentiated
to mature granulocytes, monocytes, or to both types of cells.
Accompanying differentiation, the clones exhibited normal changes in
cell surface markers, gene expression, and cellular function. Having
established and characterized these conditionally immortalized myeloid
progenitor clones, we further analyzed the effect, on differentiation,
of other leukemic oncoproteins such as AML1/ETO, PML/RAR Construction of estrogen-dependent versions of E2a/Pbx1
Cell culture
Analysis of transcriptional activation
Analysis of density-dependent growth The NIH3T3 fibroblasts were infected with helper-free retrovirus encoding wild-type and inducible versions of E2a/Pbx1. Stably expressing cells, as well as cells transduced with empty vector virus, were selected 7 days in 1 mg/mL G418 in the absence of estrogen. Equivalent numbers of cells were plated in triplicate into 60-mm dishes in the presence and absence of 1 µM -estradiol. Half-media changes
were performed every 2 days and the total number of live, adherent
cells was determined after 14 days. Fold density was calculated in
comparison to the number of cells transduced with empty-vector retrovirus.
Immunoblot analysis of E2a/Pbx1 proteins Protein from 5 × 104 cells was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Pall, Ann Arbor, MI). Membranes were blocked in tris-buffered saline-Tween (TBS-T) with 5% nonfat milk, and immunoblotting was performed using monoclonal mouse anti-human E2a (E12/E47) antibody (G193-86, Pharmingen, San Diego, CA). Primary antibody was detected with a horseradish peroxidase (HRP)-linked rabbit antimouse secondary antibody (NEB, Beverly, MA). Bound antibody was detected using SuperSignal West Pico Chemiluminescence (Pierce, Rockford, IL).Retroviral infection of primary murine marrow progenitors We isolated bone marrow mononuclear cells on a Ficoll-Paque gradient (Pharmacia, Piscataway, NJ) following harvest from the femurs and tibia of female Balb/c mice injected intraperitoneally with 5-fluorouracil (150 mg/kg) 5 days prior to harvest. Marrow progenitors were purified by negative selection of lineage-positive cells on a magnetic column with a murine progenitor antibody cocktail (Stemcell Technologies, Vancouver, BC, Canada). Progenitors were prestimulated for 48 hours in Iscoves modified Dulbecco medium (IMDM) containing 15% FBS, 50 ng/mL stem cell factor (SCF), 25 ng/mL IL-3, and 25 ng/mL IL-6.Helper-free retrovirus was prepared by calcium phosphate transfection
(Invitrogen, Carlsbad, CA) of 293T cells with MSCV retroviral constructs and an ecotropic packaging construct. Then, 25 000 marrow
progenitors were infected with 1 mL retroviral supernatant by
spinoculation (2500g, 2 hours, 22°C) in the presence of
Lipofectamine (1:1000, Gibco BRL). Following spinoculation, the cells
were cultured in RPMI media containing GM-CSF and 1 µM Single-cell clones were prepared by limiting dilution.
E2a/Pbx1-mediated conditional
myeloid (ECoM)-GM and ECoM-G clones were established from
both populations immortalized by EP Wright-Giemsa staining, nitroblue tetrazolium reduction assay, nonspecific esterase assay, and phagocytosis assay Wright-Giemsa staining (3 minutes Wright, 9 minutes 20% Giemsa) was performed on cytocentrifuge preparations of cells (Thermo Shandon, Pittsburgh, PA).Activity of NADPH oxidase was assayed by nitroblue tetrazolium (NBT)
reduction. The 2.5 × 105 cells in 200 µL growth media
were added to 800 µL 0.125% NBT (Sigma) in the presence of
2 × 10 Nonspecific esterase (NSE) activity was assayed as previously
described.20 Briefly, cytocentrifuge preparations of cells were fixed in cold phosphate-acetone-formaldehyde and stained with
Phagocytosis was assayed using fluorescein isothiocyanate (FITC)-labeled heat-killed Escherichia coli Bioparticles (Molecular Probes, Eugene, OR). The 107 BioParticles and 106 cells were incubated for 1 hour at 37°C with shaking. Following cytocentrifugation onto Superfrost Plus slides (Fisher, Pittsburgh, PA), the nuclei were counterstained with bisBenzimide (Hoechst 33258, Sigma), and the F-actin counterstained with TRITC-phalloidin (Sigma). Images were captured with a DeltaVision deconvolution microscope system and the data sets were deconvolved and analyzed using SoftWorx software (both from Applied Precision, Issaquah, WA). Flow cytometric analysis The FITC-labeled monoclonal antibodies GR-1(Ly6G) and Mac-1(CD11b) were purchased from Pharmingen and F4/80 from Serotec (Kidlington, Oxford, United Kingdom). Then, 106 cells were labeled 30 minutes at 4°C in phosphate-buffered saline (PBS)/1% FBS/0.1% NaN3, washed, and resuspended in the same buffer with 2 µg/mL propidium iodide. Flow cytometry data were acquired with the program CELLQuest on either a FACScan (Becton Dickinson, San Jose CA) or FACSCalibur bench-top flow cytometer attached to a Macintosh G3 computer. Live cells were gated for analysis by forward and side scatter signals and lack of propidium iodide staining.Northern blot analysis Cells were maintained in 15-cm dishes in the presence or absence of 1 µM-estradiol. Cytoplasmic RNA was purified (RNEasy, Qiagen),
and a portion subjected to polyA selection (Oligotex, Qiagen). Then, 15 µg cytoplasmic RNA or 5 µg polyA-selected RNA was resolved by
formaldehyde-1% agarose gel electrophoresis and the RNA transferred to
a positively charged nylon membrane (GeneScreen Plus, NEN, Boston, MA).
Membranes were dried for 2 hours at 80°C in a vacuum oven prior to
hybridization. The 32PdCTP-labeled DNA probes were prepared
from 100 ng DNA subjected to random-hexamer oligolabeling (Pharmacia).
Hybridization in Ultrahyb (Ambion, Austin, TX) and washing were carried
out at 42°C according to the manufacturer's protocols. In general,
probes for transcription factors were hybridized to blots containing polyadenylated messenger RNA (mRNA), and probes for all other gene
products were hybridized to blots containing total RNA.
Cell-cycle analysis by DNA content Washed cells were resuspended in PBS, fixed by the addition of ice-cold 100% ethanol to a final concentration of 70%, and stored overnight at 4°C. Cells were incubated with RNase A (0.1 mg/mL, 15 minutes, 37°C), followed by propidium iodide staining (50 µg/mL, 30 minutes, 4°C). DNA content (FL2-Area) was measured using the Doublet Discrimination Module (DDM) set on FL2, and MulticycleAV, Version 3.11 (Phoenix Flow Systems, San Diego, CA) was used to calculate cell-cycle phases.Retroviral infection of ECoM-G cells with heterologous oncoproteins Helper-free retrovirus was generated from wild-type E2a/Pbx1, Hoxa7, Hoxa9, Nup98/HoxA9, Hoxb8, AML1/ETO, PML/RAR , and PLZF/RAR constructs cloned into the multiple cloning site of the MSCVneo and
MSCVpac retroviral vectors. ECoM-G cells (250 000) from 3 different
clones were transduced by spinoculation (as described above) and
selected 5 days in G418 (1 mg/mL) or puromycin (1 µg/mL). The cells
were washed and plated in media without estrogen. Only those cells that
were capable of continued and indefinite proliferation in the absence
of estrogen were expanded to generate RNA for Northern analysis.
Estrogen-dependent forms of E2a/Pbx1 are produced by replacement of Pbx1 sequences with the ER HBD Fusion of the ER HBD to the N- or C-terminus of full-length E2a/Pbx1 produced proteins that were estrogen-dependent at the level of transcription, but were unstable (expressed at only 10%-30% the level of wild type), and failed to exhibit estrogen-dependent transformation (Xinyu Fu, unpublished observations, August 1997). Therefore, we replaced Pbx1 sequences N-terminal to the homeodomain, which are dispensable for the biochemical and transforming properties of E2a/Pbx1,18 with the ER HBD (as described in "Materials and methods"), creating EP 578ER and EP623ER proteins (Figure
1A). As shown below, EP578ER and EP623ER are stable, are
constitutively expressed, and demonstrate estrogen-dependent
biochemical and oncogenic functions.
Wild-type E2a/Pbx1 exhibits 2 forms of transactivation that can
be separately assessed on different luciferase reporter
constructs.18 Transactivation through TGATTGAT
motifs is accomplished by both a Hox-partner-dependent mechanism and a
Hox-independent mechanism likely mediated by E2a/Pbx1 dimerization,
whereas transactivation through TGATTTAT motifs is strictly
Hox-dependent. Estrogen responsiveness of EP E2a/Pbx1 induces proliferation and focus-formation in monolayer
cultures of NIH3T3 fibroblasts.22 Stably expressing NIH3T3 cells were established to assess the transforming function of EP EP 578ER (12 of 12 cultures) and EP623ER
(6 of 6) immortalized progenitors as efficiently as E2a/Pbx1, but only in the presence of 1 µM estrogen. Wild-type E2a/Pbx1-, EP578ER-, and EP623ER-immortalized progenitors were phenotypically identical (EP578ER shown in Figure 2A). All cell
lines were dependent on exogenous GM-CSF, which could be substituted
with IL-3. The cells were unresponsive to G-CSF, or to M-CSF,
undergoing apoptotic cell death within 24 hours when cultured in these,
or in the absence of cytokines (data not shown).
Removal of estrogen from polyclonal populations of EP The expression of cell surface markers on one population of
EP Conditionally immortalized myeloid progenitors initiate a transcriptional program of differentiation following withdrawal of estrogen In some models of myeloid differentiation, phenotypic changes are not necessarily accompanied by normal expression of terminal differentiation markers.23 To address this concern, Northern blot analysis was performed on a 9-day time course of RNA samples collected from EP578ER-immortalized cells differentiated in
the absence of estrogen (Figure 3). The
expression of genes encoding master transcriptional regulators, primary
and secondary granule proteins, components of the NADPH oxidase
complex, and other markers of myeloid differentiation was examined
(reviewed by Tenen et al,24 Yamanaka et al,25
Zhang et al,26 and Ward et al27).
In both the presence and absence of estrogen, progenitors demonstrated
stable expression of the Ets-family member PU.1, as well as members of
the CCAAT/enhancer-binding protein (c/EBP The sequential expression of primary and secondary granule genes also
accompanied differentiation. Primary granule genes neutrophil elastase
(NE) and myeloperoxidase (MPO) were rapidly up-regulated following
estrogen withdrawal. This rapid up-regulation is important in the
context of elucidating differentiation arrest by E2a/Pbx1, because it
might allow for the identification, within the NE and MPO promoters, of
transcriptional activators that are not produced, or transcriptional
repressors that are not eliminated, in the presence of active E2a/Pbx1.
Such genetic defects will provide molecular handles to identify the
direct E2a/Pbx1 target genes that mediate differentiation arrest.
Secondary granule genes lactoferrin (LF) and neutrophil gelatinase (NG)
were not expressed in the myeloblasts and were activated at day 2 and
day 7 of differentiation, respectively. Examination of 2 of the
subunits of the NADPH oxidase complex revealed that p47PHOX
was constitutively expressed, whereas gp91PHOX was not
expressed until day 4 of differentiation. The regulated expression of
gp91PHOX is consistent with the timing of robust functional
NADPH oxidase activity as assayed by NBT reduction. The cells also
demonstrated transcriptional up-regulation of genes encoding cell
surface receptors Ly6G (GR-1), CD14 (lipopolysaccharide receptor), and
the G-CSF receptor (G-CSF-R). Ly6G expression paralleled the pattern of GR-1 staining seen by flow cytometric analysis (Figure
4) and the low level of G-CSF-R
expression in progenitors is consistent with their G-CSF
unresponsiveness.
E2a/Pbx1 arrests myeloid differentiation in a DNA binding-dependent manner, possibly in cooperation with a Hox partner, or in larger complexes including other Pbx, Hox, and Meis proteins.32 Although many Hox genes, especially of the A paralog, are implicated in myeloid differentiation,33 no expression of Hoxa5, Hoxa7, or Hoxa9 was detected in progenitors, though each transcript was clearly observed in NIH3T3 fibroblasts (data not shown). Furthermore, expression of Meis1, Meis2, and Meis3 was not detected in the myeloid progenitors, although their transcripts were also easily detected in NIH3T3 fibroblasts (data not shown). Clones of EP 578ER and EP623ER allowed
us to examine whether all progenitors, seemingly identical by morphologic criteria, were committed to identical patterns of differentiation. Populations of progenitors were cloned, clonality was
verified by retroviral integration analysis using Southern blotting
(data not shown), and phenotypes were examined by light microscopy
(Figure 4A) and Wright-Giemsa staining (Figure 4B) 5 days after the
removal of estrogen. Three types of clones exhibiting E2a/Pbx1-mediated
conditional myeloid differentiation were identified: bipotential clones
that differentiated to both granulocytes and monocytes (ECoM-GM),
clones exhibiting restricted granulocytic differentiation (ECoM-G), and
one clone exhibiting restricted monocytic differentiation
(ECoM-M).
Within 4 days, ECoM-G cells differentiated homogeneously to granulocytes, whereas ECoM-M cells required 7 days for quantitative monocytic differentiation. Both ECoM-G and ECoM-M cells acquired functional NADPH oxidase activity (Figure 4C and data not shown, respectively), and the ECoM-M cells up-regulated NSE, a characteristic marker of normal macrophage development (Figure 4C). The ECoM-G cells showed a dramatic increase in GR-1 and Mac-1 staining 7 days after estrogen withdrawal, whereas the intensity of F4/80 staining was significantly reduced (Figure 4D). In contrast, the ECoM-M cells did not express GR-1 (the observed shift is due to increased autofluorescence), but were positive for, and showed increasing staining of, both Mac-1 and F4/80 during monocytic differentiation. Both ECoM-G and ECoM-M cells became functionally phagocytic, as demonstrated by the ability to engulf FITC-labeled E coli BioParticles (Figure 4E). Phenotypic changes following the removal of estrogen were accompanied by reduced proliferation and G1 cell-cycle arrest as evidenced by DNA content analysis. Although both ECoM-G and ECoM-M progenitors in estrogen had a high S-phase fraction (G1 48%/G2 8%/S 44% and G1 35%/G2 13%/S 52%, respectively), the majority of the cells had accumulated in G1 following 7 days of differentiation in the absence of estrogen (G1 96%/G2 2%/S 2% and G1 83%/G2 11%/S 6%, respectively). ECoM-G and ECoM-M cells exhibit lineage-specific and differentiation-specific gene expression The parallel models of granulocyte and monocyte differentiation permitted the examination of lineage-specific gene expression (Figure 5). ECoM-G and ECoM-M cells recapitulated well-established patterns of myeloid gene expression including the down-regulation of c-Myb and c-Myc, and the up-regulation of Egr-1, macrosialin, c-Fos, neutrophil collagenase (NC), c-Fms, and the macrophage scavenger receptor SRA-1. Zinc-finger protein and transcriptional repressor Gfi-1 was down-regulated in both ECoM-G and ECoM-M clones, whereas expression of family member Gfi-1B was down-regulated only in the ECoM-M clone (data not shown), similar to a previous report in which its down-regulation accompanied the cell-cycle arrest and up-regulation of p21 during IL-6-induced monocytic differentiation of M1-AML cells.34 Ear-2 was stably expressed throughout differentiation of both ECoM-G and ECoM-M cells in contrast to a previous report in which it was shown to be down-regulated during the G-CSF-induced granulocytic differentiation in 32Dcl3 cells and hypothesized to bind and inhibit AML1 activity.35 Similarly, Ets family member Fli-1 was also stably expressed. Expression of Fli-1 has been reported in human T-cell, B-cell, and myeloid leukemia cell lines36 and was shown to be critical for hematopoiesis in a Fli-1-deficient mouse.37 Expression of the aldoketo reductase mAKRa, whose function has not yet been elucidated, was restricted to the ECoM-G clone and was stable throughout differentiation in contrast to a previous report that showed decreased expression following the all-trans-RA (atRA)-induced granulocytic differentiation of EML-C1 and MPRO cells.38 Expression of Ets-2 was restricted to the ECoM-G clone, which was unexpected given previous reports of Ets-2 expression in normal and transformed macrophages.39
Overall, the differences in gene expression between ECoM-G and ECoM-M cells demonstrate that E2a/Pbx1 prevents differentiation but not lineage definition, and that these cell lines will be useful models to identify differences between granulopoiesis and monopoiesis. Heterologous oncoproteins arrest the differentiation of ECoM-G cells when E2a/Pbx1 is inactivated Although the ECoM cells are ideal for studying the differentiation block established by E2a/Pbx1, we wished to assay their utility in studying myeloid differentiation arrest by other oncoproteins. We introduced heterologous oncoproteins into ECoM-G clones by retroviral infection, verified expression by immunoblot analysis, and assayed for the ability to re-establish differentiation arrest following estrogen withdrawal. AML1/ETO, PML/RAR,
PLZF/RAR, Hoxa9, Hoxb8, and wild-type E2a/Pbx1 prevented the
granulocytic differentiation of specific clones, permitting their
continued indefinite proliferation in the presence of GM-CSF. The
resultant pattern of gene expression was consistent with
differentiation arrest by the new oncoprotein (Figure
6). Hoxa9, for example, permitted
up-regulation of NE, gp91PHOX, Ly6G, and the G-CSF-R to
levels similar to those of primary marrow immortalized by Hoxa9
alone.40 Similarly, whereas AML1/ETO permitted
up-regulation of gp91PHOX and Ly6G, the expression of NE
and G-CSF-R, whose activation requires AML1, remained low. This pattern
is consistent with the dominant-negative function of AML1/ETO on
AML1-responsive promoters.41,42 ECoM-G clones arrested in
differentiation by PML/RAR or PLZF/RAR showed RA sensitivity not
seen in the parental cells, providing mechanistic evidence of the
function of the second oncoprotein. Although parental cells were
completely unresponsive to treatment with 10 µM atRA, for 4 days,
both the PML/RAR and PLZF/RAR derivatives stopped proliferating
and underwent quantitative granulocytic differentiation
(Figure 7, Table
1). The ECoM-G cell lines can thus be
used for analysis of the biochemical and genetic mechanisms by which
oncoproteins prevent stage-specific myeloid differentiation.
Interestingly, certain ECoM-G clones seemed more receptive than others
to differentiation arrest, whereas the ECoM-M clone was entirely
refractory to all oncoproteins tested. We observed that Hoxa9, Hoxb8,
and AML1/ETO efficiently established differentiation arrest in multiple
ECoM-G clones, whereas PML/RAR Nup98/HoxA9 and Hoxa7 arrest the differentiation of ECoM-G cells when E2a/Pbx1 is inactivated The ECoM-G cells provide a model in which to examine suspected oncoproteins and their effects on granulocytic differentiation. Both Nup98/HoxA9 (Figure 6) and Hoxa7 (data not shown) were capable of arresting ECoM-G differentiation following estrogen withdrawal the first report to demonstrate that either protein can
block in vitro myeloid differentiation. Despite their equivalent levels
of expression, Nup98/HoxA9 was significantly less efficient than either
Hoxa7 or Hoxa9 in arresting differentiation precipitated by estrogen withdrawal. This suggested that the mechanism of differentiation arrest by Nup98/HoxA9 was fundamentally different from that of Hoxa9 or Hoxa7. The small fraction (< 1%) of ECoM-G cells that continued to proliferate expressed a smaller version of the protein by
Western analysis, indicating that some form of rearrangement had
occurred. Nup98/HoxA9, identified as the product of the t(7;11) translocation in human myeloid leukemia,43,44 has also
been shown to cause transformation of NIH3T3 fibroblasts45
and to cause AML in mice.46 The pattern of gene expression
in Nup98/HoxA9-immortalized ECoM-G cells was similar to that in
Hoxa9-immortalized cells but for the lack of gp91PHOX
expression. Hoxa7 was initially shown to be coactivated with Meis1 by
proviral integration in BXH-2 mice,47 and more recently shown to be coactivated with Meis1 in approximately 50% of human AML.48
Factor-independent ECoM cells can differentiate in the absence of GM-CSF The ECoM-G and ECoM-M clones were rendered factor-independent following infection with retrovirus encoding H-RasL61 or Bcr/Ablp190. Removal of estrogen from these cells permitted differentiation as assessed by Wright-Giemsa staining (Table 2). Although the parental ECoM-G cells in the presence of GM-CSF underwent strict granulocytic differentiation (98%), ECoM-G_Ras and ECoM-G_Bcr/Abl cells demonstrated predominant monocytic differentiation (99% and 65%, respectively). In contrast, the differentiation of ECoM-M cells was not morphologically affected by expression of Ras or Bcr/Abl, though mature monocytes expressing Ras were no longer adherent. These morphologic results were supported by flow cytometric analysis. Differentiation of ECoM-G cells expressing Ras or Bcr/Abl was accompanied by increased F4/80 and decreased GR-1 staining, whereas expression of Ras or Bcr/Abl in ECoM-M cells did not significantly alter GR-1, MAC-1, or F4/80 staining (data not shown).
E2a/Pbx1 does not induce proliferation of myeloid progenitors Although E2a/Pbx1 stimulates fibroblast proliferation, its ability to induce myeloid proliferation has not yet been investigated. If E2a/Pbx1 stimulated myeloid proliferation, then expression of BCL-2 should be sufficient to permit proliferation in the absence of GM-CSF or of other exogenous cytokines. Although Bcl-2 prevented apoptosis
of ECoM-G and ECoM-M cells following factor withdrawal, neither cell
line proliferated, indicating that E2a/Pbx1 does not stimulate myeloid
progenitor proliferation (data not shown). This suggests that
oncoproteins inducing proliferation, as well as protection from
apoptosis, are required to complement E2a/Pbx1 function in leukemogenesis.
In this paper we describe estrogen-dependent versions of E2a/Pbx1 that conditionally arrest the differentiation of clonal granulocytic, monocytic, or bipotential GM-CSF-dependent murine marrow progenitors. These ECoM clones provide models in which to (1) dissect the genetic circuitry of normal granulocytic and monocytic development, (2) identify E2a/Pbx1 target genes that contribute to differentiation arrest, (3) identify the mechanism by which other human myeloid oncoproteins interfere with the differentiation process, and (4) investigate the cooperative effects of Bcl-2, Ras, and Bcr/Abl on differentiation in vitro and on leukemogenesis in vivo. Studies of normal myeloid differentiation49 have often relied on limited numbers of highly purified progenitors and sensitive techniques such as reverse transcription-PCR to evaluate gene expression. The ECoM cell lines are an inexhaustible source of clonal myeloid progenitors that, based on transcriptional and functional criteria, recapitulate normal myeloid development as it proceeds in GM-CSF and in the absence of endogenous oncoproteins or physiologically irrelevant inducers of differentiation. Combining the advantages of primary progenitors with synchronous differentiation and the ability to confirm expression of normal and mutant oncoproteins, the ECoM cells are a system in which to dissect the critical transcriptional events that accompany maturation and dictate lineage commitment. The normal myeloid maturation following inactivation of E2a/Pbx1
demonstrates that expression of E2a/Pbx1 alone is sufficient to block
differentiation. The reversibility of transactivation, transformation,
and differentiation arrest suggests that E2a/Pbx1 does not
establish an irreversible pattern of gene expression and, that by
clinically targeting E2a/Pbx1, one might be able to reverse the in vivo
differentiation block of t(1;19) pre-B ALL cells. We have not examined
the in vivo behavior of the ECoM-G or ECoM-M clones though the cells
would presumably differentiate normally in the absence of estrogen, as
they do in vitro. Even if exogenous estrogen were supplied to maintain
the activity of EP The EP The ECoM cells are also useful for identifying the mechanisms through
which diverse oncoproteins arrest myeloid differentiation. To date, we
have shown that the differentiation of ECoM-G clones can be arrested by
Hoxa7, Hoxa9, Hoxb8, AML1/ETO, PML/RAR The observation that ECoM-G cells rearrested in differentiation by
PLZF/RAR The ECoM-G and ECoM-M cells expressing Bcl-2 did not proliferate in the absence of factor, indicating that E2a/Pbx1 alone does not induce myeloid proliferation, and is therefore unlikely to induce pre-B-cell proliferation. Although E2a/Pbx1 transforms NIH3T3 fibroblasts, we suspect that this is through a fibroblast-specific mechanism that does not occur in myeloid or pre-B lineages, and that oncoproteins that stimulate cell division will be necessary to complement the E2a/Pbx1-differentiation block in human pre-B cells. A fibroblast-specific proliferative mechanism would also explain our inability to promote the outgrowth of primary murine pre-B cells by coexpression of E2a/Pbx1 and Bcl-2 (D.B.S., unpublished observation, August 2000), and suggests that oncoproteins that induce both proliferation and protection from apoptosis may cooperate with E2a/Pbx1 in human pre-B-cell leukemia. This is consistent with a number of observations. First, the expression of E2a/Pbx1 is toxic in human pre-B-cell lines though coexpression of Bcl-2 protects against apoptosis.54 Second, T-cell leukemias in E2a/Pbx1 transgenic mice develop, with shortened latency, in cooperation with activated Pim165 or activated Notch expression.66 Third, the factor-independent proliferation of ECoM cells by expression of Ras or Bcr-Abl oncoproteins was consistent with a limited number of observations of RAS activation in ALL67 and of coexpression of E2a/Pbx1 and Bcr/Abl in pre-B ALL.68 Neither Ras nor Bcr/Abl, which mimic the downstream proliferative effects of GM-CSF signaling,69,70 prevented differentiation of the ECoM progenitors, indicating that the myeloid differentiation program was intrinsic to the cells and did not require continued GM-CSF signaling. Two recently reviewed models, the stochastic71 and the deterministic,72 have been proposed to describe the role of hematopoietic cytokines during differentiation. In the stochastic model, cytokines act as survival and proliferative factors, whereas lineage commitment decisions are intrinsic to the cell. The deterministic model suggests that the cytokine environment directs the pattern of gene expression, which determines the fate of the cell along a particular lineage. The differentiation of the ECoM cells in the absence of factor supports the stochastic model, in regard to E2a/Pbx1-immortalized myeloid progenitors, though the initial culture in GM-CSF may still have been required at earlier stages of development to "determine" the subsequent "stochastic" events. The observation in ECoM-G cells that Ras expression can bias predominant granulocytic differentiation toward the monocytic lineage has also been previously demonstrated in FDCP-mix73 as well as HL6074 and K562 cells.75 Although the ECoM cells are similar to murine cell lines derived by Hogg and coworkers using a c-Myb-ER fusion protein, clonal c-Myb-ER cell lines were not derived, nor were the cells assayed for their ability to score differentiation arrest by other oncoproteins.76 It should be noted that the expression of endogenous c-Myb in the ECoM cell lines is down-regulated late in differentiation (about 5 days after the removal of estrogen), indicating that the ECoM cells might represent an earlier stage of development whose differentiation can be disrupted by a broader spectrum of oncoproteins. The EP
We would like to thank Dan Tenen, Mary Dinauer, Renate Pilz, Martin Haas, Julie Leckstrom-Himes, Nancy Berliner, Arati Khanna-Gupta, Simon Williams, Yoshiaki Ito, Leighton Grimes, Beth Broome, and Rick Van Etten for supplying the probes for Northern analysis as well as BCL-2 and BCR/ABL expression constructs. Many thanks to Dennis Young (flow cytometry) and to James Feramisco, Stephen McMullen, and Carolan Buckmaster (deconvolution microscopy). A special thanks to Xinyu Fu and to Martina Pasillas for her excellent technical support. Much appreciation to Christopher Glass, Bruce Torbett, Katherine Calvo, Richard Lin, and Gernot Walter for critical reading of this manuscript.
Submitted February 28, 2001; accepted June 25, 2001.
Supported by Public Health Service grant CA56876. D.B.S. is supported by Department of Defense grant F49620-99-C-0054. M.P.K. is a scholar of the Leukemia and Lymphoma Society.
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: David B. Sykes, Department of Molecular Pathology, University of California San Diego School of Medicine, 9500 Gilman Dr, La Jolla, CA 92093-0612; e-mail: dsykes{at}ucsd.edu.
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Top
Abstract
Introduction
7 M TPA. The cells were incubated 25 minutes at
37°C, washed, cytocentrifuged onto a coverslip, counterstained with
safranin O (5 minutes, 0.5% safranin O in 20% ETOH), and examined for
the dark purple deposits indicative of a positive NBT reduction.
) family of
transcription factors, all of which have been shown to be essential for
normal myeloid development.
