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INTRODUCTION |
CCAAT/enhancer-binding proteins
(C/EBPs)1 are a highly
conserved family of leucine zipper DNA-binding proteins expressed in a
variety of tissues and cell types (1-7). The six mammalian C/EBPs
(
,
,
,
,
, and CHOP) bind to DNA and facilitate the activation or repression of gene transcription (1-7). C/EBPs form
homo- or heterodimers with other C/EBP family members (1-3) and also
bind to a variety of other transcription factors and proteins,
including NF-
B (8), CREB/ATF (9), AP1 (10), Ets (11), Rb (12), and
p300 (13).
C/EBP isoforms regulate proliferation, differentiation, and apoptosis
in a cell-specific manner (1-7, 14-19). For example, C/EBP
and
C/EBP
play major roles in the regulation of proliferation and
differentiation of adipocytes and hepatocytes (2, 5, 17-19). C/EBP
also functions in the regulation of proliferation and differentiation
of white blood cells, ovarian granulosa cells, and mammary epithelial
cells (6, 17-19). Like C/EBP
and C/EBP
, C/EBP
is also
associated with adipocyte differentiation (2). In addition to adipocyte
differentiation, C/EBP
also functions in the differentiation of lung
epithelial cells (20) and myelomonocytic cells (21). C/EBP
also
plays an important role in the hepatic acute phase response (22, 23).
C/EBP
, the newest member of the C/EBP family of transcription
factors, regulates differentiation of granulocytes (6).
Recent reports indicate that C/EBPs play prominent roles in
mammary gland development, differentiation, and programmed cell death
(18, 19, 24-26). Two recent reports investigated the role of C/EBP
in mammary gland biology using C/EBP
knockout mice (18, 19). In both
reports, mammary epithelial cell proliferation and differentiation were
dramatically reduced in female C/EBP
knockout mice. C/EBP
also
functions in mammary gland biology; however, instead of promoting
mammary gland proliferation and differentiation, C/EBP
is associated
with mammary epithelial cell G0 growth arrest and
programmed cell death (24-26). Most reports indicate that C/EBP
plays a relatively minor role in mammary epithelial cell biology (19,
24, 25).
The mammary gland is a unique organ system in that it attains full
functional capacity late in life, at sexual maturation (27). Mammary
epithelial cells in the adult female cycle through intervals of
quiescence, proliferation, differentiation, and programmed cell death
in response to hormonal changes of the normal estrous cycle (27). These
alterations in mammary epithelial cell fate are well described at the
morphological level but poorly understood at the molecular level.
The overall goal of this study was to investigate the role of C/EBP
in mammary epithelial cell G0 growth arrest. Even though most cells in the adult animal have exited the cell cycle and exist in
G0, few G0 regulatory genes have been described
(27, 28). A better understanding of genes that regulate cell cycle exit/G0 entry is important in understanding normal cell
biology, tissue homeostasis, and cancer. Mutational inactivation of one G0 regulatory gene, the von Hippel-Lindau (VHL) tumor
suppressor gene, has been implicated in 80% of human sporadic renal
cell carcinomas (29).
We previously showed that C/EBP
is induced in G0
growth-arrested COMMA D mouse mammary epithelial cells (24). C/EBP
induction occurs early in cell cycle exit/G0 growth arrest
and remains elevated throughout the time the cells remain in
G0. The present results extend this observation,
demonstrating G0 induction of C/EBP
in multiple
mammary-derived cell lines. In addition, the induction of C/EBP
in
mammary epithelial cells is G0-specific. Altering mammary
epithelial cell C/EBP
content by transfection with C/EBP
antisense or over expression constructs dramatically affected both
G0 growth arrest and apoptosis in response to serum and
growth factor withdrawal. These results demonstrate a key role for
C/EBP
in the regulation of major cell fate determining pathways in
mammary epithelial cells.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
The nontransformed HC 11 mouse mammary
epithelial cell line was cultured in complete growth medium (CGM)
consisting of RPMI 1640 medium (4.5 g/liter glucose) supplemented with
10% fetal bovine serum (FBS), 10 ng/ml epidermal growth factor, and 10 µg/ml insulin. COMMA D cells were maintained as described previously (24). The NMuMG mouse mammary epithelial cell line (ATCC CRL 1636) was
cultured in Dulbecco's modified Eagle's medium (4.5 g/ml glucose)
supplemented with 10% FBS and 10 µg/ml insulin. The mouse mammary
tumor cell lines Mm5MT (ATCC CRL 1637) and MMT 060562 (ATTC CCL 51)
were cultured in Dulbecco's modified Eagle's medium (4.5 g/ml
glucose) supplemented with 10% FBS. NIH 3T3 cells (ATCC CRL 1658) were
cultured in Dulbecco's modified Eagle's medium (4.5 g/ml glucose)
supplemented with 10% calf serum. The rat intestinal epithelial cell
line IEC 18 (ATTC CRL 1589) was cultured in Dulbecco's modified
Eagle's medium supplemented with 5% FBS and 5 µg/ml insulin. All
media contained 5 units/ml penicillin and 5 µg/ml streptomycin. All
medium components were purchased from Life Technologies, Inc.
Generation of Cell Lines--
The C/EBP
RNA antisense plasmid
was produced by digesting MSV/EBP
with
EcoRI-PstI, generating an ~300-base pair 5'
C/EBP
cDNA fragment containing the ribosomal binding site. To
directionally clone the C/EBP
5' fragment in the antisense
orientation in the PcDNA 3 expression vector, the C/EBP
fragment
was first subcloned in the pGem4Z plasmid (Promega, Madison, WI),
excised with EcoRI and HindIII, and ligated into
PcDNA 3 (Invitrogen, Carlsbad, CA) in the antisense orientation.
The C/EBP
overexpression plasmid was produced by ligating an
EcoRI-HindIII restriction fragment containing the
full-length C/EBP
cDNA into PcDNA 3. HC 11 cells were
transfected with the various constructs or the PcDNA 3 vector using
Transfectam (Promega), and selection and expansion of single cell
transformants was carried out in the presence of 400 µg/ml of
Geneticin (Life Technologies, Inc.). Rescue cell lines were generated
by cotransfecting the AS1 cell line with an ~300-base pair 5'
C/EBP
cDNA sense construct (PcDNA 3) and a hygromycin resistant plasmid (ratio of 10:1). Cells were selected for by growth in
G418 (400 µg/ml) and hygromycin B (250 µg/ml). Three colonies were
chosen to propagate into cell lines, and the rest of the colonies were
pooled together to create a bulk cell line.
Growth Arrest Experiments--
80% confluent cells were washed
with serum-free medium and cultured in medium supplemented with 0.1%
FBS (growth arrest medium (GAM)). At the indicated times, cell were
harvested for Northern or Western blot analysis. For cell cycle block
experiments, cells were cultured for 36 h in GAM or in CGM
containing either hydroxyurea (1 mM), nocodazole (500
g/ml), or amino acid-deficient medium (methionine- and
isoleucine-free). For [3H]thymidine experiments, cells
were plated at 50% confluence in 96-well plates. Twenty four hours
later, cells were switched to GAM. Cells were pulsed for 2 h with
[3H]thymidine (5 µCi/ml) (DuPont), harvested by
precipitation with cold 5% trichloroacetic acid, solubilized in 0.2 N NaOH, and counted by liquid scintillation counting.
Results presented are representative of three experiments with six
wells per time point.
Northern Blot Analysis--
Total RNA was isolated at the
indicated times using RNAzol B (Tel Test, Inc. Friendswood, TX).
Northern blots were performed with 30 µg of total RNA as described
(24, 25). Filters were probed with the following
32P-labeled cDNAs: C/EBP
, C/EBP
, CHOP, histone 2B
(Oncor, Gaithersburg, MD), and Gas1. Cyclophilin receptor protein was
used as a constitutive probe. Filters were visualized by PhosphorImager
cassette and densitometry analysis performed by ImageQuant software
(Molecular Dynamics)
Growth Rate Determinations--
103 cells were
plated in individual wells in a 96-well plate. After 24 h
(t = 0), the relative number of viable cells was
assessed using the CellTiter 96 aqueous cell proliferation kit
(Promega). Cell monolayers were then washed with serum-free medium and
cultured in CGM or incomplete growth medium consisting of RPMI 1640 medium plus 10, 2, or 0.5% FBS. Three and 6 days later, viable cell
numbers were assessed. All viability assays were performed following
the manufacturer's protocol. Results presented are representative of
two experiments with three wells per time point.
Western Blot Analysis--
Whole cell and cytoplasmic and
nuclear proteins were harvested as described (24). Protease inhibitors
(complete tablets, Roche Molecular Biochemicals) and kinase and
phosphatase inhibitors (1 mM NaF, 1 mM
NaVO3, 1 mM Na2MoO4, 10 nM okadaic acid) were added to protein isolation solutions.
Proteins were quantified by Bradford method. 75 µg of protein was
separated by polyacrylamide gel electrophoresis and electroblotted to
polyvinylidene difluoride membranes (Millipore, Bedford, MA). Western
blots were performed by standard methods and visualized by ECL
(Amersham Pharmacia Biotech). Antibodies and antisera used were as
follows: C/EBP
, C/EBP, p21, p16 and cyclin D1 (Santa Cruz, Santa
Cruz, CA); p27 (Transduction Laboratories, Lexington, KY); Rb and
phosphorylated Rb (New England Biolabs, Beverly, MA). Horseradish
peroxidase-conjugated anti-mouse or anti-rabbit antibodies (New England
Biolabs) were used to detect primary antibodies.
Apoptosis Experiments--
Cells were plated at near confluence
in 96-well plates and grown to 100% confluence (about 24 h).
Cells were washed with serum-free medium and cultured in GAM. Viable
cell numbers were assayed at the indicated times using the CellTiter 96 aqueous cell proliferation kit (Promega). Results are representative of
three experiment with six wells per time point. Annexin V binding
followed by flow cytometry analysis was used to determine the
percentage of apoptotic cells. At the indicated times, cell culture
media containing both detached cells and the trypsinized monolayers
were incubated with FITC-conjugated Annexin V solution (ApoAlert
Annexin V FITC kit, CLONTECH, Palo Alto, CA).
Approximately 1.5 × 104 cells were assessed by flow
cytometry and the number of FITC-positive cells, indicative of
apoptosis, were counted. Results are representative of an experiment
performed two times.
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RESULTS |
C/EBP
mRNA and Protein Is Induced in Serum-deprived Mammary
Epithelial Cells--
We previously described C/EBP
induction in
the G0 growth-arrested COMMA D mammary epithelial cell line
(24). To investigate whether G0 induction of C/EBP
was a
general characteristic of mammary cells, we investigated C/EBP
expression in three nontransformed mouse mammary epithelial cell lines,
HC11, COMMA D, and NMuMG, and two transformed mammary epithelial cell
lines, CCL 51 and Mm5MT. C/EBP
mRNA levels were extremely low in
the growing mammary-derived cells (Fig.
1A, G). After 48 h in
growth arrest medium (GAM) C/EBP
mRNA levels were induced 8-, 7-, 3-, 15-, and 20-fold in HC 11, COMMA D, NMuMG, CCL 51, and Mm5MT
cells, respectively (Fig. 1A, S). In contrast, C/EBP
mRNA levels were constitutively elevated in growing and 48 h
growth-arrested NIH 3T3 cells. C/EBP
mRNA was barely detectable
in growing and 48 h growth-arrested IEC 18 cells. Unlike C/EBP
,
relatively high levels of C/EBP
mRNA were detected in all
mammary-derived cell lines, regardless of growth status. C/EBP
mRNA levels were relatively low and also unrelated to growth status
in NIH 3T3 cells. Histone 2B mRNA levels reflected growth status;
histone 2B mRNA levels were elevated in growing cultures (Fig.
1A, G) and reduced in confluent, growth-arrested cultures
(Fig. 1A, S).

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Fig. 1.
C/EBP expression in
growing and growth-arrested mammary-derived cells, fibroblasts, and
intestinal epithelial cells. A, Northern blot analysis.
Total RNA was isolated from nearly confluent (80%) growing cells
(G). Cells were then cultured in growth arrest medium (0.1%
FBS) for 48 h (serum-starved (S)). Blots were probed
with the indicated 32P-labeled cDNA probes.
B, Western blot analysis. Cytoplasmic (C) and
nuclear (N) proteins were isolated from cells treated as
above. Proteins (75 µg) were separated by 12.5% SDS-polyacrylamide
gel electrophoresis. Filters were probed with a rabbit anti-mouse
C/EBP antibody and detected with a horseradish peroxidase-conjugated
anti-rabbit secondary antibody. Blots were visualized with the ECL
system.
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Western blots of nuclear and cytoplasmic proteins were used to
correlate C/EBP
protein content with mRNA data and to
investigate C/EBP
subcellular localization. Consistent with the
mRNA data (Fig. 1A), C/EBP
protein was barely
detectable in growing mammary-derived cells but markedly induced in
48 h growth-arrested cultures (Fig. 1B). In contrast,
C/EBP
protein levels were elevated and unchanged in growing and
growth-arrested NIH 3T3 cells. In all cell lines, the majority of
C/EBP
protein was localized to the nucleus. The northern and Western
blot data support a novel mammary-specific induction of C/EBP
during
G0 growth arrest.
C/EBP
mRNA Is Specifically Induced during
G0-mediated Growth Arrest--
To investigate cell
cycle-dependent induction of C/EBP
mRNA, HC 11 cells
were growth-arrested at the G0, G1, S, or
G2 phase of the cell cycle. HC 11 cells were cultured for
36 h in either growth arrest medium (Fig.
2A, S;, G0 block),
amino acid-deprived medium (Fig. 2A, AA; G1
block), or complete medium containing hydroxyurea (Fig. 2A,
H; S block) or nocodazole (Fig. 2A, N; G2 block). Cell cycle blocks were verified by flow cytometry analysis of
DNA content using phosphatidylinositol staining (data not shown). Compared with proliferating HC 11 cells (Fig. 2A, G),
C/EBP
mRNA was induced 7-fold during G0 growth
arrest. C/EBP
mRNA levels were relatively unchanged following
growth arrest in other phases of the cell cycle (Fig. 2). We also
investigated other stress-related and growth arrest-specific genes. The
stress response gene CHOP10 was induced in amino acid-deprived and
hydroxyurea-treated HC11 cells. Like C/EBP
, the growth
arrest-specific gene Gas1, a marker for G0
(30), was induced only during serum withdrawal conditions. These data demonstrate a G0-specific induction of C/EBP
mRNA in mammary epithelial cells, suggestive of a role for C/EBP
in the initiation and/or maintenance of G0 growth
arrest.

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Fig. 2.
G0-specific induction of
C/EBP mRNA in HC11 cells. Total RNA
was isolated from growing cells (80% confluent). Cells were then
cultured in growth arrest (0.1% FBS) (S), complete growth
medium without methionine (AA), or complete growth medium
containing hydroxyurea (H) or nocodazole (N) for
36 h to block the cell cycle in G0, G1, S,
and G2/M phases of the cell cycle, respectively.
A, blots were probed with the indicated
32P-labeled cDNA probes: C/EBP , Gas1 (growth
arrest-specific 1), CHOP10, and cyclophilin (CP).
Cyclophilin was used as a loading control. B, the harvested
cells from the various treatments were stained with propidium iodide,
and the percentage of cells in each cell cycle phase was assessed by
fluorescence-activated cell sorter analysis. The results shown are
representative of two independent experiments.
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Construction of HC 11 Antisense and Overexpression Cell
Lines--
To further investigate the role of C/EBP
in HC 11 mammary epithelial cells, we generated C/EBP
antisense and
overexpression cell lines. Antisense 1 cell line (AS1), which had the
greatest reduction in C/EBP
protein, was chosen for further
analysis. AS1 C/EBP
protein levels were reduced by approximately
90% compared with control transfected cells (Fig.
3A). This level of C/EBP
reduction was similar to that which we previously reported in antisense-treated COMMA D mammary epithelial cells (24).

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Fig. 3.
Western blot analysis of
C/EBP protein levels in HC11 control,
antisense, and overexpression cell lines. A, nuclear
proteins were isolated from growing (G) or 48-h
growth-arrested, serum-starved (S) control transfected
(control) and antisense clone 1 (AS1) cells.
Proteins (75 µg) and an in vitro transcription/translation
lysate of a C/EBP construct (TnT) were separated by
12.5% SDS-polyacrylamide gel electrophoresis. Blots were probed with
C/EBP antisera. B, nuclear proteins from growing control
transfected cells (control) or the OV cell line.
C, cytoplasmic; N, nuclear.
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Despite multiple attempts, only one C/EBP
-overexpressing colony
survived drug selection, and therefore, only a single cell line (OV)
was available for investigation. C/EBP
protein levels and
subcellular localization was analyzed by immunoblotting of the OV cell
line during normal growth. C/EBP
protein levels were increased
2.3-fold compared with growing control cells (Fig. 3B). The
constitutively expressed C/EBP
protein, like endogenously expressed
C/EBP
, was localized in the nucleus.
C/EBP
Antisense and Overexpression Influences Mammary Epithelial
Cell Proliferation under Suboptimal Growth Conditions--
We next
plated control, AS1, and OV cells at low density (1,000 cells/well) and
examined proliferation under varying growth conditions. After 3 days in
CGM, all cell lines exhibited similar increases in cell numbers (Fig.
4A). This suggests that
C/EBP
has little effect on proliferation under optimal growth
conditions. However, in suboptimal growth conditions, there were marked
differences between the AS1, OV, and control cells (Fig. 4,
B-D). AS1 cell numbers increased 12-fold after 6 days in
0.5% serum (Fig. 4D). Control and OV cell numbers increased
by only 1.8- and 2.5-fold, respectively, after 6 days in 0.5% serum
(Fig. 4D). There were similar differences in proliferation
between cell lines in media containing 10 and 2% FBS (Fig. 4,
B and C). These data support a role for C/EBP
as a "conditional" cell cycle brake. Reducing C/EBP
levels (AS1
cells) increases proliferation under suboptimal growth conditions,
whereas increasing C/EBP
levels (OV cells) decreases proliferation
under suboptimal growth conditions.

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Fig. 4.
Growth curves of HC11 control,
C/EBP antisense, and C/EBP
overexpression cell lines in complete growth medium and low serum
medium. HC11 control, AS1, and OV cells were split
(103/well) in a 96-well plate in CGM (RPMI + 10% FBS,
epidermal growth factor (10 ng/ml), insulin (10 ng/ml)). After 24 h, the medium was changed to CGM (A), RPMI + 10% FBS (no
epidermal growth factor or insulin) (B), RPMI + 2% FBS (no
epidermal growth factor or insulin) (C), or RPMI + 0.5% FBS
(no epidermal growth factor or insulin) (D). Cell numbers
were quantitated using the CellTiter 96 aqueous cell proliferation kit
(Promega). Cell numbers were determined 3 days later for CGM
(A) and 3 and 6 days later for low serum medium
(B-D). Results are representative of an experiment
performed three times with triplicate wells per time point. Error
bars represent S.D.
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C/EBP
Antisense and Overexpression Influences Cell Cycle
Exit/G0 Entry and the Expression of Cell Cycle Regulatory
Proteins--
Eighty percent confluent control, AS1, and OV cells were
switched from CGM to GAM to initiate G0. In control cells,
[3H]thymidine incorporation declined by 10% after
12 h and 61% after 24 h in GAM (Fig.
5). In OV cells,
[3H]thymidine incorporation decreased 28 and 89% after
12 and 24 h, respectively, in GAM. In contrast, AS1 cell
[3H]thymidine incorporation was unchanged after 12 and
24 h. AS1 cell [3H]thymidine incorporation declined
by only about 50% after 48 h in GAM. This demonstrates a marked
acceleration of G0 growth arrest in OV cells and a marked
delay of G0 growth arrest in AS1 cells.

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Fig. 5.
Growth arrest induction. Nearly
confluent growing HC11 control (con), C/EBP antisense
(AS1), and C/EBP OV cells were switched from complete
growth medium to growth arrest medium (0.1% FBS) (t = 0). Cells were pulsed with 5 µCi/ml [3H]thymidine and
harvested 2 h later, at the indicated times. Results are
representative of an experiment performed two times with six replicates
per time point. Error bars represent S.D.
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We next investigated the influence of C/EBP
antisense and
overexpression on phosphorylated retinoblastoma protein (P-Rb), Rb,
cyclin D1, and the cyclin-dependent kinase inhibitor p27
during cell cycle exit. Western blot analysis of whole cell lysates was performed on growing (80% confluent) HC11 control, AS1, and OV cultures (t = 0, Fig. 6).
Cells were then switched from CGM to GAM to initiate G0
growth arrest. Densitometric scanning analysis indicated that
phosphorylated Rb levels declined 2-fold in HC11 controls after 48 h of G0 growth arrest (Fig. 6). Cyclin D1 levels also
declined in controls with the onset of G0 growth arrest, reaching nearly undetectable levels after 48 h of G0
growth arrest (Fig. 6). Control cell p27 levels increased during
G0 growth arrest. In contrast, phosphorylated Rb remained
unchanged and cyclin D1 levels declined slightly in AS1 cells after
48 h in GAM. There was a modest increase in p27 levels in AS1
cells after 48 h in GAM. In OV cells, phosphorylated Rb declined
6-fold and cyclin D1 protein levels declined to nearly undetectable
levels within 24 h of culture in GAM. OV cells also displayed a
rapid increase in p27 protein levels after 24 h of culture in GAM.
These data indicate that differences in the cell cycle exit rates
measured by [3H]thymidine incorporation between the
control, AS1, and OV cells (Fig. 5) correlate with changes in cell
cycle regulatory proteins.

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Fig. 6.
Western blot analysis of HC11 cell cycle
regulatory proteins during growth arrest. Nearly confluent growing
HC11 control (control) C/EBP antisense (AS1),
and C/EBP OV cells were cultured in growth arrest medium (0.1%
FBS), and whole cell proteins were harvested at the indicated times.
Proteins (75 µg) were separated by SDS-polyacrylamide gel
electrophoresis and electroblotted onto polyvinylidene difluoride
membranes. Duplicate filters were probed with cyclin D1 antibody, p27
antibody, and an antibody for Rb and phosphorylated Rb (serine
801/810). Primary antibody were detected with a horseradish
peroxidase-conjugated secondary antibody. Blots were visualized with
the ECL system. The results shown are representative of two independent
experiments.
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C/EBP
Antisense and Overexpression Influences Mammary Epithelial
Cell Apoptosis--
Culturing postconfluent HC11 mammary epithelial
cells in medium lacking serum and growth factors (0.1% FBS) induces an
apoptotic response that parallels early events in the involuting
mammary gland (25, 31, 32). Postconfluent control, AS1, and OV cells were cultured in medium containing 0.1% FBS, and the number of viable
cells was assayed daily for 4 days. The number of viable control cells
declined gradually, reaching 60% of the original cell number after 4 days (Fig. 7A). The OV cells
followed a similar trend; however, the decline in cell viability was
more dramatic. Viable OV cells decreased to 65% of the original cell
number after 1 day and 40% by day 4. In contrast, AS1 cell viability
increased 23% after day 1. Even after 4 days, there was
only a small decline in AS1 cell numbers compared with the
t = 0 starting time point.

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Fig. 7.
HC11 Cell viability and apoptosis
detection. A, viable cell assay. HC11 control
(con) C/EBP antisense (AS1), and C/EBP OV
cells were split at near confluence into 96-well plates and grown to
confluence (approximately 24 h). Postconfluent cells (day 0) were
switched to apoptosis medium (0.1% FBS). At the indicated times,
viable cells were quantitated using the CellTiter 96 aqueous cell
proliferation kit (Promega). Results are presented as a percentage of
viable cells before culture in low serum medium and are representative
of an experiment performed three times with six replicates per time
point. B, Annexin V assay. Postconfluent control (con)
C/EBP antisense (AS1) and C/EBP OV cells were switched
to apoptosis medium. At the indicated times, cells (including detached
cells) were harvested, washed with phosphate-buffered saline, and
resuspended in a solution of FITC-conjugated Annexin
V(CLONTECH Apoalert Annexin V kit). FITC-positive
cells were immediately quantitated by fluorescence-activated cell
sorter (a minimum of 15,000 cells counted). Results are representative
of an experiment performed three times.
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The percentage of apoptotic cells was low (<4%) in confluent control,
OV, and AS1 cells before the removal of serum and growth factors (day
0) (Fig. 7B). In control cultures, the percentage of
apoptotic cells increased 4-fold after 1 day and nearly 20-fold after 4 days in 0.1% FBS. In OV cultures, the percentage of apoptotic cells
increased 7-fold after 1 day and 12-fold after 2 days in 0.1% FBS. In
contrast, there was only a slight (1.5-fold) increase in the number of
apoptotic cells after day 1 in AS1 cells, and by day 4, there was only
a 5-fold increase in apoptotic cells. There was no significant increase
in apoptosis in any cell line when confluent cell lines were maintained
in complete growth medium for 48 h (data not shown), consistent
with previous reports in HC 11 cells (32, 33). All of the cell lines
had a similar apoptotic response when treated with apoptosis-inducing
agent staurosporine, demonstrating that the programmed cell death
response was functional in all the cell lines (data not shown).
Rescue of the AS1 Phenotype by Expression of C/EBP
Sense
RNA--
AS1 cells were stably transfected with a plasmid containing
the same 300-base pair fragment of C/EBP
as the antisense plasmid but in the sense orientation. C/EBP
protein was elevated about 10-fold in rescue cell line R1 compared with AS1 parental cell line
after 48 h in GAM (Fig.
8A). When parental AS1 cells
and the rescue cell line R1 were cultured in GAM (0.5% FBS), the AS1 cells proliferated (similar to results in Fig. 4D) but the
R1 cell line did not (Fig. 8B). Because altering C/EBP
levels influences both growth arrest and apoptosis, we next
assessed cell survival (apoptosis) of postconfluent AS1 and R1 cultures
in 0.1% FBS medium. Similar to results shown in Fig. 7B,
there was only a relatively slight (18%) reduction in relative cell
numbers in the AS1 cells (Fig. 8C). In contrast, there was a
61% reduction in relative cell numbers in R1 cells. These data show
that restoring C/EBP
expression in the C/EBP
antisense cell line
AS1 corrects defects in G0 growth arrest and cell survival.
This demonstrates that C/EBP
plays a key role in mammary epithelial
cell G0 growth arrest and cell survival.

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Fig. 8.
Western blot, growth, and apoptosis analysis
of C/EBP antisense AS1 and
C/EBP rescue R1 cell lines.
A, Western blot analysis. Nuclear proteins from growing and
48-h growth-arrested parental AS1 or rescue cell line R1. Filters were
probed with a C/EBP antibody. B, growth curve of rescue
cell lines was as follows. Parental antisense (AS1) and
rescue (R1) cell lines were split (103 cells)
into a 96-well plate. 24 h later, viable cells were quantitated
using the CellTiter 96 aqueous cell proliferation kit (Promega). The
medium was then changed to medium with 0.5% FBS. Cell numbers were
determined 4 days later. Results are representative of an experiment
performed two times with triplicate wells/time point. Error
bars represent S.D. C, viable cell assay. Postconfluent
parental AS1 and R1 cells (day 0) were switched to apoptosis medium
(0.1% FBS). At the indicated times, viable cells were quantitated as
Fig. 7. Results are presented as a percentage of viable cells before
culture in low serum medium and are representative of an experiment
performed two times with three replicates per time point.
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DISCUSSION |
This study investigated the growth regulatory role of C/EBP
in
mouse mammary epithelial cells in vitro. In a previous
report, we showed that C/EBP
gene expression and DNA binding
activity is induced in the COMMA D mouse mammary epithelial cell line
during G0 growth arrest (24). In this report, we extend
this observation, showing that C/EBP
is induced during
G0 growth arrest is a general property of mammary
epithelial-derived cell lines. C/EBP
expression is unrelated to
growth status in 3T3 cells, which express constitutively high levels of
C/EBP
, and the IEC18 rat intestinal epithelial cell line, which
expresses relatively low levels of C/EBP
. This indicates that
C/EBP
functions in a mammary epithelial cell-specific G0
growth control in vitro. We and others (25, 26) have shown that C/EBP
is induced in mouse mammary gland in vivo
during stage I of postweaning mammary gland involution. Because
G0 growth arrest precedes apoptosis in many cell types,
C/EBP
may play a role in reprogramming mammary epithelial cell gene
expression in preparation for apoptosis.
The extracellular ligand and the intracellular signal transduction
pathway that results in G0 induction of C/EBP
in mammary epithelial cells have not yet been identified. Factors that induce C/EBP
gene expression in other tissues and cell types do not appear
to induce C/EBP
expression in cultured HC 11 mammary epithelial cells. For example, glucocorticoids induce C/EBP
expression in intestinal epithelial cells, lung epithelial cells and in adipocytes (20, 33, 34) but have no effect on C/EBP
mRNA levels in HC 11 cells (data not shown). Insulin induces C/EBP
mRNA in adipocytes (35); however, C/EBP
levels are low in mammary epithelial cells cultured in insulin-containing complete growth medium (24). Interleukin-6 and cAMP induce C/EBP
expression in a variety of cell
types (22, 23, 36-38), but treatment of HC 11 cells with interleukin-6
and cAMP analogues does not induce C/EBP
mRNA (data not shown).
Other candidate molecules that may act as inducers of C/EBP
include
additional cytokines of the interleukin and interferon families and
cell adhesion molecules.
Growth rates were similar in AS1, OV, and control cell lines cultured
in complete growth medium (Fig. 4A). This suggests that C/EBP
does not influence mammary epithelial cell growth under optimal growth conditions (presence of serum and growth factors). A
similar observation has been reported for VHL, the first tumor suppressor found to function in the regulation of cell cycle exit (29).
Growth rates in complete growth medium were similar between VHL-negative and VHL wild type renal carcinoma cells (29). VHL-negative cells, however, did not exit the cell cycle and enter G0
growth arrest when cultured in low serum containing (growth arrest)
medium (29). Reintroduction of wild type VHL restored appropriate
G0 growth arrest in VHL-negative cells (29). AS1 cells are
not completely C/EBP
-negative (AS1 cells express C/EBP
at about 10% of control levels), but AS1 cells do exhibit defective cell cycle
exit/G0 entry when cultured in low serum containing (growth arrest) medium. Reintroduction of wild type C/EBP
restored
appropriate cycle exit/G0 entry growth arrest and
apoptosis in AS1 cells. These results suggest that C/EBP
, like
VHL, functions in the regulation of cell cycle exit.
The difficulty we encountered in the generation of a C/EBP
overexpressing mammary epithelial cell line supports a growth inhibitory role for C/EBP
. Similar difficulties were not encountered in producing C/EBP
antisense cell lines or other C/EBP isoform expression cell lines. Once produced, however, the presence of elevated
levels of C/EBP
(OV cells) did not directly induce cell cycle
exit/G0 growth arrest if cells were cultured in optimal growth medium. This suggests that either the individual surviving cell
line (OV) had developed a compensatory mechanism to overcome the
expression of C/EBP
, or C/EBP
alone is insufficient to induce growth arrest. Additional factors, such as subcellular compartmentation and/or posttranscriptional modification of C/EBP
may be required for
full function.
C/EBP
is regulated by subcellular localization in hepatocytes (39).
In cultured mammary epithelial cells and the mammary gland in
vivo, C/EBP
protein is primarily localized to the nucleus, regardless of growth or differentiation status (24). C/EBP
protein
is also primarily localized to the nucleus in growing OV cell lines
(Fig. 3B). This suggests that subcellular localization is
not a major mechanism of C/EBP
regulation in mammary epithelial cells. The inability of C/EBP
to inhibit OV cell growth in complete growth medium could be due to a lack of phosphorylation in growing cells. Phosphorylation of C/EBP
is required for DNA binding during the hepatic acute phase response (40). In addition, C/EBPs bind DNA as
homo- and heterodimers (1-3, 8-13). Even high levels of C/EBP
may
be ineffective in blocking cell cycle progression if the appropriate
dimerization partner is absent or inactive in cells cultured under
optimal growth conditions.
Although C/EBP
overexpression in OV cells did not induce cell cycle
exit/G0 growth arrest in optimal growth medium, C/EBP
overexpression did accelerate cell cycle exit/G0 growth
arrest in suboptimal growth medium (growth arrest medium). This
indicates that mammary epithelial cells with a ready supply of C/EBP
in the nucleus (OV cells) rapidly exit the cell cycle in response to
growth arrest conditions. This suggests that C/EBP
may be limiting
for cell cycle exit in mammary epithelial cells.
The cyclin-dependent kinase inhibitor p27 functions in
G0 growth arrest in a variety of cell types, including
mammary-derived cells (41-44). Basal p27 levels increased in confluent
HC11 control and OV cultures following exposure to growth arrest medium
and the initiation of G0 growth arrest. In contrast, AS 1 cells cultured in growth arrest medium failed to induce p27 and
exhibited a marked delay in the initiation of G0 growth
arrest. These results indicate an association between mammary
epithelial cell C/EBP
levels, p27, and G0 growth arrest
in vitro. This association may extend to the mammary gland
in vivo, as C/EBP
and p27 are both induced in the mammary
gland during involution.2
C/EBP
may influence cellular p27 levels by increasing p27
gene transcription or p27 protein stabilization. p27 overexpression is
associated with apoptosis in a variety of cell lines, including breast
cancer cell lines (45, 46). Although p27 levels are primarily regulated
by changes in protein stability (42, 47), transcriptional control has
recently been reported (48). C/EBP
and C/EBP
both transactivate
the cyclin-dependent kinase inhibitor p21waf1 promoter (49), and C/EBP
has been shown
to directly stabilize the p21 protein without activating
p21waf1 gene expression (50). Consistent with
previous reports, p21 and p16 were virtually undetectable in any of the
HC 11-derived cell lines regardless of growth status (51).
Overexpression of cyclin D1 in MCF7 breast cancer cells is
associated with cell cycle progression in low serum medium (52, 53).
When C/EBP
levels were reduced (AS1 cells), cyclin D1 levels
remained elevated and cell cycle progression continued in low serum
medium. When C/EBP
levels were increased (OV cells), cyclin D1
levels rapidly declined and cell cycle progression stopped in low serum
medium. These data indicate that C/EBP
and cyclin D1 are induced
under opposing growth conditions. C/EBP
may influence cyclin D1
levels by acting as a transcriptional repressor of cyclin D1 in
G0 growth-arrested mammary epithelial cells. C/EBP
functions as a transcriptional repressor of the apolipoprotein
C-III gene during the hepatic acute phase response (40). In
addition, cyclin D1 levels are also tightly controlled at the
posttranslational level by cell cycle regulated, calpain-mediated
degradation (54).
The marked delay in the initiation of G0 growth arrest
observed in AS1 cells cultured in growth arrest medium was similar to
previous studies carried out in our laboratory with C/EBP
antisense-expressing COMMA D mouse mammary epithelial cells (24). In
both studies, reducing endogenous C/EBP
levels consistently delayed
cell cycle exit. In this report, however, we have extended the analysis
of the C/EBP
antisense-expressing HC11 cells (AS1) to include cell
cycle regulatory proteins and apoptosis. The results indicate that
C/EBP
plays an important role in regulating cell cycle exit. When
this role is compromised by reducing endogenous C/EBP
levels,
regulation of cell cycle exit/G0 entry and the execution of
the programmed cell death response are delayed.
We previously reported a transient induction of C/EBP
during stage I
of mammary gland involution, a physiological period associated with
massive apoptosis in the mammary epithelial compartment (25). In this
report we found that the percentage of cells undergoing apoptosis was
increased in the C/EBP
overexpressing OV cells and percentage of
cells undergoing apoptosis was reduced in C/EBP
antisense AS1 cells.
Apoptosis in both cell lines, however, was similar in response to
staurosporine (data not shown). This suggests that C/EBP
influences
an upstream component of the apoptotic pathway that is activated by
serum and growth factor withdrawal. Alternate pathways of apoptosis
initiation and the downstream, common cell death pathway remain intact.
These results support a direct role for C/EBP
in the regulation of
mammary epithelial cell fate after the withdrawal of serum and growth
factors. However, our results cannot completely rule out the
possibility that the observed effects of C/EBP
antisense or
overexpression on mammary epithelial cell fate may involve other C/EBP
family members, bZIP proteins, or other regulatory proteins. Most
reports, however, indicate that C/EBP
is expressed at low
levels in mammary epithelial cells and probably plays a relatively
minor role in mammary epithelial cell growth regulation (19, 24, 25).
The role of C/EBP
is uncertain. C/EBP
knockout mice exhibit
defective mammary epithelial cell proliferation and differentiation
(18, 19); however, overexpression of C/EBP
, or LIP, the dominant
negative inhibitor of C/EBP
, does not significantly alter HC11
growth control (data not shown). The roles of CHOP10 or other bZIP
proteins in mammary epithelial growth regulation are not well understood.
Mammary epithelial cell growth, differentiation, and death is
controlled by endocrine and paracrine signals (27). A better understanding of these extracellular ligands, the intracellular signaling pathways they activate, and their nuclear targets will provide a clearer picture of mammary gland growth regulation and potentially new insights into the etiology and progression of breast
cancer. The growth suppressor activity of C/EBP
is similar to that
described for the VHL gene product (29). In addition, C/EBP
also plays a role in mammary epithelial cell apoptosis. Many
well described tumor suppressor/growth arrest genes, such as
BRCA1 (55), APC (56), p53 (57),
p33ING1 (58), CHOP (59), and the
cyclin-dependent kinase inhibitor p27 (45), also
induce apoptosis. Experiments are under way to characterize
extracellular ligands, their receptors, and intracellular signaling
pathways that induce C/EBP
gene transcription in mammary epithelial cells.