4726-4733
Nucleic Acids Research, 1995, Vol. 23, No. 22
Inhibition of cell proliferation by C/EBPoc occurs in
many cell types, does not require the presence of p53
or Rb, and is not affected by large T-antigen
L. R. Hendricks-Taylor1>+ and G. J. Darlington1-2*
Departments of 1 Molecular and Human Genetics and 2Pathology, Baylor College of Medicine, One Baylor Plaza,
Houston, TX 77030, USA
Received June 6,1995; Revised and Accepted October 6, 1995
ABSTRACT
The transcription factor CCAAT/enhancer binding
protein (C/EBPoc) is expressed predominantly in
differentiated tissues and is able to induce growth
arrest and differentiation in preadipocytes. C/EBPa
expression is high in non-dividing hepatocytes, but
decreases during liver regeneration. These observations suggest that C/EBPa is inversely related to cell
proliferation. To investigate the mechanism of growth
inhibition by C/EBPa, the response of immortal
human cells to cotransfection of a C/EBPa expression
vector (CMVa) and a CMVp-galactosidase expression
vector was examined. Hep3B2, a hepatoma; Saos2,
an osteosarcoma deficient for p53 and Rb; and 639, a
fibroblast expressing SV40 T-antlgen, were examined.
Transiently transfected cells were stained for p-gal
activity to monitor their ability to undergo division. The
ability of stable transformants to form colonies was
also assessed for each cell line. Cells transfected with
CMVa remained as non-dividing cells while control
cells divided to form colonies. Mutations of the C/EBPa
sequence demonstrated that only a small, previously
uncharacterized activation domain was required for
antimitotic activity. Our results suggest that C/EBPa
may play a role In maintaining the quiescent state of
hepatocytes and other cells. Furthermore, it appears
that the effects of C/EBPa are not mediated through
p53 or Rb and are not altered by T-antigen.
INTRODUCTION
CCAAT/enhancer binding protein (C/EBPa) belongs to a family
of DNA binding proteins characterized by a basic DNA binding
domain and a leucine zipper motif that mediates dimerization
(1,2). Members of this family share significant sequence
similarity and DNA binding activities. C/EBP proteins have been
shown to bind to the enhancer core sequences of several animal
viruses and to CCAAT promoter sequences associated with a
variety of cellular genes (3-5).
Several members of the C/EBP family have been identified:
C/EBPa (1); NF-IL6 (5) or C/EBPP (6); DBP (7); CRP1 (8);
mouse Ig/EBP-1 (9) or C/EBPy (6); human NF-IL60 (10) [rat
CRP3 (8) or mouse C/EBP8 (6)]; and AGP/EBP (11) [identical to
CRP2 (8), rat IL-6DBP (12), rat LAP (13), and C/EBPp]. C/EBP
family members have been found to both homodimerize and
heterodimerize (6,8,14). C/EBP proteins play a role in the
transcription of several genes whose activities are modulated
during inflammation or the 'acute phase response' (15-17)
including albumin, transthyretin, alpha-1 antitrypsin, fibrinogen,
haptoglobin, serum amyloid A3, and C3 (4,18-22). C/EBPa also
functions in theregulationof genes involved in energy metabolism
such as stearyl-CoA desaturase 1 (SCD1) (23,24) and Glut-4 (25).
The rat C/EBPa protein has been characterized as having two
separate activation domains (14,19,26). The DNA binding and
zipper (bZIP) regions are critical for its role as a transcriptional
activator, however, the presence of either activation domain alone is
sufficient to stimulate transcription from the albumin promoter (26).
A second group has defined a putative negativeregulatoryelement
within the activation domains (27). Two proteins are translated from
the 2.7 kb C/EBPa message: the full length 42 kDa protein and a
30 kDa protein translated from the third ATG (28,29). The 42 kDa
protein appears to be a better activator of albumin transcription than
the 30 kDa protein (28). In addition, Lin el al (29) have reported
that the 30 kDa rat protein lacks the antimitotic activity of the 42 kDa
protein. We have cloned the human C/EBPa gene and find that it
has 90% homology to the rat gene with 100% identity in the DNA
binding and zipper domains.
C/EBPa expression is restricted to terminally differentiated,
non-dividing cells and is most abundant in liver, bronchial and
adipose tissues (30,31). Interestingly, while C/EBPa protein is
present at high levels in normal adult liver, its expression is
dramatically decreased (80% less) in regenerating liver (31,32). In
addition, C/EBPa is expressed at low levels during hepatocyte
proliferation in vitro and in rapidly dividing hepatoma cells (HepG2)
(19). C/EBPP is expressed both in normal hepatocytes in culture and
in adult liver, while C/EBP5 is virtually undetectable. Upon cytokine
or LPS stimulation, C/EBPP and 8 are dramatically induced; in
contrast, CEBPa levels decrease significantly (10,33,34).
More recently, rat C/EBPa has been shown to play a key role
in differentiation-induced gene expression and growth arrest in
3T3L1 pre-adipocytes (6,24,35-38). C/EBPa also suppresses
colony growth in 10T1/2 mouse fibroblasts (37). Furthermore,
C/EBPa appears to play an opposite role to that of c-myc in cell
* To whom correspondence should be addressed
+
Present address: Department of Internal Medicine, University of Iowa College of Medicine, 540 EMRB, Iowa City, IA 52242. USA
Nucleic Acids Research, 1995, Vol. 23, No. 22 4727
proliferation. C/EBPa overexpression is able torepressgrowth of
c-myc transformed adipoblasts which are normally unable to
terminally differentiate (39).
The pattern of expression of C/EBPa and its ability to induce
growth arrest and differentiation of adipocytes suggest that
C/EBPa expression is inversely related to the proliferative state
of the cell and may play a role in maintaining quiescence. These
observations led us to question whether C/EBPa contributes to
the maturation of hepatocytes and their cessation of division. It is
also of interest to know if growth inhibition by C/EBPa is specific
for certain cell types or if it is a general phenomenon. As an
approach to understanding the mechanism of cellular growth
inhibition by C/EBPa, we designed cotransfection studies to
characterize the response of human cells in culture to C/EBPa
expression. The immortal cell lines included Hep3B2, a hepatoma
cell line that has been characterized as a model for the liver acute
phase response to inflammation (40); Saos2, an osteosarcoma
deficient for p53 and Rb, two factors known to be involved in
growth and cell cycle regulation; and 639, a fibroblast line
expressing S V40 T-antigen, an inhibitor of several factors involved
in growth control. Cells were cotransfected with a C/EBP
expression vector and a CMVfi-galactosidase vector to serve as a
marker. Growth arrest was assayed by measuring the proliferation
of transiently transfected cells and colony growth of stable
transformants. C/EBPa was able to induce growth arrest in all
cell types examined. The antimitotic activities of both the 42 and
30 kDa proteins were found to be equivalent In addition, deletion
analyses of C/EBPa were performed to demonstrate that only a
small, previously uncharacterized activation domain is required
for its growth inhibitory activity.
MATERIALS AND METHODS
Materials
Enzymes were purchased from BRL and isotopes from Amersham. Oligonucleotides were synthesized by the Nucleic Acids
Core of the Department of Molecular and Human Genetics at
Baylor College of Medicine. The rat anti-C/EBPa antibody was
from Santa Cruz. CMVC/EBPP was kindly provided by T.
Kishimoto (Osaka University, Japan). C/EBP8 cDNA was a gift
from S. McKnight (Tularik, Inc., South San Francisco). PGKNeo
was provided by A. Bradley (Baylor College of Medicine,
Houston). The CMV^-gal was a gift from G. MacGregor (Baylor
College of Medicine, Houston). 639 cells were provided by O.
Periera-Smith (Baylor College of Medicine, Houston). Saos2
cells were obtained from the ATCC.
Cell culture, transfection analyses and preparation of
whole cell extracts
Hep3B2 and 639 cells were maintained in 3 parts Eagle's Minimal
Essential Medium, 1 part Waymouth MAB 87/3 (GIBCO), 2%
fetal bovine serum, 8% horse serum (Hazleton). Saos2 cells were
maintained in Dulbeco Modified Essential Medium (high glucose)
with 10% fetal bovine serum.
DNAs were prepared by CsCl centrifugation. Transient
transfections were by co-electroporation of 4 (jg CMV[}-gal
vector and 20 \x% CMVC/EBP or control vector, CMV0, using a
BioRad Gene Pulsar. Saos2 cells (6 x 105) in 350 (xl of growth
media were electroporated with Gene Pulsar settings of 500 uF
and 220 V. Hep3B2 cells, 639 cells and fibroblasts (1 x 106) were
electroporated in 350 u.1 cytomix (120 mM KC1; 0.15 mM CaCl2;
10 mM K2HPO4/KH2PO4) pH 7.6; 25 mM HEPES, pH 7.6; 2
mM EGTA, pH 7.6; 5 mM MgCl2; pH adjusted with KOH) with
settings of 960 |iF and 280 V. Stable transfections of Saos2 cells
and 639 cells were performed as above with 20 \ig CMVC/EBP
and 2 |ig PGKNeo. Stable transfection of Hep3B2 cells was by
CaPO4-mediated gene transfer. Cells were grown in selective
medium [250 |ig/ml G418 (BRL)] for two weeks prior to analysis.
Cell extracts were harvested 24 h following electroporation by
scraping in 200 |il lysis buffer (50 mM Tris, pH 8.0, 0.1 mM
EDTA, 1 mM DTT, 12.5 mM MgCl2, 20% glycerol, 0.1 M KC1,
1% Triton X-100). Supematants were collected following
centrifugation at 12 000 r.p.m. for 10 min at 4°C. The protein
concentration of each extract was calculated using the Bradford
method (BioRad).
Construction of expression vectors
CMVP-gal, PGKNeo, MSV5 and CMVp have been previously
described. The fi-gal coding region was excised from CMVji-gal
with Notl to create CMV0. The 1.1 kb coding region, 118 bp of
5' leader sequence, and 77 bp of 3' untranslated region of the
human C/EBPa gene (from +1 to +1274) were excised from a
pUC19 vector with NruUXhol, blunted using the Klenow
fragment of DNA polymerase I and cloned into the blunted Notl
site of CMV0 to create CMVa. CMVa30kD was made by
excising a SstU-Xhol fragment (+453 to +1274) from pUCC/
EBPa, which was blunted and cloned into CMV0. A 3.7 kb
C/EBPa fragment was excised from pUCC/EBPa using £coRI
and cloned into pBluescriptKSII- (Pharmacia); a 3' C/EBPa Notl
fragment was excised using the Notl site at +585 and the Notl site
in the KSII polylinker and cloned into the Notl site of CMV0 to
create CMVaN/E. A Smal fragment from +219 to +478 in
CMVa was excised and replaced with a blunt linker
(CGGAATTCCG, NEB) to restore the correct reading frame to
create CMVaAADl. The CMV promoter, SV40 splice sites, and
5' portion of C/EBPa to nucleotide +467 was excised with
EcoRl-Narl from CMVa. The 3' portion of the gene and the
SV40 poly A signal from Nar\ (+893) to the //mdlll in the
polylinker was also excised from CMVa. These two fragments
were ligated into the EcoW-Hindin sites of pUC19 (BRL) to
create CMVaAAD2. CMVaAAD 1,2 was created by excising an
Nael fragment (+371 to +853) and inserting a blunt linker
(GGAATTCC, NEB) to restore the correct reading frame. The
C/EBP6 cDNA was excised from pBluescript with EcoRl and
BamHl, blunted using Klenow, and ligated into the blunted Notl
site of CMV0 to create CMV8. All constructs were confirmed by
dideoxy sequencing.
p-galactosidase staining
Cells were washed with PBS and fixed for 5 min with cold 0.5%
gluteraldehyde (made in PBS). Cells were rinsed twice with PBS
and incubated at 37°C for 4-8 h in X-gal staining solution
(1.3 mM MgCl2, 15 mM NaCl, 44 mM HEPES, pH 7.3, 3 mM
Fe+2CN, 3 mM Fe+3CN, 0.5 mg/ml X-gal). pVgalactosidase
converts the substrate X-gal (Sigma) into a blue product that was
visualized by light microscopy.
4728 Nucleic Acids Research, 1995, Vol. 23, No. 22
Design of growth assays
For transient assays, CMVC/EBP vectors or CMV0 were
co-electroporated into cells with CMVP-gal at a ratio of 5:1. Cells
were stained for (i-gal activity at 24, 72 and 120 h following
electroporation. Cell growth was assessed by counting the number
of blue-stained cells present in each clone of cells originating from
one transfected cell within 46 microscopic fields (magnification
= lOx). Transfected cells that had undergone division formed
colonies of blue cells while non-dividing transfectants remained as
single blue cells. Colonies containing blue cells were categorized
into one of three groups: clones that remained as single cells, clones
that contained two cells, or clones that contained >2 cells.
Transfections in which >60% of the blue clones remained as single
cells at 120 h were considered to be growth inhibited. Transfection
efficiency was determined after 24 h by calculating the percentage
of blue cells present in the total cell population in five microscopic
fields.
Suppression of colony formation was assessed by counting the
number of colonies arising from each transfection after staining
with Wright's stain for visualization.
Antibody preparation
Anti-C/EBPa antisera, 2637 and anti-C/EBPP antisera, 2710,
were raised against synthetic peptides (41) at Bethyl Laboratories
(Montgomery, TX). Anti-C/EBP8 antibodies, 10354 and 10355,
were raised against an overexpressed C/EBP8 fusion protein at
the Center for Comparative Medicine at Baylor College of
Medicine.
Western blot analysis
Transfectants were harvested after 48 h by scraping in 300 (iJ lysis
buffer (1 % Triton X-100 detergent, 0.15 M NaCl, 0.05 M Tris, pH
7.4) supplemented with protease inhibitors (50 |Ag/ml aprotinin,
10 pg/ml leupeptin, 50 \iglm\ pepstatin A, 1 mM phenylmethylsulfonyl fluoride). Extracts (20 u.1) were analyzed by 10%
SDS-PAGE, transfered to nitrocellulose, and immunoblotted
with anti-C/EBPa (1:2000). Detection was with anti-rabbit IgG
antibody coupled to horseradish peroxidase (BioRad) followed
by chemiluminescence with ECL (Amersham).
Electrophoretic mobility shift (EMSA) and supershift
assays
Two complementary oligonucleotides representing the C/EBPa
binding site of the C3 promoter, bZIP-A (5'-CATGGATGGTATTGAGAAATCTG-3') and bZIP-B (5'-GATCCAGATTTCTCAATACCATC-3'), were annealed to create a double-stranded
oligonucleotide (bZIPl) which was end-labeled with
[a-32P]dCTP with Klenow. Binding reactions contained 20 (ig of
extract, 10 000 c.p.m. of probe (-35 fmol), 2.5 jig poly
(dl-dC)poly(dl-dC) (Pharmacia), 30 mM Tris-HCl (pH 8.9),
5 mM HEPES (pH 7.9), 0.66 mM EDTA, 7.5 mM MgCl2,60 mM
KC1, 1.2 mM DTT and 14% (vol/vol) glycerol in a total volume
of 20 (JJ. For supershift assays, antibodies against human C/EBPa
(ratio of 1:20), rat C/EBPa (1:20, Santa Cruz), human C/EBPP
(1:20), or C/EBP8 (1:200) or pre-immune sera (1:20) were
pre-incubated with the extracts for 10 min at 30 °C prior to the
addition of probe. Reactions were incubated at 30°C for 20 min
and analyzed on high-ionic-strength 5% polyacrylamide gels
with TG buffer (50 mM Tris-HCl, pH 7.6/375 mM glycine).
RESULTS
C/EBPa, but not C/EBPp or C/EBP8, inhibits growth
of Hep3B2 cells
Hep3B2, a hepatoma model cell line for the acute phase response,
was chosen to examine the effects of C/EBPa on liver cell
growth. These cells express low levels of C/EBPa as well as
C/EBPP and C/EBP8 (endogenous levels are much less than
those seen following transfection of C/EBP expression vectors).
Hep3B2 cells divide rapidly under normal cell culture conditions,
suggesting that they are able to overcome the inhibitory effects of
endogenous C/EBPa expression.
A CMV expression vector encoding human C/EBPa or other
family members was introduced into Hep3B2 cells to determine
whether growth arrest could be induced. Cells were co-electroporated with CMV0, CMVa, CMVp or CMV8 at a ratio of 5:1 with
CMVjJ-gal (which served as a marker for positive transfection)
to ensure that a high number of cells receiving CMVfi-gal would
also receive CMVa or control vectors. C/EBPP and C/EBP8 were
included as controls to demonstrate that any growth inhibitory
effects were due specifically to the expression of C/EBPa and to
eliminate the possibility of squelching. The parental expression
vector CMV0 was also included to control for the possibility of
effects on growth due to the presence of high levels of exogenous
DNA. All expression vectors are described in Figure 1.
Hep3B2 cells were stained with X-gal to detect P-galactosidase
activity at 24, 72 and 120 h following co-electroporation.
Transfection efficiency was equivalent (within 10%) between the
expression vectors within one experiment, but varied from
10-30% between experiments performed at different times. The
total number of viable cells remaining after transfection of each
construct was equivalent at various time points. All transfectants
contained mostly (>90%) single blue-staining cells at 24 h. Cells
transfected with CMV0, CMVP or CMV8 had clones containing
2-20 blue cells when stained after 72 and 120 h, indicating each
original transfected cell had undergone multiple rounds of
division. However, cells transfected with CMVa contained
mostly single blue cells, indicating that these cells did not divide
following transfection (Fig. 2) and clearly demonstrating growth
inhibition by C/EBPa when compared to control cells.
Colonies containing blue-stained cells 120 h following
transfection were classified into one of three groups: colonies that
consisted of a single cell, colonies that contained two cells, or
colonies that contained >2 cells. The results from three separate
growth experiments were averaged and are shown in Figure 3A.
The graph shows the percentage of the total number of
transfectant colonies in each group. In transfections with CMV0,
CMVPandCMV8, 15, 14.5 and 15.5%,respectively,of the total
number of colonies remained as single cells; the majority of the
colonies consisted of 3-20 blue cells. In contrast, cells transfected
with CMVa contained 80% single blue cells and only 7.5% of the
colonies had >2 blue cells. The high percentage of non-dividing
blue cells in CMVa transfections suggests that Hep3B2 cells that
receive the C/EBPa expression vector do not proliferate. The few
colonies that contained multiple blue cells likely represent cells
that received only the CMVpVgal construct and not CMVa.
Nucleic Acids Research, 1995, Vol. 23, No. 22 4729
DAY1
CMV
I
aia I
CMV6
DAY 5
ato
BR
L?
CMV-STOP
't
CMV
CMVoA30Kd
BR
LZ
CMV-C/EBP a
CMV
CMVaiADl
CMVaAAD2
CMVaAAD1,2
«"u 1 = promoter & vector sequences
MMWS/77A
m bZIP coding region
LZ - Leucine zipper domain
- C/EBPa activation domains coding region
BR • Basic ONA-bindtng domain
Figure 1. Schematic diagram of CMV expression vectors encoding normal and
mutant C/EBP proteins. The segment deleted or truncated is indicated by a gap
in the horizontal line corresponding to a 5'-to-3' representation of the C/EBP
coding region. The vector CMV promoter and SV40 ATG is shown as a
rectangular box adjacent to the 5' end of the C/EBP coding region, and the 3'
C/EBP basic DNA-binding (BR) and leucine zipper (LZ) regions are indicated.
The CMVcx constructs include 118 bp of 5' leader sequence prior to the 42 kDa
initiation codon. Endogenous C/EBPa ATGs capable of yielding protein
products of 42 and 30 kDa are shown. All deletions were created by subcloning
using naturally occurring restrictions sites. The corresponding amino acid
positions of both ends of a deleted segment or the 5' end of a truncated construct
are denoted beneath each vector.
Figure 2. Hep3B2 cells stained for p-gal activity following cotransfection.
Cells are shown using bright light microscopy. Cells were cotransfected with
the control CMVstop (CMV0 followed by a stop codon) or CMVa and
CMVP-gal at a ratio of 5:1. CMVP-gal serves as a marker of transfected cells.
Cells were stained for P-gal activity 24 (day 1) and 120 h (day 5) following
transfection. Single cells or clones positive for P-gal activity are visible under
bright light as darkly stained cells. Growth inhibition is demonstrated by
comparing the number of colonies containing multiple (1-gal stained cells in
CMVstop and CMVa transfections on day 5.
fected with CMVa, 97% remained as single blue-stained cells
and only 1% of the colonies consisted of >2 blue cells.
A transformed human fibroblast cell line (639) that expresses
SV40 T-antigen was also examined (not shown) and only
C/EBPa was able to induce growth arrest. The majority (86%) of
the CMVa transfected cells remained as single cells in contrast
to CMV0 (29%), CMVfj (31%) and CMV5 (27%) transfected
cells. Thus, it appears that neither p53 nor Rb are required for
growth inhibition by C/EBPa and that T-antigen is not able to
modulate the anti-mitotic activity of C/EBPa.
Deletion mutations of C/EBPa
Growth inhibition does not require the presence of p53
or Rb and is not affected by T-antigen
Cotransfections of CMVa and CMV^-gal were repeated with
five different human transformed cell lines and in each case
growth inhibition was seen. This phenomenon suggested that
perhaps C/EBPa was acting through ubiquitous factors present in
the cell. The osteosarcoma, Saos2, was examined in further detail
because it lacks two important factors involved in regulating the
cell cycle and initiating growth arrest, namely p53 and the
retinoblastoma protein, Rb. Furthermore, endogenous C/EBPa is
not expressed in this cell line.
Saos2 cells were examined following co-electroporation with
CMV0, CMVa, CMVP or CMV8, and CMV(i-gal. Transfection
efficiencies were equivalent for each construct at 24 h and varied
from 10 to 25% between individual experiments. The graph in
Figure 3B shows the average percentage of colonies from three
separate experiments that contained 1, 2 or >2 blue cells 120 h
following cotransfection. Cells transfected with CMV0, CMVp
and CMV8 had 24, 35.5 and 18.5%, respectively, of the total
staining colonies remaining as single cells. Of the cells trans-
Mutations of human C/EBPa were made to further define the
functional domains required for growth arrest of human cells and
to provide insight into possible mechanisms of action. Expression
vectors containing deletions of parts of the activation domains, a
construct initiating at the 30 kDa ATG, and a construct deleted for
all activation domains were employed. These constructs allowed
comparison with the activity of rat C/EBPa and served to further
delineate the functional domains necessary for growth arrest.
All constructs were derived from CMVa and are described in
Figure 1. CMVa30kD encodes the 30 kDa protein only, which is
lacking the previously described activation domain 1 (ADI)
(19,26). CMVaAADl deletes a region including ADI and the 30
kDa ATG. CMVaN/E is deleted for all sequence upstream of
activation domain 2 (AD2) (19,26). This construct is lacking both
initiation ATGs; translation is likely to begin from the vector ATG
which lies between SV40 splice donor and acceptor sites
upstream of C/EBPa. CMVaAAD2 is deleted for sequences
including AD2 and the 30 kDa ATG. CMVaAADl,2 is deleted
for sequences encoding ADI and AD2 as well as the sequences
between the two domains.
4730 Nucleic Acids Research, 1995, Vol. 23, No. 22
HEP3B2 CELLS
control
1
2
CMVa CMVa CMVa CMVa
30 KD N/E
A ADI
3
4
5
6
7
8
9
CMVa
AAD2
CMVa
3AD1.2
CMVS CMVp
:
10 11 12 13 14 15 16 17 18 19 20
- m
M
• Of BLUE CELLS COLONY
• CMVfl DCMV0HCMV5 BCM
SAOS2 CELLS
# OF BLUE CELLS' COLONV
Figure 5. EMSA and supershift analyses of CMVC/EBP transfectants binding
to the bZIPl C/EBP consensus binding site. Cell extracts were made 24 h
following transfection of Saos-2 cells with CMVa, CMV0, CMV8 and the
CMVa deletion mutants. The extracts used are indicated above the lanes. Lanes
1 and 2 represent non-transfected cells. Extracts were incubated with either
pre-immune serum (lanes 1, 3,5,7,9, 11, 14, 17 and 19) or antisera prior to the
addition of labeled oligonucleotide. Antisera used in supershift assays are as
follows. Lanes: 2, 4, 6, 8, 10, 12 and 15, anti-human C/EBPa; 13 and 16,
anti-rat C/EBPa (Santa Cruz); 18, anti-C/EBP5; 20, anti-C/EBPp\ The
anti-C/EBPP antisera is neutralizing and, thus, shows the loss of the complex
containing C/EBPf$ rather than a complex of slower mobility.
• CMVe a CMV p m CMV 6 • CMVa]
Figure 3. Bar graph representing the effects of C/EBP proteins on cell growth
following transfection into Hep3B2 (A) or Saos-2 (B). Cells were cotransfected
with 20 |lg of the following vectors, CMV0, CMVp\ CMV6 or CMVa and 5
Ltg of CMVP-gal. Cells were stained for (5-gal activity after 120 h, and the
number of blue-stained colonies was recorded. Colonies consisted of 1,2 or >2
blue cells and were categorized accordingly. Colonies of 1representcells that
did not divide following transfection. For each vector, the number of colonies
in a category is shown as a percentage of the total number of colonies recorded.
Data are means ± standard deviations of three separate experiments.
42 kDa —
30 kDa —
Figure 4. Western analysis of Hep3B2 CMVa deletion mutant transfectants.
Whole cell lysates were made 24 h following transfection with CMVa and the
CMVa deletion mutants. Lysates were analyzed by SDS-PAGE analysis
followed by immunoblotting with anti-human C/EBPa antibody. Lane 1,
untransfected Hep3B2; lane 2, CMVa; lane 3, CMVaAADl,2; lane 4,
CMVOAAD2; lane 5, CMVo30kDa; lane 6, CMVaAADl; lane 7, CMVaN/E.
The 42 and 30 kDa proteins are indicated.
Hep3B2 C/EBPa mutant transfectants were analyzed by
Western blot (Fig. 4). All proteins with the exception of
CMVaN/E and CMVaAADl were expressed at high levels.
Endogenous 42 and 30 kDa C/EBPa proteins can be seen in some
lanes. Multiple bands were detected in CMVaAAD2 and
CMVaAADl,2 transfectants. These bands may represent degradation products or differentially modified proteins. In addition,
these products migrated with a slower mobility than expected,
which may be the result of post-translational modifications or
conformational changes induced by the deletions. An aberrant,
slower migration than predicted by molecular weight has been
seen for many proteins including C/EBPa mutants (8,27). Low
CMVaN/E expression was not surprising due to the location of
the vector initiation ATG between splice sites. The lack of
detection of both CMVaN/E and CMVaAADl proteins may
represent a lower level of transcription from these vectors, a
decreased stability of the protein products, or a decreased affinity
of the antibody for these mutant proteins. Low level expression
of both vectors is clearly seen in the more sensitive EMSA
analysis of Saos2 transfectants (Fig. 5).
EMSA analyses (Fig. 5) were conducted with extracts from
Saos2 transfectants that contain no endogenous C/EBP to ensure
that each construct was still able to bind DNA. Untransfected
Saos2 cells showed no C/EBP-specific complex (lanes 1 and 2).
CMVa-transfectants had the expected protein—DNA complex
(lanes 3 and 4). The CMVoc3OkD (lanes 5 and 6) protein-DNA
complex with a slightly faster mobility than that of CMVa.
CMVaN/E (lanes 7 and 8) and CMVaAADl (lanes 9 and 10)
showed reduced levels of C/EBPa protein-DNA complexes that
migrated with a faster mobility due to the deletion of ADI.
CMVa, CMVa30kD, CMVaN/E and CMVaAADl were all
supershifted by the anti-human C/EBPa antibody. CMVaAAD2
and CMVaAADl,2 both had intense C/EBPa-specific complexes (lanes 11 and 14) that migrated much faster than CMVa.
Neither extract gave a supershift with the anti-human C/EBPa
Nucleic Acids Research, 1995, Vol. 23, No. 22 4731
HEP3B2 CELLS
f OF BLUE CELLS COLONY
D CMVa30kD
• CMV«N,'E
• CMVMAADI
D CMVU^A02
B CMVOAAD1,2|
SAOS2 CELLS
All deletion constructs with the exception of CMV<xAADl,2,
which contains primarily only the bZIP region, appear to inhibit
cell division. Results from CMVaAADl and CMVccAADl,2
limit activation domain 1 to between amino acids 84 and 116.
Inhibition by both CMVaN/E and CMVaAAD2 limits activation
domain 2 to between amino acids 154 and 245. The lack of
growth inhibition by CMVaAADl,2 indicates that C/EBPa
growth arrest requires a functional activation domain within the
deleted region. Functional C/EBPa proteins (i.e. those inhibiting
growth) containing deletions of either AD 1 or 2 indicate that only
one activation domain is required for activity; these two domains
have little sequence homology, suggesting that the actual amino
acid sequence present is not critical. These deletion studies further
delimit the activation domains described for the rat C/EBPa
protein and demonstrate that they are conserved in the human. In
addition, a comparison of vectors encoding the 42 and 30 kDa
proteins indicates that these two proteins function equally well in
growth inhibition. The growth arrest properties of the human 30
kDa protein are in contrast to those of the rat C/EBPa 30 kDa
protein in adipocytes (29).
Stable transfections of C/EBPa do not form colonies
# OF BLUE CELLS .' COLONY
QCMVaiA02
BCMVajiAOI.2
Figure 6. Bar graph representing the effects of C/EBPa deletion mutants on cell
growth following transfection into Hep3B2 (A) or Saos-2 (B). Cells were
cotransfected with CMVoc deletion mutants and CMVfJ-gal at a ratio of 5:1.
Cells were stained for p-gal activity after 120 h, and the number of blue-stained
colonies was recorded. Colonies consisted of 1, 2 or >2 blue cells and were
categorized accordingly. Colonies of 1 represent cells that did not divide
following transfection. For each vector, the number of colonies in a category is
shown as a percentage of the total number of colonies recorded. Data are means
± standard deviations of three separate experiments.
Colony suppression assays were used to further characterize the
C/EBP expression vectors. CMV0, CMVa, CMVa mutants,
CMVP and CMV5 were transfected into Hep3B2 cells or Saos2
cells at a ratio of 10:1 with PGKNeo. The number of surviving
colonies was assessed following 2 weeks of G418 selection
(Table 1). Many colonies containing 50-150 cells were seen for
CMV0, CMVaAADl,2, CMV(3 and CMV5. However, few
colonies developed in cells transfected with CMVa or the other
CMVa mutants, demonstrating that both the 42 and 30 kDa
C/EBPa and all mutants containing either activation domain
inhibit these cells from dividing to form stable colonies.
Table 1. Colony suppression assay
Plasmid
No. colonies"
Cell type
Hep3B2
antibody (lanes 12 and 15), demonstrating that they were both
lacking AD2 in which the peptide epitope resides. These extracts
did, however, have supershifted complexes (lanes 13 and 16) when
incubated with the anti-rat C/EBPa antibody developed against the
DNA-binding domain; thus, the proteins present in each complex
were C/EBPa-derived proteins. Finally, CMV8 (lanes 17 and 18)
and CMVp (lanes 19 and 20) extracts had intense C/EBP-specific
protein-DNA complexes . Similar protein—DNA complexes were
seen for Hep3B2 transfectants (not shown).
The results of three separate transient growth assays from
co-electroporation of the deletion constructs and CMV(3-gal for
both Hep3B2 and Saos2 cells were averaged and are shown in
Figure 6. CMVa, CMVa30kD, CMVaAADl, CMVaN/E and
CMVocAAD2 transfections contained from 62 to 88% individual
blue cells and few colonies with >2 blue cells when stained 120
h following cotransfection. However, transfections with CMVaAADl,2 gave results similar to those seen for control experiments with only 10-20% of the total colonies remaining as
individual blue cells.
Saos2
CMV(|>
76
180
CMVP
S3
CMV5
CMVa
CMVa30 kD
CMVaN/E
CMVaAADl
CMVaAAD2
74
20
159
210
5
21
15
28
35
108
140
CMVaAl,2
i
12
aAverage number of colonies from two experiments arising after 2 weeks of
G418 selection following cotransfection of 20 \ig of plasmid and 2 (ig of
PGKNeo.
DISCUSSION
The results presented in this report demonstrate a specific growth
inhibitory function for C/EBPa in many cell types. C/EBPa was
able to induce growth arrest when introduced into the hepatoma
cell line Hep3B2. C/EBPa is expressed in the liver at late stages
in animal development (30,31). While C/EBPa expression is
4732 Nucleic Acids Research, 1995, Vol. 23, No. 22
high in non-dividing adult liver, mRNA expression is decreased
both in regenerating liver and dividing hepatocytes (31,32). A
similar pattern of expression is seen for developing adipoblasts,
and premature expression of C/EBPa in pre-adipoblasts induces
growth arrest (37,38). C/EBPa may play a similar role in liver
development. The pattern of expression of C/EBPa and its ability
to inhibit Hep3B2 cell division suggest that C/EBPa may be
involved in maintaining the antiproliferative state of liver cells.
It is not clear how Hep3B2 cells, which express endogenous
C/EBPa, are able to divide rapidly in culture. These cells may
produce a non-inhibitory C/EBPa that acts as a dominant
negative by inhibiting wild-type C/EBPa or by binding factors
and/or DNA more efficiently than normal C/EBPa. Both of these
possibilities have been seen for some mutated p53 proteins
(42-44). It is also possible that Hep3B2 cells express normal
C/EBPa, but have developed an ability to escape growth arrest
However, growth arrest arising from exogenous C/EBPa indicates that the cell cannot overcome C/EBPa inhibition when
excess protein is present A similar result has been seen for
proliferating cell types that express endogenous MyoD but that are
growth arrested when exogenous levels of MyoD are introduced
(45,46).
Expression vectors encoding C/EBPP and C/EBP8 were not
growth inhibitory in any of the cell types tested. The lack of
growth inhibition by CMVp was somewhat surprising since Buck
et al. (47) have reported that the mouse C/EBP(3 homolog LAP
has anti-mitotic activity. However, their LAP construct was not
full length and did not express LIP, the transcriptional inhibitor
derived from the same transcript as LAP. Our construct contains
the full length sequence that would give rise to both LAP and LIP
transcripts, although we are uncertain as to the ratio expressed in
the transfectants. The lack of growth inhibition by other C/EBP
proteins demonstrates that this phenotype is specific to C/EBPa
and not due to a general squelching phenomenon (48,49) or DNA
toxicity.
CMVa induced growth arrest in a variety of cell types,
including fibroblasts, HeLa cells and rat epithelial IEC6 cells (not
shown). These results suggested that C/EBPa was influencing
growth through ubiquitous factors present in most cells. To
further examine this possibility, CMVa was introduced into the
osteosarcoma Saos2, which is deficient for p53 and Rb. CMVa
induced growth arrest in these cells despite their lack of normal
cell cycle regulation, suggesting that the effects of C/EBPa are
not mediated through p53 or Rb. The fibroblast cell line 639,
which expresses SV40 T-antigen, was also examined. T-antigen
has been shown to inactivate other growth inhibitory factors such
as p53, Rb and Cipl (50,51). However, the antimitotic activity of
C/EBPa was not altered by T-antigen in the 639 cell line.
Deletion mutations were made in human C/EBPa to further
characterize its growth arrest properties. An expression vector
(C/EBPaAADl,2) encoding only the bZIP region did not induce
growth arrest in any cell type, demonstrating that some form of
activation domain is required for growth inhibition. This result
suggests that C/EBPa is not acting as a dominant negative
through the dimerization domain to inhibit growth. Deletions
including the two identified activation domains were made to
confirm, and further delineate, the two domains identified in the rat
protein. Mutants that contained only activation domain 1 or 2,
which contain no sequence similarity, appeared to be able to
induce growth arrest equally well, suggesting that the activation
domain sequence is not critical for anti-mitotic activity.
Therefore, the DNA binding domain and adjacent NH3regionare
likely critical for determining the specificity of the antimitotic
activities of C/EBPa. The 30 kDa protein were as potent a growth
inhibitor as the full length human C/EBPa. This result is in
contrast to those of Lin et al. (29) who found that only the mouse
42 kDa protein, but not the 30 kDa protein, was antimitotic in
3T3-L1 cells. It is not clear why the human 30 kDa protein
behaves differently. However, human C/EBPa has not been
tested in 3T3-L1 cells.
In conclusion, the studies outlined here demonstrate that
C/EBPa induces growth arrest in a variety of cell types, including
some that do not express the endogenous C/EBPa gene under
normal circumstances. The ability of C/EBPa to inhibit growth
of the hepatoma Hep3B2 suggests that C/EBPa may play a role
in maintaining the quiescent state of hepatocytes. Inhibition of
Saos2 cells by C/EBPa implies that the mechanism of growth
arrest does not involve p53 or RB. C/EBPa induced growth arrest
of 639 cells indicates that its function is not attenuated by SV40
T-antigen. Deletion mutants of C/EBPa suggest that C/EBPa may
be acting as a positive transcription factor since both activation
domains are able to function independently. The mechanism by
which C/EBPa induces growth arrest is currently under study in
our laboratory. It seems likely that C/EBPa brings about growth
arrest indirectly by acting to influence the expression of other gene
products that are required for inhibition of proliferation.
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