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A Novel Temporal Expression Pattern of Three C/EBP Family Members in
Differ entia t i ng My el om onocyt i c Cell s
By Linda M. Scott, Curt I. Civin, Pernille Rorth, and Alan D. Friedman
Members of the CCAAT/enhancer binding protein (C/ EBP]
family have been shown to regulate the terminal differentiation of adipocytes and hepatocytes. In these cell lineages,
high levels of C/EBPa are found only in mature, nondividing
cells. UsingWestern blotting and immunohistochemicalstaining, we have determinedthe temporal order of expression for
C/EBPa, C/EBPp, and C/ EBPG in differentiatingmyelomonocytic marrow cells. These studies show a unique temporal
pattern of C/EBP isoform expression in the myeloid lineage.
In particular, C/EBPa expression is very high in proliferative
myelomonocytic cells, and diminishes during phenotypic
maturation. While we have detected C/EBPa, C/EBPp, and
C/EBPG in multiple myeloid leukemia cell lines, and C/EBPa
in normal myeloid cells and in de novo human myeloid
leukemias, we have not detected these C/EBP isoforms in
either erythroid or lymphoid cells. Finally, we show that
C/EBPa. C/EBPp, and C/EBPS protein and messenger RNA
levels correlate in maturing granulocytic cells. The formation
of tissue-specific combinations of C/EBP homodimers and
heterodimersmay allow this family of transcriptionfactors to
regulate different sets of genes in adipocytes, hepatocytes,
and myelomonocytes.
o 1992by The American Society of Hematology.
T
function of granulocytic differentiation. Although variability of this cell line in culture resulted in some quantitative
differences between experiments, a surprisingly different
pattern from that described for adipocyte development was
evident. Most notably, C/EBPa was found to be expressed
at a high level in dividing myeloblasts and to diminish to low
levels during their terminal differentiation into polymorphonuclear leukocytes (PMNs). We will discuss the implications of this novel temporal pattern of C/EBP isoform
expression.
HE CCAAT/enhancer-binding protein (C/EBP) family of transcription factors consists of several proteins
with highly homologous dimerization and DNA contact
domains. The founding member of this family, C/EBPa,
was originally purified from rat
This protein binds
DNA as an obligate homodimer via a basic region-leucine
zipper (bZIP) domain.'-6 The C/EBP family also includes
C/EBPP (also known as NF-IL6, LAP, IM-DBP, AGP/
EBP, and CRP2),7-12CIEBPG (also known as CRP3),"J2
and C/EBPy (also known as Ig/EBP-1).I3 The various
C/EBP family members can homodimerize, but can also
heterodimerize and maintain DNA-binding activity.11-13
Members of the C/EBP family, especially C/EBPu, have
been implicated in regulating the terminal differentiation of
several mammalian cells. Although present at low levels in
hepatoma cell lines, C/EBPa is expressed at high levels in
mitotically quiescent hepatocytes, in which it is believed to
regulate a variety of hepatocyte-specific genes.14J5 Similarly, although absent from 3T3-Ll preadipocytes, C/EBPa
is expressed at high levels when these cells differentiate into
nondividing adipocytes.11J6C/EBPa is capable of activating
several "fat-specific'' genes in 3T3-Ll cells, including steroylCoA desaturase and the insulin-responsive glucose transp ~ r t e r . ' ~ JSelective
*
inhibition of C/EBPa expression in
differentiating adipocytes by expression of antisense
C/EBPa RNA reduced the ability of these cells to form
lipid droplets, a phenotype of their terminally differentiated ~ t a t e . ' In
~ ,contrast
~~
to C/EBPa, C/EBPP and C/EBPG
are expressed highest in the early stages of adipocyte
formation." The ability of C/EBP family members to
heterodimerize may allow complex regulation of hepatocyte and adipocyte development.21
In both hepatocytes and adipocytes, high levels of C/EBPa
expression have been found only in nondividing cells.
Moreover, when C/EBPa is ectopically expressed in dividing preadipocytes, mitotic proliferation ceases.22Therefore,
it has been proposed that C/EBPu may regulate a genetic
program that blocks cell divi~ion.~~J2
We have now discovered that members of the C/EBP
family are also expressed in myelomonocytic cells of human
and rodent bone marrow. Using the myelomonoblastic
murine cell line 32D C13, a valuable model of granulocytic
myeloid differentiati0n,2~,~~
we have examined the expression profile of C/EBPa, C/EBPP, and C/EBPS as a
Blood, Vol 80, No 7 (October 1). 1992: pp 1725-1735
MATERIALS AND METHODS
Cell culture, marrow cells, and human leukemia cells. 32D C13
cells23 were maintained at 37"C, 5% C o t in Iscove's modified
Dulbecco's medium (IMDM) supplemented with 10% heatinactivated fetal bovine serum (HI-FBS) and 5% WEHI-3B supernatent as a source of interleukin-3 (IL-3).25 For induction of
granulocytic differentiation, cells were washed twice with phosphatebuffered saline (PBS) and placed in IMDM/lO% HI-FBS supplemented with 500 U/mL granulocyte colony-stimulating factor
(G-CSF; Amgen, Thousand Oaks, CA). Cell morphology was
assessed by Wright-Giemsa ~taining.2~
HL-60, SP2, U937, PLB985, KGla, HEL, K.562, Molt-3, Daudi, and WEHI 274.1 cells were
maintained in RPMII10% HI-FBS. P388D1 and IC-21 cells were
grown in Dulbecco's minimum essential medium (DMEM)/ 10%
HI-FBS. Cytotoxic T-cell lymphocytic leukemia (CTLL) cells were
provided by A. Hess (John Hopkins Oncology Center), and
From the Division of Pediatric Oncology, Zhe Johns Hopkins
Oncology Center, Baltimore, MD; and the Department of Embyology,
Camegze Institution of Washington, Baltimore, MD.
SubmittedApnl IO, 1992; accepted June 15,1992.
Supported by grants (to A.D.F.) from the W.W. Smith Charitable
Trust, the Searle Scholars ProgramlChicago Community Trust, and
the National Institute of Health (CA01326), by Grant No. CH-480
from the American Cancer Society (to C.I.C.), and by a grant from the
Danish Medical Research Council (toP.R.).
Address reprint requests to Alan D. Friedman, MD, Division of
Pediatnc Oncology, Johns Hopkins Oncology Center, Room 3-109,
600 N Wove St, Baltimore, MD 21287.
The publication costs of this article were defrayed in part by page
charge payment. Thul article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.sectton 1734 solely to
indicate thul fact.
0 I992 by The American Society of Hematology.
0OO4-4971/92l8007-0020$3.00l0
1725
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SCOTT ET AL
1726
induced 3T3-Ll cells were provided by Z. Cao (Carnegie Institution).
Normal human marrow cells were obtained from the posterior
iliac crest of volunteer donors. Approval was obtained from the
Institutional Review Board for these studies. Informed consent
was provided according to the Declaration of Helsinki. CD15+ cells
were isolated as described.2628 In brief, marrow was first layered
onto a cushion of Ficoll-Hypaque (Pharmacia, Uppsala, Sweden),
and mononuclear cells were collected from the interface after
centrifugation at 5OOg for 30 minutes at 22°C. The cells were then
washed twice and incubated for 30 minutes at 4°C in RPMI/10%
HI-FBS, with immunomagnetic Dynabeads M-450 (Dynal, Great
Neck, NY), 5 beads per cell, to which had been adsorbed CD15
monoclonal antibody MOA^).^^ Beads were collected with a
magnet and washed, and protein extraction buffer (see below) was
added. The morphology and number of cells in the initial and
unbound samples were determined by Wright-Giemsa staining of
cytospin slides.
Human leukemia cells, obtained from bone marrow or peripheral blood at diagnosis or relapse and stored in liquid nitrogen,
were thawed, washed three times in the presence of DNase1 (100
UlmL), subjected to Ficoll-Hypaque centrifugation as above to
remove dead cells and debris, washed, counted, and extracted.
Each sample contained greater than 95% leukemic blasts, as
verified by morphologic examination.
Protein extraction and Western blotting. For protein extraction,
cells were pelleted, washed with PBS, and extracted with 3 mL of
10 mmol/L Tris, pH 7.5, 2 mmol/L EDTA, 1% sodium dodecyl
sulfate (SDS). The extract was then sonicated and concentrated by
precipitation with four volumes of acetone at -20°C. The precipitates were resuspended in sample buffer by boiling for 5 minutes
and applied to 10% polyacrylamide gels,)O 4 x lo6 cell equivalents
per lane. After electrophoresis. the proteins were transferred to
nitrocellulose?1 The integrity and levels of transferred proteins
were determined by staining the blots with Ponceau S (0.2% in 2%
trichloroacetic acid [TCA]) followed by destaining with 5% glacial
acetic acid.
Filters were then washed extensively with Tris-buffered salineTween (TBS-T, 150 mmol/L NaCI, SO mmol/L Tris, pH 7.5, 0.3%
Tween-20), blocked for 20 minutes with TBS-T/5% nonfat dried
milk (NFDM), incubated at room temperature for 1 hour with
antisera diluted 1:1,000 in TBS-T/0.25% NFDM, washed three
times with the same solution, incubated with horseradish peroxidase (HRP)-conjugated donkey antirabbit Ig (1:15,000), washed
with TBS-T three times, and developed with ECL reagents
(Amersham, Arlington Heights, IL) as described by the manufacturer. Developed filters were then exposed to Kodak XAR film
(Eastman Kodak, Rochester, NY).
C/EBPa peptide antisera3-16and the C/EBPP and C/EBPG
antisera” were kindly provided by 2. Cao and S. McKnight
(Carnegie Institution). The C/EBPa protein antiserum was prepared by expression of CIEBPAl-2 protein3z in Escherichia coli
using the pT5 expression system33 and purified to apparent
homogeneity by heat treatment and clearing of bacterial lysate,
followed by chromatography on S-Sepharose. The protein was then
concentrated and used to immunize rabbits (Spring Valley Laboratories, Woodbine, MD). The myeloperoxidase (MPO) antiserum34
was kindly provided by W. Nauseef (University of Iowa).
Immunohistochemical staining. Immunohistochemical staining
was performed as described.35In brief, lo5cells were cytospun onto
glass slides and fixed for 30 minutes on ice in 4% paraformaldehyde/
PBS. Slides were then rinsed (and stored at 4°C) with PBT (PBS,
0.2% bovine serum albumin [BSA], 0.05% Tween-20), blocked
with PBT containing 2% goat and 2% human AB+ sera, aspirated,
and antisera diluted 1:500 in the same solution were then added for
1 hour. Slides were then washed and incubated in PBS/60%
methanol/3% H z O for
~ 5 minutes to inactivate endogenous peroxidase. After washing, the slides were then developed using biotinylated antirabbit Ig and avidin-biotin/HRP (Vector Labs, Burlingame, CA) as described by the manufacturer. Slides were then
incubated with diaminobenzene (1 mg/mL) and H202 (0.1%) for
30 minutes, washed, incubated with 20 mmol/L CuSO4/86 mmol/L
NaCl for 5 minutes, washed, and counterstained with 0.001% Fast
Green FCF (Sigma, St Louis, MO) in 1% acetic acid.
RNA extraction and Northern blotting. Total cellular RNA was
prepared using the acid guanidinium thiocyanate p r ~ c e d u r eFor
.~~
Northern blotting, RNA samples ( lo7 cell equivalents) were
separated on 1% agaroseiformaldehyde gels and transferred3’ to a
nylon membrane (Genescreen; New England Nuclear, Boston,
MA). Filters were prehybridized at 68°C in a solution3s containing
0.5 mol/L NaP04, pH 7.0, 1 mmol/L EDTA, 7% SDS, 10%
dextran sulfate, and hybridized similarly after the addition of
probes labeled with 32Pby random priming.39 CIEBPa, CIEBPP,
C/EBPG, MPO, and mouse P5-tubulin cDNAs used for labeling
have been d e s ~ r i b e d . ”Filters
, ~ ~ were then washed to a stringency
of 0.1 x SSC/O.5% SDS at 68°C and exposed to Kodak XAR film at
-70°C.
RESULTS
CIEBPa CIEBPP, and CIEBPS expression during granulopoiesis in 320 C13 cells. 32D C13 cells divide with a
generation time of approximately 18 hours in the presence
of IL-3. Upon removal of IL-3 and exposure to G-CSF, they
undergo, asynchronously, a 6- to 14-day program of differentiation, resulting in terminally differentiated granulocytes. Cell division continues, with a generation time of 24
hours, during the first half of this program, and then cell
division ceases.
32D C13 cells growing in IL-3 were washed and placed in
G-CSF-containing media. Differentiation was monitored
by morphologic examination of the cells (Fig 1A). Uninduced cells were large myeloblasts. Several of the cells have
phagocytosed dust particles. After 1 day in G-CSF, the cells
diminished in size and had begun chromatin condensation.
By day 4, promyelocytes predominated, with prominent
primary granules. By day 6, myelocytes, bands, and PMNs
were evident.
Protein extracts from this culture were analyzed for the
presence of CIEBPa (Fig lB), using two different antisera.
One antiserum was raised against the entire CIEBPa (top
panel), and the other was raised against an internal
C/EBPa peptide unique to that protein (middle panel).
C/EBPa, a 42-Kd protein, was present at high levels in the
uninduced cells, increased approximately twofold during
the first day of differentiation, was maintained for several
days, and then diminished to low levels by day 6 of
differentiation. Occasionally, C/EBPa resolved into closely
spaced bands, perhaps alternatively modified forms (eg, Fig
lB, top panel; and also see Figs 2, 3, and 5). On the other
hand, these multiple bands were not evident in some blots
(Fig lB, middle panel; and Fig lC, top panel). This
discrepancy may result from variability between cultures
and/or differences in gel conditions. The integrity of the
cellular proteins in these extracts was confirmed by staining
the blots with Ponceau S (Fig lB, bottom panel). Protein
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ClEBP EXPRESSION IN MYELOID CELLS
1727
A
4- I
"d.
I
Fig 1. Expression of ClEBPa in differentiating 320 C13 cells and
comparison with ClEBPp and ClEBPS expression and with ClEBP
expression in 3T3-Ll cells. 320 C13 cells growing in IL-3 were induced
for granulocytic differentiation by exposure t o G-CSF. (A) Cell morphology was determined, by Wright-Giemsa staining of cytospins, for
uninduced cells (IL-3) and for cells exposed t o G-CSF for 1,4, or 6 days
(Gl, G4, or G6, respectively). (e)Total cellular protein extracts were
obtained daily, and C/EBPa levels were determined by Western blotting using an antisera raised against bacterially expressed C/EBPa (top
panel), or against an internal C/EBPa peptide (middle panel). MPO
levels were determined using an antisera raised against human MPO
(bottom panel). Protein integrity and levels in these extracts were
assessed by staining one of these blots with Ponceau S before blocking
and incubation with antiserum (bottom panel). (C) A set of protein
extracts obtained from a second culture of induced 320 C13 cells was
probed with an antiserum raised against a C-terminal ClEBPa peptide
(top panel) or with antisera specific for ClEBPB or ClEBPG (middle
panel). Extracts from an equivalent number of 3T3.Ll cells were also
analyzed; these extracts were taken from cells induced t o form adipocytes for either 7 days (top panel) or 2 days (middle panel). Total cellular
proteins were visualized by Ponceau S staining (bottom panel). Extracts
from 4 x lo" cells were loaded in each lane. The positions of the
molecular weight markers are indicated.
68 kd
42kd
CIEBPt
CEBPa
42kd
42kd
CIEBPG
1
-26kd
-68kd
42kd
concentrations were similar (within twofold) in these extracts.
Finally, the same extracts were analyzed for the level of
MPO (Fig lB, bottom panel), a protein present in primary
myeloid granules. The large subunit of murine MPO,
migrating with an apparent molecular weight of 67 Kd,
achievedits highest l&el per cell on days 3 and 4. The late
decrease in MPO expression may reflect the diminished
MPO messenger RNA (mRNA) expression observed in
fully differentiated 32D C13 cells." The two bands detected
between 25 and 27 Kd on the MPO blot represent direct
binding of the secondary antibody to endogenous proteins
(as will be shown in the bottom panel of Fig 3B). They were
detected with seven different primary antisera and they
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SCOlT ET AL
serve, to some extent, as another control for protein
integrity.
Granulocytic differentiation was also induced in a second
culture of 32D C13 cells. Aliquots were removed periodically and used to prepare total cellular protein. These
extracts were analyzed for C/EBPa using a third specific
antiserum, raised against a C-terminal peptide also unique
to that protein (Fig lC, top panel). These extracts were also
analyzed for C/EBPP and C/EBPG using antisera raised
against peptides specific for those proteins (Fig lC, middle
panels). For comparison, extracts from an equivalent number of induced 3T3-Ll preadipocytes were also analyzed.
Again, C/EBPa was expressed at high levels early, but not
late, in the granulocytic differentiation program; these high
levels were similar to the maximum obtained by C/EBPa,
on day 7, in induced 3T3-Ll cells. Thus, C/EBPa was
detected in 32D C13 cells using three different antisera.
In contrast, C/EBPP, a 31-Kd protein, increased during
granulocytic differentiation of 32D C13 cells. C/EBPP
achieved levels similar to the maximum obtained by this
C/EBP isoform, on day 2, in induced 3T3-Ll cells. Crossreactive material located between 25 and 27 Kd again
represents direct binding of the secondary antibody. Finally, C/EBPG, a 29-Kd protein, was expressed at a very low
level in 32D C13 cells maintained in IL-3. When these cells
were transferred to media containing G-CSF, the level of
this C/EBP isoform first increased markedly and then
diminished somewhat as the cells progressed to terminally
differentiated granulocytes. Induced 32D C13 cells expressed C/EBPG at levels higher than the maximum found
in 3T3-Ll cells. Of note, these data do not allow determination of the absolute levels of C/EBPa, C/EBPP, and
C/EBPG, nor the ratios of these levels, as each antiserum
has a different affinity for its corresponding epitope. In
A
NI
P1
P2
D1
D2
NI
addition, the radiographic exposure times of the various
Western blots in this report have not been normalized to an
internal standard. However, as extract corresponding to
equal cell numbers were loaded in all lanes, comparisons
are valid between lanes on any given blot.
The integrity and level of intracellular proteins recovered
in these extracts was again monitored by Ponceau S staining
(Fig lC, bottom panel). Total cellular protein diminished
approximately twofold during granulocytic differentiation.
Given the modest decrease in total cellular protein in this
experiment, and given the large decrease in nuclear volume
during granulocytic differentiation, it is difficult to be
certain that C/EBPa nuclear concentration actually diminishes in 32D C13 cells as they differentiate. This uncertainty
will be addressed directly using immunohistochemicalstaining (see below). On the other hand, the nuclear concentrations of C/EBPP and C/EBPG evidently increase during
granulocytic differentiation, as will be confirmed for
C/EBPP immunohistochemically.
CIEBPa expression during differentiation of HL-60 cells.
The expression of C/EBPa was also determined in a human
model of myeloid differentiation, HL-60 cells.40HL-60 cells
were induced to differentiate along either the monocytic
lineage with phorbol ester (phorbol myristate acetate
[PMA]) or along the granulocytic lineage with dimethylsulfoxide (DMSO). Extracts were prepared on days 0,1, and 2
and analyzed for C/EBPa expression (Fig 2A, left). Total
protein integrity and levels in these extracts were also
assessed (Fig 2A, right). C/EBPa levels were high in
uninduced, rapidly dividing HL-60 cells, and diminished
markedly during both monocytic and granulocytic diffcrentiation of these cells. The diminution is more rapid than in
32D C13 cells; this difference could be accounted for if
uninduced HL-60 cells are more mature than uninduced
P1
ClEBPtr
B
ClEBPa
NI Dlh 2h 4h
8h
24h Pfh 2h
P2
D1
D2
I
4h 8h
24h
Fig 2. Expression of ClEBPa
in differentiating HL-80 cells. (A)
Uninduced HL-60 cells (NI) or
HL-60 cells induced for monocytic differentiation with 50
nmollL phorbol ester (PMA) for
1 (Pl) or 2 (P2) days or for granulocytic differentiation with 1.2%
DMSO for 1 ( D l ) or 2 (D2) days
were analyzed for ClEBPa expression (left) as in Fig 1B (top
panel). By Wright's staining, NI
cells were promyelocytic, P1 and
P2 cells were monocytic, and D1
and D2 cells were myelocytic and
metamyelocytic. Total cellular
proteinswere visualized by Ponceau S staining (right). (6) A second culture was induced similarly, and C/EBPa expression
determined for uninduced cells
(NI)and for cells induced for 1,2,
4,s. or 24 hourswith DMSO (Dlh
through 24h) or with phorbol ester (Plh through 24h).
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C/EBP EXPRESSION IN MYELOID CELLS
32D C13 cells. The small increases noted on day 2 may
result from the growth of PMA- and DMSO-resistant
subpopulations of cells.
To determine whether the observed marked decrease in
CIEBPa expression is an immediate-early (within several
hours) response to induction, additional HL-60 cultures
were treated with either DMSO or PMA and protein
extracts from hours 0, 1, 2, 4, 8, and 24 were prepared and
analyzed for C/EBPa expression (Fig 2B). No decrease in
C/EBPa was observed during the first 4 hours of induction,
though marked reduction was again observed by 24 hours.
Of note, our CIEBPP and C/EBP8 antisera detected
C/EBPP or C/EBPS in murine (see below) but not in
human (data not shown, A.D.F., June 1991) cell lines. This
observation suggests that these antisera, raised against
peptides from the murine forms of these proteins, react in a
species-specific manner. Indeed, the CIEBPP epitope is
not conserved between humans and
CIEBPcu, CiEBPfi and CIEBPS are expressed in myelomonocytic, but not in lymphoid or elythroid, leukemia cells.
To determine whether CIEBPa, C/EBPP, and CIEBPS are
present in other hematopoietic cell lines, extracts from
several murine leukemic cell lines were prepared and
analyzed (Fig 3A). None of these C/EBP isoforms were
detected in uninduced murine erythroleukemia (MEL)
(erythroid lineage) cells, SP2 (B-lineage) cells, or CTLL
(T-lineage) cells. A very low level of C/EBPa was evident in
8-day induced MEL cells. Two additional bands located just
below C/EBPP and CIEBPs again represent nonspecific
binding of the secondary antibody to endogenous proteins,
as shown in the bottom panel of Fig 3A, wherein these
extracts have been probed with secondary antibody alone.
The degree of nonspecific binding to these proteins varied
between extracts and between experiments.
C/EBPa was detected in the immature myelomonocytic
cell line, WEHI 274.1 (and in induced 32D C13 cells), but
not in the more mature macrophage cell lines P388D1 and
IC-21. This pattern is reminiscent of the diminished expression of CIEBPa observed during differentiation of 32D C13
and HL-60 cells (Figs 1 and 2). C/EBPP was expressed
most highly in the more mature P388D1cells, again reminiscent of the increased expression of C/EBPP observed as
32D C13 cells differentiated into mature granulocytes (Fig
1C). Finally, C/EBPS was present in WEHI 274.1 cells, as
well as in 32D C13 cells.
We also examined the expression of C/EBPa in a variety
of human leukemia cell lines (Fig 3B). This protein was
present in all four human myelomonocytic cell lines examined (U937, HL-60, PLB-985, and KGla). However,
CIEBPa was absent from both human erythroid (HEL and
K562) cell lines, from the B-lineage cell line (Daudi), and
from the T-lineage cell line (Molt-3).
In addition, we determined the expression of C/EBPa in
human myeloid and lymphoid leukemias, using cryopreserved marrow or peripheral blood samples obtained from
patients at diagnosis or relapse (Table 1). Nine of 10
myeloid leukemias expressed this transcription factor,
whereas C/EBPa was not detected in any of the seven
lymphoid leukemias examined.
1729
C/EBPa is expressed in normal myeloid cells. To determine whether C/EBPa is present in normal myelomonocytic cells, a virtually pure population of developing myeloid
cells was isolated from human marrow, extracted, and
analyzed for C/EBPa by Western blotting (Fig 3C). CIEBPa
was detected in these cells.
To further examine the lineage-specific expression of
CIEBPa, cytospins of human marrow were incubated with
either normal rabbit serum or C/EBPa antiserum, and
bound antibodies were detected immunocytochemically
(Fig 4a and b). The predominant CIEBPa staining was in
cells with eccentric, kidney-shaped nuclei, representing
fairly mature granulocytic cells. Very few of the cells with
round nuclei (the erythroid and lymphoid cells) showed
specific staining. Interestingly, all of the fully mature PMNs
(arrow, lower right) and some of the small band forms
stained at background levels. This staining pattern suggests
that there is diminished expression of CIEBPa in these
most mature granulocytic cells. As will be shown, immunohistochemical staining of induced 32D C13 cells also
demonstrated decreased CiEBPa levels in the nuclei of
these mature cells.
CIEBPa and C/EBPpare present in the nuclei of 3.20 C13
cells. One mechanism that might allow dividing myeloblasts to tolerate high levels of CIEBPa would be to
sequester this protein in the cytoplasm, as can occur for
C/EBPP.41 As the majority of myeloid cells in human
marrow (such as in Fig 4b) are more mature, nondividing
cells, we addressed this possibility using 32D C13 cells.
Cytospins of uninduced, 4-day- and 8-day-induced 32D
C13 cells were stained with either normal rabbit serum (Fig
4c and data not shown), C/EBPa antiserum (Fig 4d through
f), or C/EBPP antiserum (Fig 4g through i). Bound antibodies were again detected immunocytochemically. Rabbit
serum gave only light staining. Both C/EBPa and C/EBPP
were detected only in the nucleus of positively staining cells.
In this experiment, the 32D C13 cells differentiated at a
slower rate than those shown in Fig 1; PMNs first became
evident on day 8. The 32D C13 cells manifested significant
variability in their rate of differentiation, perhaps accounting for quantitative differences in C/EBP isoform, MPO,
and total protein levels at particular points in time between
experiments. Nevertheless, a consistent qualitative temporal pattern of C/EBP isoform expression was evident in
these cells, as will be discussed. Although quantification of
protein expression is imprecise using immunohistochemical
staining, large changes in staining intensity are qualitatively
meaningful. Interestingly, although uninduced and 4-dayinduced 32D C13 cells stained similarly for CIEBPa (Fig 4d
and e), by day 8 all of the PMNs and some of the small band
forms stained only at background level. These results are
consistent with the diminishing average level of C/EBPa
per cell detected by Western blotting as 32D C13 cells
differentiate (Fig 1B). The presence of large, immature
cells on day 8, which show heavy staining for C/EBPa, is a
consequence of the slower maturation of this culture.
CIEBPP staining increased steadily as the cells matured
(Fig 4g through i), again consistent with the results of
Western blotting (Fig 1C).
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SCOTT ET AL
1730
5
A
42kd-
C/EBPa
Fig 3. Expression of C/EBPa. CIEBPP, and C/EBPS in murine cell
lines, and of ClEBPa in human cell lines and in normal human myeloid
cells. (A) Protein extracts from the murine cell lines MEL (erythroid), SP2
(6-lineage), CTLL (T-lineage), IC-21 and P338D1 (macrophage), and
WEH1274.1 (myelomonoblastic) and 32D C13 induced for 6 days with
G-CSF (32D-G6) were analyzed for C/EBPaand, in some cases, C/EBPB
and C/EBPS expression by Westem blotting (top three panels). MEL
cells were either uninduced (MEL-NI) or induced for erythroid differentiation by 8 days of exposure t o 2% DMSO (MEL-DO). Two bands were
detected between 25 and 27 Kd in many lanes due t o nonspecific
binding of the secondary antibody t o endogenous proteins. This is
shown in the bottom panel wherein several of these extracts were
hybridized only with this second antibody. CRM, crossreactive material.
(9) Protein extracts from the human leukemia cell lines U937, HL-60,
KGla and PLB-985 (myelomonoblastic), HEL and K562 (erythroid),
Daudi (6-lineage), and Molt-3 (T-lineage) were analyzed similarly for
C/EBPa expression. (C) Myeloid cells were isolated from human marrow by adsorption onto immunomagnetic beads t o which had been
bound monoclonal anti-CD15 antibodies. Protein extracts from these
cells (CD15) and from beads with bound antibodies alone (Ab) were
again analyzed for C/EBPa expression. The CD15- cells were a viltually
pure population of myelomonocytic cells, with approximately 4%
myeloblasts, 8% promyelocytes, 28% myelocytes, 40% metamyelocytes, 16% bands,and4% PMNs.
ClEBPB
26kd-
C/EBPG
26kd.
26kd-
--
CRM
_.
v)
" Z I 9
B
5
c
n
a
b
0
I
C/EBPa
Regulation of C / E B P s CIEBPP, and CIEBPG levels
duringgranulopoiesis. To address the regulation of expression of C/EBP family members during granulopoiesis, we
probed Northern blots containing total cellular RNA from
an equal number of uninduced and induced 32D C13 cells
with radiolabeled cDNAs specific for either C/EBPa,
C/EBPP, C/EBPG, MPO, or tubulin (Fig SA). For comparison, an aliquot of cells from the same cultures used to
prepare the RNAs was used to prepare total cellular
proteins, and these cells were analyzed for C/EBPa,
CIEBPP, and C/EBPG, MPO, and total protein expression
(Fig SB). Monitoringof cell morphology showed that PMNs
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
1731
C/EBP EXPRESSION IN MYELOID CELLS
Table 1. C/EBPu Expression in Human Leukemias
Patient
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
-
CIEBPu
Diagnosis
AM L
AM L
AM L
AM L
AM L
AM L
AM L
AM L
AML
AML*
BALL
B-ALL
B-ALL
B-ALL
B-ALL
T-ALL
T-ALL
~_____
Surface Phenotype
DR, CD11b, 15,33
CD33
DR, CD11b, 15,33
DR, CD15.34
DR, CD15,33,34
DR, CD7. 15,34
DR, CD7
Unknown
DR,CDllb, 15
DR, CD7.33,34
DR, CD19,22,34
DR, CD10,19,22
CD10,19,22,34
DR, CD19,22,34
DR, CD19
CD2. 5, 7,34
CD2.5, 7,8
Expression
++++
++++
++++
+++
++
++
+
+
+
-
~
Marrow or peripheral blood cells were obtained from patients at
diagnosis or relapse and stored in liquid nitrogen. Surface phenotypes,
which help establish the diagnoses, were determined by FACS analysis26,28before freezing. Cells were then thawed and analyzed for C/EBPa
expression by Western blotting.
Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphocytic leukemia.
'Relapse sample from a patient initially diagnosed as ALL.
first became evident at day 10 in this experiment. Total
protein diminished twofold to threefold during granulocytic
differentiation, similar to the experiment described in Fig
1C. C/EBPa, CIEBPP, and C/EBPS mRNA and protein
levels correlated fairly well throughout 32D C13 cell differentiation, suggesting predominant pretranslational regulation of these three C/EBP isoforms.
MPO mRNA was induced by day 4, and tubulin mRNA
diminished somewhat as the 32D C13 cell matured. MPO
protein levels paralleled MPO mRNA levels and remained
high on day 10 of induction. Of note, MPO protein did not
show a diminution as it had in Fig lA, perhaps reflecting
the fact that the 10-day-induced culture in this experiment
was not as mature (10% PMNs) as the 6-day-induced
culture in the former experiment (50% PMNs).
DISCUSSION
Whereas some transcription factors, such as M ~ o and
D ~
O ~ t - 2 appear
, ~ ~ to be truly restricted to a single tissue,
others, such as GATA-1,44,45and HNF-1,46,47have been
found in several cell types. CIEBPa, C/EBPP, and C/EBPG
fall into this latter group. We have shown herein that these
three C/EBP isoforms are highly expressed in myelomonocytic cells. This finding is consistent with the recent description of C/EBP DNA-binding activity in chicken myeloid,
but not erythroid or lymphoid, cells.48 C/EBPs are also
expressed in hepatocytes and adipocytes, and perhaps in
lung and intestinal tissues as well, but have not been
detected in kidney, brain, heart, testis, or spleen.11J2J6
Although we also did not detect C/EBPa in lymphoid or
erythroid cells, a subset of these cells may well express this
protein. Indeed, the mRNA for C/EBPy is expressed most
highly in B lymphocyte^.^^
C/EBPa has been implicated in the regulation of both
hepatic-~pecific~~J~
and adipocyte-specific gene^.'^,'^ The
presence of several DNA-binding proteins capable of
binding the C/EBP sites within these genes (eg, see Mueller
et aP9) has made it difficult to ascertain which protein(s)
actually mediates transcription through these DNA elements. However, recent experiments using antisense RNA
expression have directly implicated C/EBPa as a regulator
of a d i p o g e n e s i ~ . ~ ~ , ~ ~
The inability of preadipocytes to divide when overexpressing C/EBPLX~~
suggests that this transcription factor might
also activate genes that inhibit cell division. Expression of
C/EBPa even overcame the proliferative and antidifferentiative effects of overexpressed Myc in preadipocytes, and
induced cessation of cell division and differentiati~n.~~
In
contrast, C/EBPa's expression pattern in differentiating
myeloid cells suggests that it may even activate genes, such
as those encoding protein hormones, which stimulate cell
division in these cells. For example, in chicken myeloid
cells,@a C/EBP-family member has been implicated in the
activation of the gene encoding chicken myelomonocytic
growth factor (cMGF), a homologue of human G-CSF.51
Moreover, the human G-CSF promoter contains a C/EBP
binding site active in macrophage^^^ that binds C/EBPf3.53
C/EBPa, along with C/EBPp and CIEBPG, is one of only
a few tissue-restricted transcription factors known to be
present in immature myeloid cells (see Hromas et aIs4for a
recent review of hematopoietic transcription factors). C-myb
is predominantly expressed in hematopoietic cells,55 can
induce myeloid-specific
and is required for fetal
hematopoiesi~.~~
CCAAT-displacement protein is a ubiquitous repressor that participates in the regulation of the
myeloid-specific gp91-phox gene.58 MZF-1, a zinc-finger
protein that plays a role in granulocyte de~elopment,5~
is
only expressed in later-stage granulocytic c e k m The activity of a helix-loop-helix protein(s) has recently been shown
to be required for the early stages of 32D C13 cell
differentiatione61
Three models of how C/EBPa might participate in the
activation of different sets of genes in myeloid, adipocyte,
and hepatic cells are: (1) a different set of C/EBPa's several
trans-activating domains32might be active in each tissue; (2)
~ CiEBPa might dimerize with a different subset of C/EBP
family members in each tissue; or (3) C/EBPa might
dimerize with a predominant partner, but interact with
additional, tissue-restricted transcription factors in each
lineage.
The ability of C/EBP family members to heterodimerize
readily in ~ i t r o l suggests
~ . ~ ~ that they could do so in vivo as
well. The expression pattern of C/EBPa, C/EBPP, and
CIEBPS in differentiating preadipocytes suggests that PP,
66, and PS dimers could predominate early and that aa
homodimers could predominate late in adip0genesis.l'
Determination of the pattern of C/EBP isoform expression
in maturing 32D C13 myeloblasts is confounded somewhat
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S C O T ET AL
1732
Fig 4. In situ antibody staining of human manow and of 32D
C13 cells. Cytospins of human
marrow were stained with either
normal rabbit serum (a) or with
rabbit anti-C/EBPa protein antiserum (b). Bound antibodieswere
detected immunocytochemically
and the cells were then counterstained with Fast Green FCF. The
arrow at the lower right in (b)
indicates a PMN. (c) 32D C13
cells were Induced with GCSF
for 4 days and stained with normal rabbit serum. (d through f l
320 C13 cells were induced for 0,
4, or 8 days and stained with
a n t i 4 1EBPa protein antiserum.
(g through i) 32D C13 cells were
induced for 0, 4, or 8 days and
stained with anti-C/EBPp antiserum. Photomicrographs are at
40x.
by the variability of this cell line. Maturation of an entire
culture to PMNs required anywhere from 8 to 14 days. The
levels of C/EBPu and C/EBPP in undinduced 32D C13
cells was also variable, as were the quantitative changes in
the expression levels of these isoforms during granulocytic
differentiation. These quantitative differences could be
partly explained if the maturation level of uninduced cells is
itself variable. More “mature” uninduced 32D C13 cells
B
A
113
ClEBPa
04
G7
I13
G10
’
MPC
04
113 04 07 010
67 GI0
Y,s)rr,
CEBPa
113 04 GI G10
MU
TUl
ClEBPG
Fig 5. CIEBPa. CIEBPp, and CIEBP8 mRNA expression In d l f f e r e n t l ~ n g32D C13 wlls and comparison with the levels of the corresponding
proteins. 32D C13 cells growing In IL-3 wore Induced for granulocytic differentiation by exposure t o G-CSF. Initially, and after 4,7, or 10 d a y ,
aliquots were removed and used t o prepare both total cellular RNA and protein. (A) C/EBPa, CIEBPp, C/EBPG, MPO, and tubulin (Tub) mRNA
expression was determined by Northern blotting. RNA from lo7 cells were loaded In each lane. Blots were stripped between probes by incubation
In boiling water for 5 minutes. (B) C/EBPcr. C/EBPp. C/EBPS, and MPO protein expression was determined by Western blotting. Total cellular
proteins were visualized by Ponceau S staining (bottom right).
From www.bloodjournal.org by guest on August 11, 2017. For personal use only.
1733
C/EBP EXPRESSION IN MYELOID CELLS
100
CIEBPa
50
2
u
>
u
CIEBPP
A
<r
CIEBPG
x
2I-
100
CIEBPP
50
CIEBPG
2
u
w
n
CIEBPa
NI
TD
TIME OF DIFFERENTIATION
Fig 6. Temporal pattern of C/EBPu, C/EBPp, and C/EBPG expression in differentiating 3T3-Ll preadipocytes“ and in differentiating
32D C13 myeloblasts. The absolute levels of these three proteins,
within each cell type, are unknown. The temporal pattern for each
C/EBP isoform in 32D C13 cells can only be considered an approximation given the variability of these cells in culture. NI, noninduced; TD,
terminally differentiated.Arrows indicatethe approximatetime when
proliferationceases in each pathway.
would differentiate into PMNs faster (eg, Figs 1and 2) than
would less “mature” uninduced cells (eg, Figs 4 and 5).
Perhaps the levels of C/EBPa and C/EBPP are lower in the
more “immature,” compared with the more “mature”
uninduced 32D C13 cells.
Despite these caveats, a consistent qualitative temporal
pattern of C/EBP isoform expression in differentiating 32D
C13 myeloblasts emerges from the data presented. The
nuclear concentration of C/EBPa first increases, being
expressed maximally in cells that retain the capacity for cell
division, and eventually diminishes, at least in some of the
most mature cells; C/EBPP increases steadily throughout;
and C/EBPG increases markedly early during induction and
then remains fairly constant, or may diminish mildly. This
pattern of C/EBP isoform expression in granulocytic differentiation is contrasted with that described for adipocyte
developmentll in Fig 6. Our results with differentiating
myeloblasts therefore suggest that act, PP, and ctP dimers
could predominate in immature myelomonocytic cells; that
all possible combinations could exist at intermediate stages
of differentiation; and that Pp, 66, and PS dimers could
predominate in terminally differentiated cells. In addition,
other C/EBP family members, such as C/EBPy, may be
present in these cell types, allowing for the formation of
additional heterodimers.
Because the C/EBP family appears likely to play a role in
regulating myelopoiesis, we are also interested in determining how the levels of these transcription factors are themselves regulated. Our data suggest that regulation of mRNA
production or turnover could account for the changes in
C/EBPa, C/EBPP, and C/EBPG levels observed during
granulopoiesis.
Finally, our observation of CiEBPa expression in myeloid, but not lymphocytic, human leukemias has potential
clinical utility, as assessing C/EBPa expression may aid in
diagnosis, and inhibition of CIEBPa expression in myeloid
leukemia cells may prove therapeutic.
ACKNOWLEDGMENT
We thank Steven McKnight and Zhaodan Cao for their encouragement and generous provision of antisera and CIEBP cDNAs.
We also thank W. Nauseef for MPO antiserum; J. Suzow, S. Amin,
J. Hebb, and T. Trischmann for technical assistance; M. Kastan for
guidance on antibody staining; and A. Hess for CTLL cells.
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1992 80: 1725-1735
A novel temporal expression pattern of three C/EBP family members
in differentiating myelomonocytic cells
LM Scott, CI Civin, P Rorth and AD Friedman
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