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HEMATOPOIESIS
From the Division of Pediatric Oncology, Johns Hopkins
University, Baltimore, MD.
Potential redundancy among members of the CCAAT/enhancer-binding
protein (C/EBP) family in myeloid cells is indicated by the ability of
C/EBP The CCAAT/enhancer-binding protein (C/EBP)
transcription factors homodimerize and heterodimerize via their
C-terminal leucine zipper domains and bind DNA as obligate dimers via
the adjacent basic regions.1,2 C/EBP Within hematopoiesis, full-length C/EBP C/EBP The presence of several C/EBPs in the myeloid lineages suggests that
related family members might compensate in vivo for the lack of a
single isoform, just as GATA family members partially compensate for
the lack of GATA-1.23 Potential redundancy is evident from
the ability of C/EBP To determine the effect of global inhibition of C/EBP-regulated genes
on granulopoiesis, we developed a potent dominant-inhibitory protein,
Kruppel-associated box (KRAB)-C/EBP Cell culture, transduction, and proliferation assays
Plasmids and transient transfection
Western, Northern, and FACS analyses Preparation of total cellular protein and RNA, Western blotting, and filter stripping were performed as described.41 Each figure represents the same blot probed sequentially. Murine and human ER antisera (MC-20 and HC-20; Santa Cruz Biotechnology, Santa Cruz, CA) were employed at 1:1000. The murine myeloperoxidase (MPO), lactoferrin (LF), lysozyme, PU.1, G-CSFR, C/EBP , and
 -actin cDNA probes have been described.40,42-44
Assessment of G-CSFR expression was performed as
described.44 In brief, human G-CSF was biotinylated by
means of a kit (Pierce, Rockford, IL). Then, 2 × 106
cells were washed and incubated in 100 µL on ice with 5 µg/mL biotin-G-CSF in the presence or absence of 1000-fold excess, unlabeled G-CSF. The cells were then washed 3 times, incubated with 10 µg/mL streptavidin-phycoerythrin (PE), washed, fixed with 1%
paraformaldehyde in 10 mM Hepes, pH 7.4, and analyzed by FACS. CD11b
expression was assessed with the use of FITC-anti-CD11b antibody or
rat immunoglobulin G2b isotype control, with the use of 2 µL of each to stain 106 cells in 100 µL (BD Pharmingen,
San Diego, CA).
Transduction and analysis of murine hematopoietic progenitors MIG-KER was transfected into Phoenix-A cells, and supernatant
collected 48 hours later was used to transduce  CRE cells in the
presence of 4 µg/mL polybrene. At 7 days later, GFP+
cells were isolated by flow cytometry and expanded. GFP+
cells were then again selected, to yield a uniformly GFP+
packaging line. Marrow cells for transduction were obtained from the
long bones of C57BL/6 mice that had been treated by tail vein injection
with 150 mg/kg 5-fluorouracil (5-FU) 3 days earlier. Fetal liver cells
were obtained at day 14.5 of gestation from timed matings and were
rendered into a single suspension by passage through a 25-gauge needle.
Isolated marrow cells were prestimulated for 48 hours with 10 ng/mL
IL-3, 50 ng/mL IL-6, and 100 ng/mL stem cell factor (SCF) in IMDM with
10% HI-FBS and penicillin (100 units/mL)/streptomycin (100 µg/mL), and then cocultured with irradiated CRE-MIG-KER
cells in the same media in the presence of 8 µg/mL polybrene for an
additional 72 hours in 100-mm dishes. To stimulate erythroid
progenitors from fetal liver, we employed DMEM with 15% HI-FBS, 1%
bovine serum albumin (BSA), 1.9 mM NaHC03, 0.1 mM
-mercaptoethanol, 128 µg/mL transferrin, 1 µM dexamethasone, 1 µM -estradiol, 3 U/mL erythropoietin (Epo), and 100 ng/mL SCF, as
described.45 During this 72-hour period, 3 mL BOSC23
supernatant obtained from cells transiently transfected with MIG-KER
was added to the coculture at both 24 and 48 hours. Transduced cells were then rinsed with media and recovered in prestimulation media for 1 day. GFP+ marrow or fetal liver cells were then isolated by
flow cytometry and cultured in Marrow-Gro methylcellulose (Quality
Biologicals, Gaithersburg, MD), including IMDM, 32.5% HI-FBS, 0.11 mM
-mercaptoethanol, 1.1% BSA, penicillin (100 units/mL)/streptomycin
(100 µg/mL), and either 20 ng/mL G-CSF, 10 ng/mL macrophage
CSF (M-CSF), 10 ng/mL GM-CSF, 10 ng/mL IL-3, or the combination of 3 U/mL Epo and 5 µg/mL insulin. Each 35-mm dish was seeded with 2 to
4 × 103 transduced marrow cells or
2 × 104 transduced fetal liver cells, with 200 nM 4HT or
the ethanol vehicle, and colony numbers were assessed on days 8 through
10 by light microscopy. Several colonies of each type were plucked, cytospun, and subjected to Wright-Giemsa staining to confirm the accuracy of morphologic assignments.
C/EBP WT-ER, C/EBP WT-ER, and the ER segment alone were
introduced into 32D cl3 cells by retroviral transduction. Subclones obtained by limiting dilution were assessed for expression of the
introduced proteins by Western blotting, with the use of extracts corresponding to equivalent numbers of cells (Figure
1A). A slowly migrating, nonspecific band
is evident in each extract, including the extract derived from parental
32D cl3 cells. The 2 subclones expressing full-length C/EBPWT-ER
also express a prominent shorter form (indicated by an asterisk in
Figure 1A) reactive with the ER antisera. As the ER segment is at the
C-terminal end of the fusion protein, this shorter form probably
results from the use of an internal ATG.4 Only
one subclone was obtained in which the expression of the ER segment
alone was similar to that of C/EBPWT-ER and C/EBPWT-ER.
Morpologically, the cell lines expressing C/EBP Of note, C/EBP Development of a regulated, dominant-negative C/EBP To determine the consequences of repressing genes regulated by C/EBP family members, we generated a fusion between an 89-amino acid KRAB transrepression domain (K), the C/EBP DNA-binding domain (), and a variant of the murine ER ligand-binding domain that is
activated by 4HT, but not by estradiol. KER is diagrammed in Figure
2A. Also shown is the diagram of a
variant, KVER, in which the first 2 leucines of the C/EBP segment's
leucine zipper have been mutated to valine. KVER is expected to be
incapable of both dimerization and DNA-binding.1 The 32D
cl3 cells were stably transduced with KER and KVER. Exogenous
protein was detected by Western blotting in several subclones (Figure
2B). C/EBP activities in 32D-KER-1 and 32D-KVER-2 cells were
assessed by transient transfection of p(C/EBP)2TKLUC, a
reporter plasmid containing 2 C/EBP-binding sites, a minimal thymidine
kinase promoter, and the luciferase cDNA. In the presence of 4HT,
KER reduced the activity of p(C/EBP)2TKLUC in 32D cl3
cells approximately 50-fold, whereas KVER had no effect (Figure 2C).
KER also specifically inhibited activation by exogenous C/EBP in
NIH 3T3 cells (not shown). KVER did not affect 32D cl3 proliferation in
IL-3 or in G-CSF, and KER only mildly affected their proliferation
in IL-3 (see below and data not shown). Therefore, KER is a potent
inhibitor of endogenous C/EBPs in the presence of 4HT and does not
introduce nonspecific toxic effects.
A dominant-negative C/EBP reduces endogenous G-CSFR expression in 32D cl3 cells The proliferation of 32D-KVER and 32D-KER cells was
assessed in IL-3 or G-CSF, in the presence or absence of 4HT (Figure 3A). The 4HT did not alter the growth of
32D-KVER cells, whereas 32D-KER cells exposed to 4HT mildly slowed
their proliferation in IL-3 and rapidly died in G-CSF. KVER cells
differentiated to neutrophils in the presence of 4HT and G-CSF (Figure
3B). The finding that KVER did not alter the differentiation of 32D cl3 cells indicates that the KRAB domain does not produce nonspecific toxicities. In the absence of 4HT, the KER cell lines differentiated to neutrophils in response to G-CSF, whereas in the presence of 4HT
they developed an apoptotic nuclear morphology by day 2 (Figure 3C).
Induction of apoptosis in 32D-KER cells transferred to G-CSF in the
presence of 4HT was confirmed with the use of FITC-annexin V (not
shown). Apoptosis was not induced when KER was activated in 32D cl3
cells proliferating in IL-3; the slowing observed resulted from modest
inhibition of G1 to S progression (not shown). Induction of
MPO RNA by G-CSF was prevented by activation of KER but not KVER,
confirming that KVER with its KRAB domain did not prevent differentiation in response to G-CSF (Figure 3D).
As C/EBP
A dominant-negative C/EBP blocks differentiation in response to exogenous G-CSFR The 32D-KER-1 and 32D-KVER-2 cells were transduced with the
human G-CSFR. Then, two 32D-KER/GR sublcones (designated KER/GR and KER/GR2) and one 32D-KVER/GR subclone were isolated and
subjected to further analysis. Expression of exogenous G-CSFR was
confirmed by means of biotin-G-CSF and FACS analysis (Figure 4B, right
panels and data not shown for the 32D-KER/GR2 clone). Exogenous
G-CSFR is expressed more than 10-fold higher than endogenous G-CSFR in these lines, and its expression was not affected by addition of 4HT
(Figure 4B).
To assess their proliferation, 32D-K
To further assess differentiation, 32D-K The 32D-K Dominant inhibition of C/EBPs does not prevent induction of CD11b or PU.1 by G-CSF The 32D-KER/GR cells were cultured in G-CSF with and without
4HT, and CD11b surface expression was assessed daily by FACS analysis.
Activation of KER did not inhibit, and in fact increased, the
average CD11b expression in 2 separate experiments. Results from a
representative experiment are shown in Figure
6A. CD11b expression was not affected by
addition of 4HT to 32D-KVER/GR cells in G-CSF (not shown). The
32D-KER/GR2 cells were cultured in G-CSF with and without 4HT, and
total cellular RNAs were prepared on days 0, 2, 4, or 7. These RNAs
were subjected to Northern analysis for MPO, PU.1, and -actin
(Figure 6B). In the absence of 4HT, MPO levels were strongly induced by
G-CSF, whereas even basal expression of MPO was eliminated by addition
of 4HT, as was seen with 32D-KER/GR cells. Despite potent inhibition
of endogenous C/EBP activities, G-CSF induced PU.1 RNA in
32D-KER/GR-2 cells (Figure 5D) and in 2 experiments with
32D-KER/GR cells (not shown). Induction of PU.1 may account for the
observed induction of CD11b by G-CSF even in the presence of
activated KER.
Dominant C/EBP inhibition reduces the number of myeloid colonies from marrow To assess the effect of KER on normal hematopoietic myeloid
progenitors, murine marrow was transduced with MIG-KER, which expresses both KER and EGFP from a single mRNA. The CRE-MIG-KER packaging line produced 5 × 105 infectious particles per
milliliter, on the basis of transduction of NIH 3T3 cells. Typically,
approximately 20% of marrow cells expressed high levels of GFP (Figure
7A). This fraction was isolated by flow
cytometry and then cultured in methylcellulose with G-CSF, M-CSF,
GM-CSF, or IL-3, with and without 4HT. 4HT did not affect the growth of
myeloid colonies, CFU-Gs, CFU-Ms, and CFU-GMs, from untransduced
marrow, but markedly reduced the yield of each of these myeloid CFUs
from marrow cells transduced with KER, in 2 experiments. Results
from a representative experiment are shown in Figure 7B. As our yield
of erythroid burst-forming units (BFU-Es) from transduced marrow cells
was very low, we assessed the affect of KER on the growth of BFU-Es
from transduced day-14.5 fetal liver cells (Figure 7B). BFU-E yields
were similar with and without 4HT in 2 determinations.
Lysozyme and C/EBP ER inhibited MPO induction by G-CSF, but C/EBPWT-ER did not
induce MPO RNA in the presence of cycloheximide, an inhibitor of RNA
translation,25 suggesting that C/EBPs are necessary but not sufficient, for activating the MPO gene during granulopoiesis. As
KER also prevented or reduced lysozyme, C/EBP, C/EBP, and C/EBP induction by G-CSF, we assessed the effect of activating C/EBPWT-ER or C/EBPWT-ER for 8 hours in 32D cl3 cells on the expression of these RNAs, with and without cycloheximide. Extension of
cycloheximide exposure beyond 8 hours results in nonspecific toxicity.
The endogenous C/EBP RNA was not induced and the C/EBP RNA was
only minimally increased by C/EBP-ER within 8 hours (not shown), as
previously shown for the G-CSFR and LF RNAs.25 In contrast, both the lysozyme and C/EBP RNAs were rapidly and strongly induced by C/EBPWT-ER. C/EBP was also strongly induced by
C/EBPWT-ER, whereas lysozyme was only mildy induced by this C/EBP
isoform (Figure 8). Even in
cycloheximide, estradiol induced the C/EBP RNA in both cell lines
and in a second experiment with C/EBPWT-ER cells, but not to the
same extent as it induced in the absence of cycloheximide. As
cycloheximide alone consistently reduced actin RNA expression, 28S and
18S RNAs are shown (Figure 8) as a control for equality of loading.
Cycloheximide increased the basal expression of lysozyme RNA in both
cell lines. C/EBPWT-ER induced lysozyme strongly and
C/EBPWT-ER induced lysozyme weakly even in cycloheximide,
similar to the inductions seen with estradiol alone. Cycloheximide
prevented induction of MPO RNA in each cell line, confirming the
activity of this reagent in these experiments. Actinomycin D, an
inhibitor of RNA polymerase, prevented induction of both C/EBP and
lysozyme by C/EBPWT-ER (Figure 8), suggesting that the increases
observed with estradiol are due to direct gene activation, and not to
increased RNA stability. The difference in basal expression of MPO and
C/EBP between the 32D-WT-ER and 32D-WT-ER-1 cells employed
in this experiment is typical of clonal variation seen among 32D cl3
subclones. Such variation is also evident when one subclone is cultured
at different times, as is evident if one compares MPO expression in
32D-WT-ER cells in Figures 1 and 8.
The major finding of this study is that C/EBPs are required for granulopoiesis beyond their ability to induce the expression of cytokine receptors. In addition, we have taken advantage of our ability to regulate C/EBP activities in 32D cl3 cells, both positively and negatively, to determine the role that C/EBPs play in the expression of several endogenous myeloid differentiation markers and transcriptional regulators. C/EBPs activities increase when 32D cl3 myeloblasts are transferred
from IL-3 to G-CSF,14 and overexpression of
either C/EBP To further characterize the role of endogenous C/EBPs in
granulopoiesis, we developed 32D cl3 lines expressing a
dominant-negative C/EBP, K Activation of K In the presence of exogenous G-CSFR signals, K We surveyed the effect of C/EBP inhibition and activation on a
group of myeloid differentiation markers and transcriptional regulators. The lysozyme and C/EBP As with the G-CSFR RNA, C/EBP Unexpectedly, K As with C/EBP K In summary, we have perturbed granulopoieis by globally inhibiting
C/EBP-regulated genes. The multiplicity of C/EBPs made it necessary to
employ a dominant-inhibitory protein for this purpose, and this
complicates interpretation of the observations, owing to the
possibility of protein-protein interactions. Despite this, we conclude
that C/EBPs contribute to granulopoiesis via induction of
lineage-specific markers, including MPO, lysozyme, and LF, via
induction of transcription factors, including PU.1 and C/EBP
We thank W. Wang for technical assistance, J. Flook for assistance with flow cytometry, and L. Cheng for the MIG vector.
Submitted October 11, 2001; accepted December 7, 2001.
A.D.F. was supported by National Institutes of Health grant R01 HL62274. A.D.F. is a Scholar of the Leukemia and Lymphoma Society and also receives support from the Children's Cancer Foundation.
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: Alan D. Friedman, Johns Hopkins University, Cancer Research Bldg, Rm 253, 1650 Orleans St, Baltimore, MD 21231, MD; e-mail: adfrdman{at}jhmi.edu.
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© 2002 by The American Society of Hematology.
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  Y. Chen, R. M. B. Costa, N. R. Love, X. Soto, M. Roth, R. Paredes, and E. Amaya C/EBP{alpha} initiates primitive myelopoiesis in pluripotent embryonic cells Blood, July 2, 2009; 114(1): 40 - 48. [Abstract] [Full Text] [PDF]
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 D. Wang, I. Paz-Priel, and A. D. Friedman NF-{kappa}B p50 Regulates C/EBP{alpha} Expression and Inflammatory Cytokine-Induced Neutrophil Production J. Immunol., May 1, 2009; 182(9): 5757 - 5762. [Abstract] [Full Text] [PDF]
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R. Bedi, J. Du, A. K. Sharma, I. Gomes, and S. J. Ackerman Human C/EBP-{epsilon} activator and repressor isoforms differentially reprogram myeloid lineage commitment and differentiation Blood, January 8, 2009; 113(2): 317 - 327. [Abstract] [Full Text] [PDF]
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 S. Iida, R. Watanabe-Fukunaga, S. Nagata, and R. Fukunaga Essential role of C/EBPalpha in G-CSF-induced transcriptional activation and chromatin modification of myeloid-specific genes. Genes Cells, April 1, 2008; 13(4): 313 - 327. [Abstract] [Full Text] [PDF]
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M. Geletu, M. Y. Balkhi, A. A. Peer Zada, M. Christopeit, J. A. Pulikkan, A. K. Trivedi, D. G. Tenen, and G. Behre Target proteins of C/EBP{alpha}p30 in AML: C/EBP{alpha}p30 enhances sumoylation of C/EBP{alpha}p42 via up-regulation of Ubc9 Blood, November 1, 2007; 110(9): 3301 - 3309. [Abstract] [Full Text] [PDF]
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S. Agrawal, W.-K. Hofmann, N. Tidow, M. Ehrich, D. v. d. Boom, S. Koschmieder, W. E. Berdel, H. Serve, and C. Muller-Tidow The C/EBP{delta} tumor suppressor is silenced by hypermethylation in acute myeloid leukemia Blood, May 1, 2007; 109(9): 3895 - 3905. [Abstract] [Full Text] [PDF]
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D. Wang, J. D'Costa, C. I. Civin, and A. D. Friedman C/EBP{alpha} directs monocytic commitment of primary myeloid progenitors Blood, August 15, 2006; 108(4): 1223 - 1229. [Abstract] [Full Text] [PDF]
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C. Guerzoni, M. Bardini, S. A. Mariani, G. Ferrari-Amorotti, P. Neviani, M. L. Panno, Y. Zhang, R. Martinez, D. Perrotti, and B. Calabretta Inducible activation of CEBPB, a gene negatively regulated by BCR/ABL, inhibits proliferation and promotes differentiation of BCR/ABL-expressing cells Blood, May 15, 2006; 107(10): 4080 - 4089. [Abstract] [Full Text] [PDF]
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 I. Paz-Priel, D. H. Cai, D. Wang, J. Kowalski, A. Blackford, H. Liu, C. A. Heckman, A. F. Gombart, H. P. Koeffler, L. M. Boxer, et al. CCAAT/Enhancer Binding Protein {alpha} (C/EBP{alpha}) and C/EBP{alpha} Myeloid Oncoproteins Induce Bcl-2 via Interaction of Their Basic Regions with Nuclear Factor-{kappa}B p50 Mol. Cancer Res., October 1, 2005; 3(10): 585 - 596. [Abstract] [Full Text] [PDF]
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 P. F. Johnson Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors J. Cell Sci., June 15, 2005; 118(12): 2545 - 2555. [Abstract] [Full Text] [PDF]
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 D. Vradii, S. K. Zaidi, J. B. Lian, A. J. van Wijnen, J. L. Stein, and G. S. Stein Point mutation in AML1 disrupts subnuclear targeting, prevents myeloid differentiation, and effects a transformation-like phenotype PNAS, May 17, 2005; 102(20): 7174 - 7179. [Abstract] [Full Text] [PDF]
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 A. Numata, K. Shimoda, K. Kamezaki, T. Haro, H. Kakumitsu, K. Shide, K. Kato, T. Miyamoto, Y. Yamashita, Y. Oshima, et al. Signal Transducers and Activators of Transcription 3 Augments the Transcriptional Activity of CCAAT/Enhancer-binding Protein {alpha} in Granulocyte Colony-stimulating Factor Signaling Pathway J. Biol. Chem., April 1, 2005; 280(13): 12621 - 12629. [Abstract] [Full Text] [PDF]
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A. D. Friedman A microarray window into granulocyte development and function Blood, February 15, 2005; 105(4): 1379 - 1379. [Full Text] [PDF]
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S. Gery, D. J. Park, P. T. Vuong, D. Y. Chih, N. Lemp, and H. P. Koeffler Retinoic acid regulates C/EBP homologous protein expression (CHOP), which negatively regulates myeloid target genes Blood, December 15, 2004; 104(13): 3911 - 3917. [Abstract] [Full Text] [PDF]
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V. Heath, H. C. Suh, M. Holman, K. Renn, J. M. Gooya, S. Parkin, K. D. Klarmann, M. Ortiz, P. Johnson, and J. Keller C/EBP{alpha} deficiency results in hyperproliferation of hematopoietic progenitor cells and disrupts macrophage development in vitro and in vivo Blood, September 15, 2004; 104(6): 1639 - 1647. [Abstract] [Full Text] [PDF]
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M. Schwieger, J. Lohler, M. Fischer, U. Herwig, D. G. Tenen, and C. Stocking A dominant-negative mutant of C/EBP{alpha}, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse Blood, April 1, 2004; 103(7): 2744 - 2752. [Abstract] [Full Text] [PDF]
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S. Gery, A. F. Gombart, Y. K. Fung, and H. P. Koeffler C/EBP{epsilon} interacts with retinoblastoma and E2F1 during granulopoiesis Blood, February 1, 2004; 103(3): 828 - 835. [Abstract] [Full Text] [PDF]
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 T. Kummalue and A. D. Friedman Cross-talk between regulators of myeloid development: C/EBP{alpha} binds and activates the promoter of the PU.1 gene J. Leukoc. Biol., September 1, 2003; 74(3): 464 - 470. [Abstract] [Full Text] [PDF]
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H. Liu, J. R. Keefer, Q.-f. Wang, and A. D. Friedman Reciprocal effects of C/EBPalpha and PKCdelta on JunB expression and monocytic differentiation depend upon the C/EBPalpha basic region Blood, May 15, 2003; 101(10): 3885 - 3892. [Abstract] [Full Text] [PDF]
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A. Khanna-Gupta, T. Zibello, H. Sun, P. Gaines, and N. Berliner Chromatin immunoprecipitation (ChIP) studies indicate a role for CCAAT enhancer binding proteins alpha and epsilon (C/EBPalpha and C/EBPepsilon ) and CDP/cut in myeloid maturation-induced lactoferrin gene expression Blood, May 1, 2003; 101(9): 3460 - 3468. [Abstract] [Full Text] [PDF]
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J. Kim, Y. Ogata, and R. A. Feldman Fes Tyrosine Kinase Promotes Survival and Terminal Granulocyte Differentiation of Factor-dependent Myeloid Progenitors (32D) and Activates Lineage-specific Transcription Factors J. Biol. Chem., April 18, 2003; 278(17): 14978 - 14984. [Abstract] [Full Text] [PDF]
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M. Mizuki, J. Schwable, C. Steur, C. Choudhary, S. Agrawal, B. Sargin, B. Steffen, I. Matsumura, Y. Kanakura, F. D. Bohmer, et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific Flt3 mutations Blood, April 15, 2003; 101(8): 3164 - 3173. [Abstract] [Full Text] [PDF]
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B.-T. H. Truong, Y.-J. Lee, T. A. Lodie, D. J. Park, D. Perrotti, N. Watanabe, H. P. Koeffler, H. Nakajima, D. G. Tenen, and S. C. Kogan CCAAT/Enhancer binding proteins repress the leukemic phenotype of acute myeloid leukemia Blood, February 1, 2003; 101(3): 1141 - 1148. [Abstract] [Full Text] [PDF]
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C. Schuster, K. Forster, H. Dierks, A. Elsasser, G. Behre, N. Simon, S. Danhauser-Riedl, M. Hallek, and M. Warmuth The effects of Bcr-Abl on C/EBP transcription-factor regulation and neutrophilic differentiation are reversed by the Abl kinase inhibitor imatinib mesylate Blood, January 15, 2003; 101(2): 655 - 663. [Abstract] [Full Text] [PDF]
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R. Zheng, A. D. Friedman, and D. Small Targeted inhibition of FLT3 overcomes the block to myeloid differentiation in 32Dcl3 cells caused by expression of FLT3/ITD mutations Blood, December 1, 2002; 100(12): 4154 - 4161. [Abstract] [Full Text] [PDF]
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P. Zhang, E. Nelson, H. S. Radomska, J. Iwasaki-Arai, K. Akashi, A. D. Friedman, and D. G. Tenen Induction of granulocytic differentiation by 2 pathways Blood, May 29, 2002; 99(12): 4406 - 4412. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
/
and CHOP
are dominant inhibitory by virtue of their ability to dimerize with
other C/EBPs and their lack of intact basic
regions.
) or with
4HT (
). Viable cell counts performed daily are shown. (B) Cells from
these same cultures were cytospun and subjected to Wright-Giemsa
staining. Cells are shown in IL-3, after transfer to G-CSF in the
absence of 4HT for 2, 4, or 6 days (G2, G4, G6), or after transfer to
G-CSF in the presence of 4HT for 1, 2, 4, or 8 days (G1, G2, G4, G8).
Original magnification ×100. (C) The 32D-K
/+
d3). Total cellular RNAs prepared on days 0, 1, 2, 3, 4, 5, and 6 were subjected to Northern blot analysis, 20 µg per lane, for
MPO, lysozyme (Lys), LF, C/EBP
