|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-04-1288.
NEOPLASIA
From the Department of Internal Medicine III,
University Hospital Grosshadern, Ludwig-Maximilians-University Munich
and GSF-National Research Center for Environment and Health, Munich,
Germany; and Harvard Institutes of Medicine, Harvard
Medical School, Boston, MA.
The transcription factor PU.1 plays a pivotal role in normal
myeloid differentiation. PU.1 The Ets family of transcription factors plays a key
role in the growth, survival, differentiation, and activation of
hematopoietic cells. This family of proteins is characterized by
presence of an 85 amino acid,1 winged helix-turn-helix
DNA-binding domain. PU.1 is one of the most important Ets transcription
factors.2 Its expression is limited to hematopoietic
cells, including primitive CD34+ cells, macrophages, B
lymphocytes, neutrophils, mast cells, and early
erythroblasts.2,3 In vitro studies suggest that PU.1 regulates the activity of a number of myeloid- and lymphoid-specific promoters and enhancers.4-10 PU.1 is a key transcription
factor for normal myeloid development as demonstrated by a complete
block of myeloid development in PU.1 AML1 is a member of the Runt-like transcription factors
(Runx-1, -2, and -3) named after the Runt
protein that regulates segmentation during
Drosophila embryogenesis.14-16 AML1 appears to
act as an "organizing" factor for many promoters and enhancers by
interacting with various coactivators and DNA-binding transcription
factors.17-22 The AML1 gene is one of
the most frequently translocated or mutated genes in human
cancer.23-25 The t(8;21)(q22;q22) translocation fuses
residues 1-177 of AML1 (including the DNA-binding domain) to
nearly all of ETO (also known as
CBF2T1).26 ETO is the human homolog of
Drosophila NERVY protein.27-29 The t(8;21)
belongs to the most common chromosomal abnormalities in AML, accounting
for 10% of all AML cases and 40% of the AML French-American-British (FAB) M2 phenotype.30-33 AML1 activates transcription from
enhancer core motifs (TGT/cGGY), which are present in a number of genes relevant to myeloid development, including the macrophage
colony-stimulating factor (M-CSF) receptor, granulocyte-macrophage
colony-stimulating factor (GM-CSF), myeloperoxidase, and neutrophil
elastase.34-39 Like AML1, AML1-ETO can act as a
transcriptional activator,40-43 but it is also a
transcriptional repressor in other contexts.44 Only one
allele of AML1 is altered in leukemia cells expressing t(8;21), and
AML1-ETO can efficiently repress AML1-dependent transcriptional activation. Therefore, AML1-ETO has been postulated to act as a
dominant inhibitor of AML1 function.34,37,44
Recently, we have shown that AML1-ETO blocks CCAAT enhancer-binding
protein (C/EBP Cell lines and cell culture
Bone marrow cells were isolated from the femurs of Balb/C mice. The
femurs were removed and stripped of the soft tissue and crushed to
release cells within marrow cavity. The red blood cells were lysed with
a 0.15-M solution of ammonium chloride. The pelleted cells were
subjected to low-density mononuclear cell separation by incubating with
density gradient (Histopaque 1083; Sigma, St Louis, MO) for 10 minutes and centrifuged at 600 rpm for 30 minutes, washed twice in
phosphate-buffered saline (PBS), followed by culturing in Iscove
modified Dulbecco medium (IMDM; Stem Cell Technologies, Vancouver, BC,
Canada) supplemented with 10% FBS (Stem Cell Technologies), 50 ng/mL stem cell factor (R & D Systems, Minneapolis, MN), 50 ng/mL interleukin 6 (IL-6; R & D Systems), and 50 ng/mL Flt-3 ligand
(Flt-3L; R & D Systems).
Coimmunoprecipitation assay
Western blot After plating in 100-mm plates, 293T cells were transfected using the LipofectAMINE Plus kit (Gibco) as per the manufacturer's protocol. At 24 hours after transfection, cells were harvested and lysed in RIPA lysis buffer, and immunoblot for PU.1 was performed with 100 µg protein as described earlier.46-48 To generate protein lysates, 1 × 106 F9, Kasumi-1, or 293T cells were lysed and nuclear extracts were prepared and immunoblot was performed with 100 µg protein for c-Jun (Santa Cruz Biotechnologies; catalog no. sc54). Mouse bone marrow cells transduced with PU.1, AML1-ETO, or respective empty vectors were similarly lysed (RIPA lysis) and 100 µg protein was used for immunoblot analysis for PU.1 and AML1-ETO (anti-ETO antibody, Santa Cruz Biotechnologies; catalog no. sc9737). Mouse monoclonal anti- -tubulin purchased from Roche
(Mannheim, Germany; catalog no. 1111876) was used for
immunoblot assay as internal control. Protein A-peroxidase-conjugated
for antirabbit (Amersham Pharmacia, Freiburg, Germany; catalog
no. NA 9120), or antigoat peroxidase-conjugated immunoglobulins (Dako,
Hamburg, Germany; code no. p0449) were used as secondary antibodies.
Reporter constructs and expression plasmids The human monocyte-specific M-CSF receptor promoter with or without AML1-binding site, p(PU.1)4TK, and p(mutPU.1)4TK (PU.1-binding sites and mutated PU.1-binding sites subcloned into pTK61luciferase) were described earlier.46 As an internal control plasmid for transient transfection assay, we used the pRL-null construct driving a Renilla luciferase gene (Promega, Madison, WI).49 Other vectors used were pECE-PU.1-murine, pcDNA.1-PU.1, pGEX-2TK-PU.1 or34, pS3H-c-Jun, and pSP6-c-Jun, as described previously.46,50
AML1B-pCMV5 and CBF-pCMV5 were described earlier.42
AML1-ETO-pcDNA3 was constructed by enzymatic digestion of
AML1-ETO-pCMV542 with XbaI and subcloning the
resulting 2258-bp fragment into the XbaI site of pcDNA3
plasmid (Invitrogen, Karlsruhe, Germany).
Transfection assays Transient transfections in 293T or F9 cells were carried out with LipofectAMINE transfection kit (Gibco) in 24-well plates as described earlier.46,47,49 U937 cells were transiently transfected by electroporation in RPMI medium at 980 µF and 280 V. Firefly luciferase activities from the constructs M-CSF receptor promoter luciferase, pXP2, p(PU.1)4TK, p(mutPU.1)4TK, and Renilla luciferase activity from internal control plasmid pRL-null were determined 24 hours after transfection using Dual Luciferase Reporter Assay System (Promega). Results are given as means + SEMs from at least 3 independent experiments.Protein interaction assays c-Jun and AML1-ETO were transcribed in vitro and translated in the presence of [35S]-methionine (Amersham Pharmacia) using the T7/SP6-coupled reticulocyte system (Promega) in accordance with the manufacturer's instruction. Glutathione-S-transferase (GST) precipitation assays were performed as described earlier.46,48EMSA  32P-adenosine triphosphate (ATP; Amersham
Pharmacia)-labeled double-stranded oligonucleotides of PU.1
DNA-binding site51 and AML1-binding site52
for electrophoretic mobility shift assay (EMSA) were prepared. The
assay was performed with in vitro-translated proteins as mentioned
earlier.11,47 For supershift experiments 3 µL of either
anti-PU.1 or anti-ETO antibodies were added to the reaction mixture.
Retroviral transduction assay Ecotrophic Phoenix cells (5 × 106) were plated in 10-cm plates and transfected with 5 µg PINCO-GFP, PINCO-AML1-ETO-GFP, pGsam-PU.1-ires-NGFR, or pGsam-ires-NGFR vectors using LipofectAMINE transfection kit (Gibco). At 24 hours after transfection, the transfection medium was replaced with IMDM (supplemented with 10% FBS, 50 ng/mL stem cell factor, 50 ng/mL IL-6, and 50 ng/mL Flt-3L) for collection of the virus particles. After the viral particle production, freshly isolated mouse bone marrow cells were incubated with viral medium on fibronectin-coated plates and centrifuged for 30 minutes at 1000g (this step was repeated every 12 hours).53 At 60 hours after first transduction, nerve growth factor receptor-positive (NGFR+) or enhanced green fluorescence protein-positive (EGFP+) cells were isolated by fluorescence-activated cell sorting (FACS) analysis (Becton Dickinson, Heidelberg, Germany). To detect the expression of NGFR on the cell surface, cells were stained with mouse antihuman NGFR (Chemicon, Hofheim, Germany; catalog no. MAB5246) followed by phycoerythrin (PE)-conjugated rabbit antimouse immunoglobulins (mouse IgG R-phycoerythrin [RPE]; Dako; catalog no. R0439). Then, 1 × 104 transduced cells sorted for NGFR positivity were plated in 1.2 mL mouse colony-forming medium (Stem Cell Technologies). After 3, 6, and 12 days of plating live cells were counted by trypan blue staining.Patient material and FACS analysis Bone marrow cells from AML-M2 patients with or without t(8;21) were obtained after informed consent was given by the patients. Mononuclear cells were isolated from the bone marrow by density gradient centrifugation with Histopaque (Sigma). FACS analysis was performed with CD11b (Pharmingen, Hamburg, Germany; catalog no. 555388), CD14 (Pharmingen; catalog no. 555397), and CD64 (Pharmingen; catalog no. 555527).Transfection of Kasumi-1 cells and FACS analysis Kasumi-1 cells were electroporated as mentioned for U937 cells electroporation with pGsam-PU.1-ires-NGFR or pGsam-ires-NGFR vectors and sorted 24 hours after transfection for NGFR positivity by FACS (with anti-NGFR antibody from Chemicon, catalog no. MAB5246, and mouse IgG RPE from Dako, catalog no. R0439). Five days after sorting for NGFR expression, morphologic changes were observed by Wright-Giemsa staining of cells. Then, 1 × 106 NGFR+ Kasumi-1 cells were incubated with 10 µL recombinant PE-conjugated mouse monoclonal CD11b (Pharmingen; catalog no. 555388) or fluorescein isothiocyanate (FITC)-conjugated mouse monoclonal CD14 (Pharmingen; catalog no. 555397) in 100 µL PBS for 60 minutes on ice, washed in PBS followed by analysis on a FACScan flow cytometer (Becton Dickinson) using Cellquest software. The cells were also analyzed for the isotype controls, PE-conjugated mouse IgG1 (Pharmingen, catalog no. 554680) for CD11b-PE and FITC-conjugated mouse
IgG1 (Pharmingen; catalog no. 555748) for CD14-FITC. At 24 hours
after transfection, 5 × 104 NGFR+ cells were
plated in a 6-well plate and passaged with fresh medium every 24 hours.
Cell count for live cells was performed by trypan blue staining every
24 hours.
AML1-ETO interacts with PU.1 in vivo and inhibits its transcriptional activity To determine whether PU.1 interacts with AML1-ETO, coimmunoprecipitation assays were performed in Kasumi-1 cells, a human cell line containing t(8;21). PU.1 coprecipitated with both AML1 and ETO antibodies but not with IgG control, suggesting that PU.1 interacts with AML1-ETO in vivo (Figure 1Ai). A similar experiment was performed using a PU.1-specific antibody: AML1-ETO coprecipitated with PU.1, but not with rabbit IgG control (Figure 1Aii).To investigate the functional impact of this in vivo interaction, we performed transient transfection assays in 293T cells. An M-CSF receptor promoter luciferase reporter construct, which was transactivated 12-fold by PU.1 and 28-fold by PU.1/c-Jun, is completely down-regulated by AML1-ETO (Figure 1Bi). AML1-ETO had no effects on serum response element (pSRE)/Ras activity nor on the empty vector (pXP2) as negative controls. The expression levels of cotransfected PU.1 did not change in the presence of AML1-ETO indicating that the transactivating capacity, but not the expression of cotransfected PU.1, was down-regulated (Figure 1Bii). AML1 does not affect transactivation capacity of PU.1 or PU.1/c-Jun The M-CSF receptor promoter has adjacent AML1- and PU.1-binding sites.45 AML1-ETO retains the 177 N-terminus amino acids of AML1, suggesting that AML1 might also have an influence on transactivation of PU.1 or PU.1/c-Jun. Therefore, we addressed if AML1 had any functional impact on the transactivation capacity of PU.1 or PU.1/c-Jun using a promoter containing only PU.1-binding sites (p(PU.1)4TK). Transient transfection assays in 293T cells were performed with p(PU.1)4TK and expression plasmids of PU.1, c-Jun, AML1, and core-binding factor (CBF). Results (Figure 1C) show
that AML1 did not affect the PU.1 or PU.1/c-Jun transactivation capacity. In the same experiment AML1 could transactivate the M-CSF
receptor promoter 4-fold in the presence of CBF (data not shown).
AML1, PU.1, c-Jun, and CBF had no effects on control vectors
(p(mutPU.1)4TK and pXP2) in the experiments above (data not
shown).
AML1-ETO inhibits the coactivation of PU.1 by c-Jun We have earlier shown that c-Jun can coactivate transactivation of PU.1 in a JNK-independent manner.46 PU.1 induced strong transactivation of p(PU.1)4TK in 293T cells (Figure 1C). This is possibly due to high expression of its coactivator c-Jun in these cells. Immunoblot assay for c-Jun indicated that 293T cells have high amounts of c-Jun (Figure 2A lane 1) comparable to Kasumi-1 cells (Figure 2A lane 3). However, F9 cells had no detectable c-Jun protein (Figure 2A lane 2). Therefore, further experiments were carried out in F9 cells, which served as a model cell line for understanding how AML1-ETO might interfere with the capacity of c-Jun in coactivating PU.1. PU.1/c-Jun could transactivate p(PU.1)4TK (Figure 2B) and also the M-CSF receptor promoter (Figure 2C) in F9 cells as described earlier.46 In the presence of AML1-ETO, the capacity of PU.1/c-Jun in transactivating the target promoters (Figure 2B-C) was down-regulated. c-Jun up-regulated the p(PU.1)4TK promoter in 293T cells (Figure 1B) and F9 cells (Figure 2B-C), which might be due to presence of noncanonical sites in the promoter construct or unknown factors in these 2 cell lines collaborating with c-Jun. A similar effect was also reported earlier.48 However, this does not influence the final conclusion.
AML1-ETO displaces c-Jun by binding to the
34 region of the DNA-binding domain of
PU.1.48 Therefore, we performed protein-protein
interaction assays using GST-34 and found
that AML1-ETO also binds to GST-34 (Figure 3B). In competitive protein-protein interaction assays on
increasing the AML1-ETO protein, c-Jun protein bound to
GST-34 was reduced (Figure 3B). These
results indicate that AML1-ETO competes c-Jun away from binding to the
34 domain of PU.1. Thus, the c-Jun
coactivation function of PU.1 is down-regulated and this in turn
down-regulates transcriptional activity of PU.1.
AML1-ETO does not change the DNA binding of PU.1 The protein-protein interactions described in Figure 3 demonstrate that AML1-ETO directly interacts with PU.1. The physical interaction of AML1-ETO/PU.1 might down-regulate the DNA-binding capacity of PU.1. To address this possibility, we performed an EMSA using in vitro-translated PU.1 and AML1-ETO and oligonucleotide probes having respective DNA-binding sequences.51,52 In vitro-translated PU.1 binds specifically to the PU.1-binding oligonucleotide (Figure 4). Even in presence of AML1-ETO, no change of DNA binding of PU.1 was observed (Figure 4), indicating that AML1-ETO blocks the transactivation capacity, but not DNA binding of PU.1. In the same experiment in vitro-translated AML1-ETO was found to bind to the AML1 probe (data not shown).
AML1-ETO down-regulates PU.1 transcriptional activity in myeloid cells All the above transfections were performed in nonmyeloid 293T or F9 cells. We asked whether the same effects were also observed in myeloid cells. Therefore, we performed transient transfection assays in myelomonocytic U937 cells. U937 cells were transfected with wild-type M-CSF receptor promoter, M-CSF receptor promoter without AML1-binding site, minimal promoter having PU.1-binding sites (p(PU.1)4TK), minimal promoter with mutated PU.1-binding sites (p(mutPU.1)4TK) as control, and empty vector with or without AML1-ETO expression plasmid. We observed that all the promoters were down-regulated by AML1-ETO without any effect on the empty vectors (Figure 5A). These data confirm that AML1-ETO down-regulates the transcriptional activity of PU.1 in myeloid cells also. U937 cells express high levels of PU.1 and C/EBP ; therefore
AML1-ETO might not only down-regulate PU.1 but also C/EBP. Therefore
only 50% down-regulation of the promoters transfected into U937 cells could be seen. Furthermore, there might be proteins in myeloid cells
(in contrast to 293T or F9 cells) that might interfere with the
capacity of AML1-ETO to block PU.1 function.
Low expression of PU.1 target genes in patients with t(8;21) To further understand if the down-regulation of the PU.1/c-Jun transactivation capacity by AML1-ETO leads to down-regulation of the PU.1 target genes, we performed FACS analysis of PU.1 target cell surface markers.7,51 In AML-M2 patients with t(8;21), CD14, CD11b, and CD64 were 4.6-, 5.4-, and 5.8-fold less expressed in comparison to patients with normal M2 karyotype (Figure 5B). Regulation of CD11b promoter by PU.1 has been shown51 and further analysis (by TRANSFAC analysis to identify potential transcription factor-binding sites in a promoter) of the promoter revealed potential AML1-binding sites were present (data not shown). Down-regulation of CD11b might also be due to down-regulation of AML1 in addition to PU.1's transactivation capacity by AML1-ETO. Similar analysis of CD14 and CD64 promoters showed (data not shown) that these gene promoters have PU.1-binding sites but no C/EBP-, AML1-, or MEF-binding sites.
Therefore, CD14 and CD64 down-regulation could be due to specific
down-regulation of PU.1's transactivation capacity by AML1-ETO in
these patients.
AML1-ETO causes proliferation of mouse bone marrow cells by inhibiting PU.1 To investigate the functional consequences of AML1-ETO down-regulating the transactivation capacity of PU.1, we transduced mouse bone marrow cells with PU.1 (pGsam-NGFR-PU.1) and AML1-ETO (PINCO-AML1-ETO-GFP). The cells transduced with AML1-ETO rapidly increased in number over 12 days, as did the cells overexpressed with AML1-ETO and PU.1 (Figure 6A). The cells transduced with PU.1 showed no increase in cell number (Figure 6A). Furthermore, transduction of AML1-ETO blocks PU.1-induced monocytic differentiation in mouse bone marrow cells (data not shown). The expression of transduced genes is shown in Figure 6 B-C. Densitometric quantification of the PU.1 protein expression in the same experiment revealed down-regulation of endogenous PU.1 expression on overexpression of AML1-ETO (Figure 6B). This could be due to AML1-ETO preventing the autoregulation of PU.1.54 The expression of AML1-ETO was also quantified (data not shown).
Overexpression of PU.1 initiates differentiation in t(8;21)+ Kasumi-1 cells Our data so far shows that AML1-ETO interacts with PU.1 at the34 region in the DNA-binding domain of
PU.1 and displaces c-Jun from binding and coactivating PU.1 (Figures
2-3). Moreover, overexpression of AML1-ETO down-regulated the PU.1
expression in mouse bone marrow cells (Figure 6B). It is important to
note that Kasumi-1 cells shows high levels of c-Jun protein expression (Figure 2A). Hence, we asked whether overexpression of PU.1 could overcome the functional block of PU.1 by AML1-ETO. Transient
overexpression of PU.1 (pGsam-NGFR-PU.1) in t(8;21)-bearing Kasumi-1
cells was performed. FACS sorting (for NGFR) of the transfected cells
showed the PU.1 expression, which was further shown by immunoblot
analysis of sorted cells for PU.1 expression (Figure
7B). Fourfold overexpression was observed
after transfection (Figure 7B).
Five days after transfection of PU.1, morphologic changes (Figure 7A) were observed by Wright-Giemsa staining of cells. PU.1-transfected cells differentiated to the monocyte like cells, whereas the empty vector (pGsam-NGFR) transfected cells showed no morphologic change. The PU.1-transfected Kasumi-1 cells also showed an increase in cell surface markers CD11b (Figure 7C; marker for myeloid differentiation) and CD14 (Figure 7D; marker for the monocytic lineage). At 24 hours after transfection, the NGFR-sorted cells were further plated and counted for live cells every 24 hours. In PU.1-transfected cells a decrease in cell number was observed (Figure 7E).
The importance of PU.1 in myeloid differentiation is well
established. Recently we have reported that PU.1 is mutated in patients with AML13 similar to C/EBP We observed that AML1-ETO down-regulates the transcriptional activity
of PU.1 in myeloid cells (Figure 5A) and physically interacts at the
We observed that physical interaction between AML1-ETO and PU.1 did not
abolish the DNA- binding capacity of PU.1 (Figure 4A), although
AML1-ETO interacted with the PU.1 DNA-binding domain. Interestingly, in
PU.1's crystal structure,1 the
AML1B and AML1-ETO have been shown to transactivate the M-CSF receptor,42 suggesting that interaction between AML1B and AML1-ETO could be important for leukemogenesis. To investigate the importance of AML1-ETO/PU.1 interaction in leukemogenesis, transactivation, proliferation, and differentiation assays were performed in cells expressing wild-type AML1B protein. In the presence of AML1-ETO, the M-CSF receptor promoter was down-regulated and similarly the AML1 site mutated M-CSF receptor promoter and minimal promoter containing only PU.1-binding sites in U937 cells (Figure 5A). This could be explained by a dual function of AML1-ETO in regulation of the M-CSF receptor expression. During normal myeloid differentiation, M-CSF receptor expression is required for G1-to-S phase transition, which could be down-regulated by AML1-ETO through the functional interaction with PU.1, and then AML1-ETO cooperates with AML1B to up-regulate the M-CSF receptor expression for transformation and proliferation of abnormal progenitor cells. In patients with t(8;21), expression of the cell surface markers CD11b,
CD14, and CD64 was less in comparison to patients without t(8;21)
(Figure 5B). CD14 and CD64 promoters have putative PU.1 binding sites
but not AML1-, C/EBP Because AML1 Overexpression of PU.1 in t(8;21)+ Kasumi-1 cells
differentiates them toward the monocytic lineage (Figure 7).
Morphologically, cells did not appear to be terminally differentiated
even though the cell surface markers CD11b and CD14 were increased in
expression. It has been earlier shown that short-term activation of
PU.1 in multipotent hematopoietic cells leads to immature
eosinophils.63 However, stable overexpression of PU.1
could lead to myeloid lineage in hematopoietic progenitor
cells.63 Therefore, higher and stable expression of PU.1
in Kasumi-1 cells might be needed to terminally differentiate toward
the monocytic lineage. The cell number of PU.1-transfected Kasumi-1
cells decreased over a course of time (Figure 7E) showing that PU.1
functions as an antiproliferative factor on overexpression in
Kasumi-1 cells. However, this mechanism needs to be further elucidated.
Our data suggest that the ectopic expression of PU.1 in
Kasumi-1 cells overcomes the functional block by AML1-ETO.
PU.1 and C/EBP
We thank Dr Atsushi Iwama, University of Tsukuba, Japan for providing pGsam-PU.1-ires-NGFR and PGsam-ires-NGFR retroviral vectors.
Submitted April 30, 2002; accepted July 30, 2002.
Prepublished online as Blood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-04-1288.
Supported by a DFG (German research foundation) grant to G.B. (no. 2042/2-1).
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: Gerhard Behre, Department of Internal Medicine III, University Hospital Grosshadern, Ludwig-Maximilians-University Munich, Marchioninistr 15, D-81377 Munich, Germany; e-mail: gerdbehre{at}aol.com.
1. Kodandapani R, Pio F, Ni CZ, et al. A new pattern for helix-turn-helix recognition revealed by PU.1 ETS-domain-DNA complex. Nature. 1996;380:456-460[CrossRef][Medline] [Order article via Infotrieve].
2.
Tenen DG, Hromas R, Licht JD, Zhang DE.
Transcription factors, normal myeloid development and leukemia.
Blood.
1997;90:489-499
3.
Chen H, Zhang P, Voso MT, et al.
Neutrophils and monocytes express high levels of PU.1 (Spi-1) but not Spi-B.
Blood.
1995;85:2918-2928 4. Anderson KL, Perkin H, Surth CD, et al. Transcription factor PU.1 is necessary for development of thymic and myeloid progenitor-derived dendritic cells. J Immunol. 2000;164:18551861. 5. Behre G, Zhang P, Zhang DE, Tenen DG. Analysis of the modulation of transcriptional activity in leukemogenesis. Methods. 1999;17:231-237[CrossRef][Medline] [Order article via Infotrieve]. 6. Hohaus S, Petrovick MS, Voso MT, et al. PU.1 (Spi-1) and C/EBP alpha regulate expression of the granulocyte-macrophage colony-stimulating factor receptor alpha gene. Mol Cell Biol. 1995;15:5830-5845[Abstract].
7.
Olweus J, Thompson PA, Lund-Johansen F.
Granulocytic and monocytic differentiation of CD34hi cells is associated with distinct changes in the expression of the PU.1 regulated molecules, CD64 and macrophage colony-stimulating factor receptor.
Blood.
1996;88:3741-3754
8.
Smith LT, Hohaus S, Gonzalez DA, Dziennis SE, Tenen DG.
PU.1 (Spi-1) and C/EBP alpha regulate the granulocyte colony-stimulating factor receptor promoter in myeloid cells.
Blood.
1996;88:1234-1247
9.
Zhang DE, Hetherington CJ, Chen H, Tenen DG.
The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.
Mol Cell Biol.
1994;14:373-381 10. Zhang DE, Hohaus S, Voso MT, et al. Function of PU.1 (Spi-1), C/EBP, and AML1 in early myelopoiesis regulation of multiple myeloid CSF receptor promoters. Curr Top Microbiol Immunol. 1996;211:137-147[Medline] [Order article via Infotrieve]. 11. McKercher SR, Torbett BE, Anderson KL, et al. Targeted disruption of the PU.1 gene results in multiple hematopoietic abnormalities. EMBO J. 1996;15:5647-5658[Medline] [Order article via Infotrieve].
12.
Scott EW, Simon MC, Anastasi J, Singh H.
Requirement of transcription factor PU.1 in the development of multiple hematopoietic lineages.
Science.
1994;265:1573-1577
13.
Mueller BU, Pabst T, Osato M, et al.
Heterozygous PU.1 mutations are associated with acute myeloid leukemia.
Blood.
2002;100:998-1007
14.
Crute BE, Lewis AF, Wu Z, Bushweller JH, Speck NA.
Biochemical and biophysical properties of core binding factors 2 (AML1) DNA binding domain.
J Biol Chem.
1996;271:26251-26260 15. Daga A, Tighe JE, Calabi F. Leukemia/Drosophila homology. Nature. 1992;356:484-489[Medline] [Order article via Infotrieve]. 16. Golling G, Li L, Pepling M, Stebbins M, Gergen JP. Drosophila homologous of the proto-oncogene product PEBP2/CBF beta regulate the DNA binding properties of the Runt. Mol Cell Biol. 1996;16:932-942[Abstract]. 17. Hernandez-Munian C, Krangel MS. c-Myb and core binding factor PEBP2 display functional synergy but bind independently to adjacent sites in the T-cell receptor enhancer. Mol Cell Biol. 1995;15:3090-3099[Abstract].
18.
Look AT.
Oncogenic transcription factors in human acute myeloid leukemias.
Science.
1997;278:1059-1067
19.
Ness SA, Kowenz-Leutz E, Casini T, Graf T, Leutz A.
Myb and MF-M: combinatorial activators of myeloid genes in heterologous cell types.
Genes Dev.
1993;7:749-759 20. Ogawa E, Inuzuka M, Maruyama M, et al. Molecular cloning and characterization of PEBP2 beta, the heterodimeric partner of novel Drosophila runt-related DNA binding protein PEBP2. Virology. 1993;194:314-331[CrossRef][Medline] [Order article via Infotrieve]. 21. Sun W, O'Connell M, Speck NA. Characterization of a protein that binds multiple sequences in mammalian type C retrovirus enhancers. J Virol. 1993;67:1986-1995. 22. Wang S, Wang Q, Crute BE, et al. Cloning and characterization of subunits of the T-cell receptor and murine leukemia virus enhancer core-binding factor. Mol Cell Biol. 1993;13:3329-3332.
23.
Nucifora G, Birn DJ, Erickson P, et al.
Detection of DNA rearrangements in the AML1 and ETO loci and an AML1-ETO fusion mRNA in patients with t(8;21) acute myeloid leukemia.
Blood.
1993;81:883-888
24.
Osato M, Asou N, Abdalla E, et al.
Biallelic and heterozygous point mutations in the Runt domain of the AML1/PEBP2 25. Song WJ, Sullivan MG, Legare RD, et al. Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukemia. Nat Genet. 1999;23:166-175[CrossRef][Medline] [Order article via Infotrieve]. 26. Era T, Asou N, Kunisada T, et al. Identification of two transcripts of AML1-ETO-fused gene in t(8;21) leukemic cells and expression of wild-type ETO gene in hematopoietic cells. Genes Chromosomes Cancer. 1995;13:25-33[Medline] [Order article via Infotrieve].
27.
Erickson P, Gao J, Chang KS, et al.
Identification of break point in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript, AML1-ETO, with similarity to Drosophila segmentation gene, runt.
Blood.
1992;80:1825-1831 28. Miyoshi H, Kozu T, Shimizu K, et al. The t(8;21) translocation in acute myeloid leukemia results in production of an AML1-MTG8 fusion transcript. EMBO J. 1993;12:2715-2721[Medline] [Order article via Infotrieve]. 29. Okuda T, van Deursen J, Hiebert SW, Grosveld G, Downing JR. AML1, the target of multiple chromosomal translocations in human leukemia, is essential for normal fetal liver hematopoiesis. Cell. 1996;84:321-330[CrossRef][Medline] [Order article via Infotrieve]. 30. Bitter MA, Le Beau MM, Rowley JD, et al. Associations between morphology, karyotype, and clinical features in myeloid leukemias. Hum Pathol. 1987;18:211-225[Medline] [Order article via Infotrieve]. 31. Downing JR. The AML1-ETO chimeric transcription factor in acute myeloid leukemia: biology and clinical significance. Br J Haematol. 1999;106:296-308[CrossRef][Medline] [Order article via Infotrieve]. 32. Lowenberg B, Downing JR, Burnett A. Acute myeloid leukemia. N Engl J Med. 1999;30:1051-1061.
33.
Miyoshi H, Shimizu K, Kazu T, Maseki N, Kaneko Y, Ohki M.
t(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene, AML1.
Proc. Natl. Acad. Sci. U S A.
1991;88:10431-10434 34. Frank R, Zhang J, Uchida H, Meyers S, Hiebert SW, Nimer SD. The AML1-ETO protein blocks transactivation of GM-CSF promoter by AML1B. Oncogene. 1995;11:2667-2674[Medline] [Order article via Infotrieve].
35.
Nuchprayoon L, Meyers S, Scott LM, Suzow J, Hiebert S, Friedman AD.
PEBP2/CBF, the murine homolog of human myeloid AML1 and PEBP2 36. Pabst T, Mueller BU, Schoch C, et al. AML1-ETO downregulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia. Nat Med. 2001;7:444-451[CrossRef][Medline] [Order article via Infotrieve]. 37. Yergeau DA, Hetherington CJ, Wang Q, et al. Embryonic lethality and impairment of hematopoiesis in mice heterozygous for AML1-ETO fusion gene. Nat Genet. 1997;15:303-306[CrossRef][Medline] [Order article via Infotrieve].
38.
Zhang DE, Fujioka K, Hetherington CJ, et al.
Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).
Mol Cell Biol.
1994;14:8085-8095 39. Zhang DE, Hetherington CJ, Meyers S, et al. CCAAT enhancer binding protein (C/EBP) and AML1 (CBFalpha2) synergistically activates the macrophage colony stimulating factor receptor promoter. Mol Cell Biol. 1996;16:1231-1240[Abstract]. 40. Frank RC, Sun X, Berguido FJ, Jakubowaik A, Nimer SD. The t(8;21) fusion protein, AML1-ETO transforms NIH3T3 cells and activates AP-1. Oncogene. 1999;18:1701-1710[CrossRef][Medline] [Order article via Infotrieve].
41.
Klampfer L, Zhang J, Zelenetz AO, Uchida H, Nimer SD.
The AML1-ETO fusion protein activates transcription of BCL-2.
Proc Natl Acad Sci U S A.
1996;93:14059-14064
42.
Rhoades KL, Hetherington CJ, Rowley JD, et al.
Synergistic up-regulation of the myeloid-specific promoter for macrophage colony-stimulating factor receptor by AML1 and the t(8;21)fusion protein may contribute to leukemogenesis.
Proc Natl Acad Sci U S A.
1996;93:11895-11900
43.
Rhoades KL, Heterington CJ, Harakawa N, et al.
Analysis of the role of AML1-ETO in leukemogenesis using inducible transgenic mouse model.
Blood.
2000;96:2108-2115 44. Meyers S, Lenny N, Hiebert SW. The t(8;21) fusion protein interferes with AML1B-dependent transcriptional activity. Mol Cell Biol. 1995;15:1974-1982[Abstract].
45.
Mao S, Franks RC, Zhang J, Miyazaki Y, Nimer SD.
Functional and physical interactions between AML1 proteins and ETS protein: MEF; implications for pathogenesis of t(8;21) positive leukemias.
Mol Cell Biol.
1999;19:3635-3644
46.
Behre G, Whitmarsh AJ, Coghlan MP, et al.
c-Jun is a JNK-independent coactivator of PU.1 transcription factor.
J Biol Chem.
1999;274:4939-4946
47.
Behre G, Singh SM, Liu H, et al.
Ras signaling enhances the activity of C/EBPalpha to induce granulocytic differentiation by phosphorylation of serine 248.
J Biol Chem.
2002;277:26293-26299
48.
Reddy V, Iwama A, Iotzava G, et al.
The granulocytic inducer C/EBPalpha inactivates the myeloid master regulator PU.1: possible role in lineage commitment decisions.
Blood.
2002;100:483-490 49. Behre G, Smith LT, Tenen DG. Use of a promoterless Renilla luciferase vectors as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation. Biotechniques. 1999;26:24-26[Medline] [Order article via Infotrieve].
50.
Zhang P, Behre G, Pan J, et al.
Negative cross-talk between hematopoietic regulators: GATA proteins repress PU.1 function.
Proc Natl Acad Sci U S A.
1999;96:8705-8710
51.
Pahl HL, Scheibe RJ, Zhang DE, et al.
The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter.
J Biol Chem.
1993;268:5014-5020
52.
Meyers S, Downing JR, Hiebert SW.
Identification of AML-1 and t(8;21) translocation protein (AML1-ETO) as sequence-specific DNA-binding proteins: the runt homology domain is required for DNA binding and protein-protein interactions.
Mol Cell Biol.
1993;13:6336-6345
53.
Grignani F, Kinsella T, Mencarelli A, Gergen JP.
High efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein.
Cancer Res.
1998;58:14-19 54. Chen H, Ray D, Zhang P, et al. PU.1 (Spi-1) autoregulates its expression in myeloid cells. Oncogene. 1995;11:1549-1560[Medline] [Order article via Infotrieve]. 55. Pabst T, Mueller BU, Zhang P, et al. Dominant negative mutations of C/EBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia. Nat Genet. 2001;27:263-270[CrossRef][Medline] [Order article via Infotrieve].
56.
Petrovick MS, Hiebert SW, Friedman AD, et al.
Multiple functional domains of AML1: PU.1 and C/ EBPalpha synergize with different regions of AML1.
Mol Cell Biol.
1998;18:3915-3925 57. Szabo E, Francis J, Birrer MJ. Alterations in differentiation and apoptosis induced by bufalin in c-Jun overexpressing U937 cells. Int J Oncol. 1998;12:403-409[Medline] [Order article via Infotrieve].
58.
Klampfer L, Lee TH, Hsu W, Vilcek J, Chen-Kiang S.
NF-IL6 and AP-1 cooperatively modulate the activation of TSG-6 gene by tumor necrosis factor alpha and interleukin-1.
Mol Cell Biol.
1994;14:6561-6569
59.
Doyle GAR, Pierce RA, Parks WC.
Transcriptional induction of collagenase-1 in differentiated monocyte-like U937 cells is regulated by AP-1 and upstream C/EBPbeta site.
J Biol Chem.
1997;272:11840-11849
60.
Zagariya A, Mungre S, Lovis R, et al.
Tumor necrosis factor alpha gene regulation: enhancement of C/ EBPbeta-induced activation by c-Jun.
Mol Cell Biol.
1998;18:2815-2824
61.
Okada H, Watanabe T, Niki M, et al.
AML1(
62.
Mulloy JC, Cammenga J, MacKenzie KL, et al.
The AML1-ETO fusion protein promotes the expansion of human stem cells.
Blood.
2002;99:15-23
63.
Nerlov C, Graf T.
PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitors.
Genes Dev.
1998;12:2403-2412
© 2003 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  Y. Satoh, I. Matsumura, H. Tanaka, S. Ezoe, K. Fukushima, M. Tokunaga, M. Yasumi, H. Shibayama, M. Mizuki, T. Era, et al. AML1/RUNX1 Works as a Negative Regulator of c-Mpl in Hematopoietic Stem Cells J. Biol. Chem., October 31, 2008; 283(44): 30045 - 30056. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Chyla, I. Moreno-Miralles, M. A. Steapleton, M. A. Thompson, S. Bhaskara, M. Engel, and S. W. Hiebert Deletion of Mtg16, a Target of t(16;21), Alters Hematopoietic Progenitor Cell Proliferation and Lineage Allocation Mol. Cell. Biol., October 15, 2008; 28(20): 6234 - 6247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. K. Cheng, L. Li, S. H. Cheng, K. M. Lau, N. P. H. Chan, R. S. M. Wong, M. M. K. Shing, C. K. Li, and M. H. L. Ng Transcriptional repression of the RUNX3/AML2 gene by the t(8;21) and inv(16) fusion proteins in acute myeloid leukemia Blood, October 15, 2008; 112(8): 3391 - 3402. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-R. J. Yeh, K. M. Munson, Y. L. Chao, Q. P. Peterson, C. A. MacRae, and R. T. Peterson AML1-ETO reprograms hematopoietic cell fate by downregulating scl expression Development, January 15, 2008; 135(2): 401 - 410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Papetti and A. I. Skoultchi Reprogramming Leukemia Cells to Terminal Differentiation and Growth Arrest by RNA Interference of PU.1 Mol. Cancer Res., October 1, 2007; 5(10): 1053 - 1062. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Wichmann, L. Chen, M. Heinrich, D. Baus, E. Pfitzner, M. Zornig, O. G. Ottmann, and M. Grez Targeting the Oligomerization Domain of ETO Interferes with RUNX1/ETO Oncogenic Activity in t(8;21)-Positive Leukemic Cells Cancer Res., March 1, 2007; 67(5): 2280 - 2289. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Liew, K. D. Rand, R. J. Y. Simpson, W. W. Yung, R. E. Mansfield, M. Crossley, M. Proetorius-Ibba, C. Nerlov, F. M. Poulsen, and J. P. Mackay Molecular Analysis of the Interaction between the Hematopoietic Master Transcription Factors GATA-1 and PU.1 J. Biol. Chem., September 22, 2006; 281(38): 28296 - 28306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nishida, N. Hosen, T. Shirakata, K. Kanato, M. Yanagihara, S.-i. Nakatsuka, Y. Hoshida, T. Nakazawa, Y. Harada, N. Tatsumi, et al. AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1 Blood, April 15, 2006; 107(8): 3303 - 3312. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Rosenbauer, S. Koschmieder, U. Steidl, and D. G. Tenen Effect of transcription-factor concentrations on leukemic stem cells Blood, September 1, 2005; 106(5): 1519 - 1524. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Walter, J. S. Park, R. E. Ries, S. K. M. Lau, M. McLellan, S. Jaeger, R. K. Wilson, E. R. Mardis, and T. J. Ley Reduced PU.1 expression causes myeloid progenitor expansion and increased leukemia penetrance in mice expressing PML-RAR{alpha} PNAS, August 30, 2005; 102(35): 12513 - 12518. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. T. Porse, D. Bryder, K. Theilgaard-Monch, M. S. Hasemann, K. Anderson, I. Damgaard, S. E. W. Jacobsen, and C. Nerlov Loss of C/EBP{alpha} cell cycle control increases myeloid progenitor proliferation and transforms the neutrophil granulocyte lineage J. Exp. Med., July 5, 2005; 202(1): 85 - 96. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Choudhary, J. Schwable, C. Brandts, L. Tickenbrock, B. Sargin, T. Kindler, T. Fischer, W. E. Berdel, C. Muller-Tidow, and H. Serve AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations Blood, July 1, 2005; 106(1): 265 - 273. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Zhong, A. Takeda, R. Nazari, H. Shio, G. Blobel, and N. R. Yaseen Carrier-independent Nuclear Import of the Transcription Factor PU.1 via RanGTP-stimulated Binding to Nup153 J. Biol. Chem., March 18, 2005; 280(11): 10675 - 10682. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-Y. Wang, G.-B. Zhou, T. Yin, B. Chen, J.-Y. Shi, W.-X. Liang, X.-L. Jin, J.-H. You, G. Yang, Z.-X. Shen, et al. AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: Implication in stepwise leukemogenesis and response to Gleevec PNAS, January 25, 2005; 102(4): 1104 - 1109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. D. Cook, B. J. McCaw, C. Herring, D. L. John, S. J. Foote, S. L. Nutt, and J. M. Adams PU.1 is a suppressor of myeloid leukemia, inactivated in mice by gene deletion and mutation of its DNA binding domain Blood, December 1, 2004; 104(12): 3437 - 3444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Tiacci, S. Pileri, A. Orleth, R. Pacini, A. Tabarrini, F. Frenguelli, A. Liso, D. Diverio, F. Lo-Coco, and B. Falini PAX5 Expression in Acute Leukemias: Higher B-Lineage Specificity Than CD79a and Selective Association with t(8;21)-Acute Myelogenous Leukemia Cancer Res., October 15, 2004; 64(20): 7399 - 7404. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Dohner, K. Tobis, T. Bischof, S. Hein, R. F. Schlenk, S. Frohling, H. Dohner, B. U. Mueller, T. Pabst, M. Osato, et al. Mutation analysis of the transcription factor PU.1 in younger adults (16 to 60 years) with acute myeloid leukemia: a study of the AML Study Group Ulm (AMLSG ULM) Blood, November 15, 2003; 102(10): 3850 - 3851. [Full Text] [PDF]
|
|
|
|
|
|
M. Rehli, H.-H. Niller, C. Ammon, S. Langmann, L. Schwarzfischer, R. Andreesen, and S. W. Krause Transcriptional Regulation of CHI3L1, a Marker Gene for Late Stages of Macrophage Differentiation J. Biol. Chem., November 7, 2003; 278(45): 44058 - 44067. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. S. Choe, F. Radparvar, I. Matushansky, N. Rekhtman, X. Han, and A. I. Skoultchi Reversal of Tumorigenicity and the Block to Differentiation in Erythroleukemia Cells by GATA-1 Cancer Res., October 1, 2003; 63(19): 6363 - 6369. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. U. Mueller, T. Pabst, M. Osato, N. Asou, L. M. Johansen, M. D. Minden, G. Behre, W. Hiddemann, Y. Ito, and D. G. Tenen Heterozygous PU.1 mutations are associated with acute myeloid leukemia Blood, March 1, 2003; 101(5): 2074 - 2074. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2003 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
/
/