From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720
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ABSTRACT |
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The preceding paper (Cha, H. H., Cram,
E. J., Wang, E. C., Huang, A. J., Kasler, H. G.,
and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000-0000(478563) defined a glucocorticoid responsive region within
the promoter of the p21 CDK inhibitor gene that contains a putative
DNA-binding site for the transcription factor CCAAT/enhancer binding
protein- (C/EBP
). Wild type rat BDS1 hepatoma cells as well as
as4 hepatoma cells, which express antisense sequences to C/EBP
and
ablate its protein production, were utilized to investigate the role of
this transcription factor in the glucocorticoid regulation of p21 gene
expression. The stimulation of p21 protein levels and promoter
activity, as well as inhibition of CDK2-mediated retinoblastoma protein
phosphorylation, by the synthetic glucocorticoid, dexamethasone,
required the expression of C/EBP
. Overexpression of C/EBP
in as4
cells rescued the dexamethasone responsiveness of the p21 promoter.
Site-directed mutagenesis of the p21 promoter revealed that
dexamethasone stimulation of p21 promoter activity required
the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBP
and p21 protein expression and promoter activities. Our results have established a functional link between the
glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of rat hepatoma cells and the
transcriptional control of p21 by a cascade that requires the steroid
induction of C/EBP
gene expression.
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INTRODUCTION |
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Treatment with glucocorticoids, one class of steroid hormones, can inhibit both the in vivo and in vitro growth of many different types of normal and transformed cells. Normal hepatocytes and certain hepatoma cell lines are acutely sensitive to the anti-proliferative effects of glucocorticoids (1-9). We have previously established that in specific types of steroid responsive rat hepatoma cells (10, 11) and rat mammary tumor cells (12), glucocorticoids induce an early G1 block in cell cycle progression suggesting the existence of a unique hormone-regulated G1 restriction point in these transformed cells. Furthermore, the loss of G1 cell cycle control has been implicated in the uncontrolled proliferation of a variety of neoplastically transformed cells (13, 14). Given that steroid receptors are transcriptional regulators (15-20), this G1 cell cycle arrest is likely to be controlled in part by a glucocorticoid-mediated transcriptional cascade in which the glucocorticoid receptor directly alters the transcription of a small subset of genes which then regulate the subsequent expression and/or activity of specific sets of downstream proteins. Important final targets of this glucocorticoid growth suppression pathway are likely to be G1-acting components of the cell cycle which define a critical checkpoint in cell cycle progression. However, the molecular basis for the functional relationship between the glucocorticoid control of early events within this signaling cascade and the final cell cycle arrest of hepatoma cells is poorly understood.
To investigate the cellular signaling pathways mediating the
glucocorticoid growth arrest of epithelial cells, we have isolated glucocorticoid growth suppressible and non-suppressible hepatoma cell
proliferation variants derived from the rat Reuber hepatoma (2, 9).
Characterization of these hepatoma cell variants revealed that the
anti-proliferative effect of glucocorticoids is a
receptor-dependent process that does not affect cell
viability, decrease total cell number, or induce an apoptotic response
(3, 4). Moreover, glucocorticoids induce an early G1 block
in cell cycle progression within one cell doubling time in BDS1
hepatoma cells (4). By using this cell system, we have defined some of
the earliest transcriptional events associated with the G1 arrest of the tumor cell line. Most significantly, the CCAAT/enhancer binding protein-
(C/EBP
)1 gene expression
is specifically required for the glucocorticoid-mediated G1
cell cycle arrest of hepatoma cells. Ablation of C/EBP
protein by
expression of antisense sequences precluded glucocorticoids from
inducing the G1 cell cycle arrest and overexpression of
C/EBP
suppressed hepatoma cell growth in the absence of
glucocorticoids (11). In addition to the well established role
of the C/EBP family of transcription factors in normal liver
function (21, 22), the glucocorticoid stimulation of C/EBP
gene
expression is a rapid response that represents an early and crucial
intermediate in the glucocorticoid-stimulated anti-proliferative
cascade that governs the cell cycle of liver-derived epithelial tumor
cells (11).
G1-acting cell cycle components may be important downstream
targets of the glucocorticoid growth suppression pathway in rat hepatoma cells. For example, C/EBP, or other steroid responsive transcriptional regulators, may inhibit the transcription of components necessary for cell cycle progression, such as the cyclins or
cyclin-dependent kinases (CDK), or stimulate expression of
cell cycle inhibitors that inactivate specific CDKs (23). In the
preceding accompanying paper (57), we established that glucocorticoids
stimulate p21waf1/cip1 promoter activity through multiple
elements within a glucocorticoid responsive region of promoter. One of
the glucocorticoid responsive fragments contains a canonical C/EBP
DNA-binding site. Because C/EBP
expression is required for
glucocorticoids to induce a G1 cell cycle arrest of
hepatoma cells (11), we examined if C/EBP
expression is linked to
the glucocorticoid stimulation of p21 promoter activity. In this study,
by using glucocorticoid responsive rat hepatoma cells in which we
ablated C/EBP
expression with antisense sequences for this
transcription factor, as4 cells (11), and a glucocorticoid-resistant
hepatoma cell line, EDR1 (2), we demonstrate that the glucocorticoid
stimulation of p21 promoter activity and induction of protein levels
required the regulated expression of the C/EBP
transcription
factor.
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EXPERIMENTAL PROCEDURES |
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Materials--
Dulbecco's modified Eagle's/F12 (1:1) medium,
fetal bovine serum, calcium- and magnesium-free phosphate-buffered
saline, and trypsin-EDTA were supplied by BioWhittaker (Walkersville,
MD). Dexamethasone was obtained from Sigma. [3H]Thymidine
(84 Ci/mmol), [3H]acetyl coenzyme A (200 mCi/mmol),
[-32P]dCTP (3,000 Ci/mmol), and
[
-32P]dATP (3,000 Ci/mmol) were obtained from NEN Life
Science Products. Anti-p21, anti-C/EBP
, anti-CDK2, and horseradish
peroxidase-conjugated donkey anti-goat antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish
peroxidase-conjugated goat anti-rabbit antibodies were purchased from
Bio-Rad. The enhanced chemiluminescence protein detection system and
the Multiprime DNA labeling kit were purchased from Amersham Corp. The
chimeric p21 promoter CAT reporter plasmid containing 2.326 kilobase
pairs of p21 promoter sequences upstream of the RNA start site linked
to the bacterial chloramphenicol acetyltransferase (CAT) gene or the
firefly luciferase (luc) gene were generous gifts from Dr.
Bert Vogelstein (Molecular Genetics Laboratory, Johns Hopkins Oncology
Center, Baltimore) and has been described previously (24). The C/EBP
promoter luciferase reporter plasmid was generously provided by Dr.
Kleanthis G. Xanthopoulos (Center for Biotechnology, Karolinska
Institute, Sweden) and has been described previously (25). The C/EBP
eukaryotic expression vector (pCD-mC/EBP) and parental blank expression
vector (pCD) were generously provided by Dr. Heinz Baumann (Department
of Molecular and Cellular Biology, Roswell Park Cancer Institute,
Buffalo, NY) and have been described previously (26).
Hepatoma Cell Lines and Methods of
Culture--
Glucocorticoid-sensitive BDS1 cells are epithelial tumor
cells derived from the rat Reuber hepatoma (2). The as4 and vector control (vc) cell lines were derived by single cell subcloning of BDS1
cells transfected with the pBCMG-AS antisense C/EBP expression vector or the pBCMGneo parental vector, respectively. The EDR1 glucocorticoid-resistant cells are epithelial tumor cells derived from
the rat Reuber hepatoma and were selected for their inability to be
growth-arrested by glucocorticoids (2). All of the hepatoma cell lines
were routinely grown in Dulbecco's modified Eagle's medium/F-12/10%
fetal bovine serum at 37 °C in humidified air containing 5%
CO2. The stably transfected cell lines were maintained in
400 µg/ml G418. Cell culture medium was routinely changed every 48 h. Dexamethasone was added to a final concentration of 1 µM as indicated.
Glucocorticoid Treatment of Rats and Analysis of Liver Gene
Expression--
Two-month-old female virgin Lewis rats (Harlan
Sprague-Dawley, Indianapolis, IN) were anesthetized with an
intraperitoneal injection of ketamine, xylazine, and acepromazide (55 mg/kg body weight). A subscapular silastic capsule containing 20 mg of
cortisol, or cholesterol as a control, was aseptically placed
subcutaneously in an incision at the dorsal midline which was
subsequently closed with wound clips. After 2 weeks, the rats were
killed by anesthesia overdose, and the liver was isolated and
quick-frozen in liquid nitrogen. Approximately 0.5 g of frozen
tissue were minced with a sterile razor blade and lysed with 1 ml of
RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) containing
aprotinin, pepstatin, and leupeptin. Following centrifugation at
1.4 × 104 rpm for 20 min, the protein content for
each lysate was determined by the Bradford assay (Bio-Rad). For each
sample, 30 µg of protein were mixed with 15 µl of sample buffer
(62.5 mM Tris-HCl, pH 6.8, 8% glycerol, 5%
-mercaptoethanol, 3% SDS, 0.01% bromphenol blue) prior to
resolution by electrophoresis and Western blotting (described below).
Assay of DNA Synthesis by [3H]Thymidine Incorporation-- Triplicate samples of asynchronously growing BDS1, vc, and as4 cells were treated with dexamethasone for the indicated times and pulse-radiolabeled as described in the accompanying paper (57).
Western Blot Analysis--
Western blots were prepared
essentially as described in the accompanying paper (57) with the
following procedural variations for blotting with the anti-C/EBP
antibodies. For each sample, 30 µg of protein were mixed with 15 µl
of sample buffer and fractionated on 10% polyacrylamide, 0.1% SDS
resolving gels by electrophoresis. Blots were subsequently incubated in
TBST at room temperature with 1 mg/ml rabbit anti-C/EBP
for 1 h. Horseradish peroxidase-conjugated goat anti-rabbit secondary
antibodies were diluted in TBST, 1% non-fat dry milk 1:10000, and
membranes were incubated with the diluted antibodies for 1 h at
room temperature.
Transfection Procedures--
Logarithmically growing hepatoma
cells were transfected by electroporation essentially as described in
the preceding paper (57) with the following changes. In all
transfection experiments, the cells were electroporated with 14 µg of
pBLCAT2 empty vector or 16 µg of p21 promoter-CAT reporter construct
alone or with 10 µg of DNA encoding a C/EBP expression vector and
plated into pre-warmed Dulbecco's modified Eagle's medium/F12, 10%
fetal bovine serum. For the assays involving stably transfected cell
lines (as4, vc), cells were cultured with medium supplemented with 400 µg/ml G418.
Reporter Gene Assays-- All CAT and luciferase assays were conducted exactly as described in the preceding paper (57).
PCR Mutagenesis of the C/EBP DNA-binding Site in the 199-bp
1.383/
1.184 Fragment of the p21waf1/cip1
Promoter--
The ATCCTCTGCAATTT wild type C/EBP
DNA-binding site at
1.270 in the p21 promoter was mutated to
ATCCTCCCATGGTT. Two separate PCR reactions were set up to
amplify a 135-bp 5
fragment with mutations at the 3
end within the
C/EBP binding site and a 90 bp-3
fragment with mutations at the 5
end
within the C/EBP binding site using
1383/
1184 p21-tkCAT as a
template. The C/EBP sites in the 5
and 3
fragments are overlapping by
26 nucleotides and can subsequently be annealed together to serve as
templates for further amplification of a full-length 199-bp fragment
containing selective point mutations in the C/EBP binding site. The 5
and 3
fragments were amplified in independent reactions containing 1 ng of
1333/
1184p21-tkCAT, 25 pmol of sense and antisense
oligonucleotide primers, 250 µM nucleotide (62.5 µM dTTP, dATP, dCTP, and dGTP), 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.75, 0.1% Triton X-100, 100 µg/ml
bovine serum albumin (BSA), and 2.5 units of cloned Pfu DNA
polymerase (Stratagene, La Jolla, CA) in a total reaction volume of 50 µl and overlaid with 50 µl of mineral oil. DNA was amplified for 30 cycles (denaturation at 95 °C for 30 s, annealing at 55 °C
for 30 s, and extension at 75 °C for 30 s). The following
specific primers were used for PCR and sequencing: 5
fragment,
pC/EBPmutR-5
-CTTTTAAAAACCATGGGAGGATGGAT-3
; 3
fragment, pC/EBPmutF-5
-ATCCATCCTCCCATGGTTTTTAAAAG-3
.
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RESULTS |
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Dexamethasone Stimulation of p21 Protein Levels and Inhibition of
CDK2 Activity Requires the Expression of the C/EBP Transcription
Factor--
We have previously demonstrated that the G1
block in cell cycle progression in rat hepatoma cells induced by the
synthetic glucocorticoid, dexamethasone, requires the steroid-regulated expression of C/EBP
. This analysis was accomplished using hepatoma cells in which C/EBP
expression had been ablated by stable
transfection of an antisense C/EBP
expression vector, generating the
as4 cells, and with vc hepatoma cells transfected with an empty
expression vector (11). As described in the accompanying paper (57), glucocorticoids stimulate the expression of the p21 CDK inhibitor gene
which contains a canonical C/EBP DNA-binding site in its promoter.
Therefore, as4 and vc hepatoma cells, as well as the BDS1 parental cell
line, were utilized to functionally examine the mechanistic
relationships between C/EBP
expression and the regulated expression
of the p21 CDK inhibitor gene within the glucocorticoid growth
suppression response.
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Dexamethasone Activation of p21 Promoter Activity Can Be
Functionally Complemented by Overexpression of C/EBP in as4 Cells
That Lack Induction of This Transcription Factor--
The preceding
paper (57) demonstrated that the dexamethasone stimulation of p21 gene
products in BDS1 hepatoma cells resulted from the transcriptional
stimulation of the p21 gene. The requirement of C/EBP
transcription
factor expression for the induction of p21 protein, the induction of
C/EBP
by dexamethasone (11), and the presence of a canonical C/EBP
DNA-binding site at
1270 bp in the p21 promoter implicated this
transcription factor in the activation of p21 promoter activity. To
test directly this possibility, as4, vc, and BDS1 hepatoma cells were
transiently cotransfected with a C/EBP
expression vector (11, 26)
and a chimeric reporter plasmid containing 2326 bp of the p21 promoter upstream of the transcription start site linked to the CAT reporter gene. Hepatoma cells transfected with an empty expression vector served
as a negative control. Analysis of CAT activity in cells treated with
or without dexamethasone for 48 h revealed that in vc and BDS1
hepatoma cells, this p21 promoter fragment conferred glucocorticoid
responsiveness to the CAT reporter gene (Fig.
3, right and left
panels). In contrast, in as4 cells, which lack C/EBP
,
dexamethasone treatment failed to induce p21 promoter activity
(Fig. 3, middle panel). Importantly, cotransfection of a
C/EBP
expression vector into as4 cells rescued the defective glucocorticoid induction of p21 promoter activity (Fig. 3, middle panel) and caused a modest increase in the absolute level of both the basal and steroid-induced levels of p21 promoter activity in BDS1
or vc hepatoma cells (Fig. 3, left and right
panels). This functional complementation of
glucocorticoid-inducible p21 promoter activity demonstrates the
requirement for expression of the C/EBP
transcription factor in this
steroid response.
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Dexamethasone-stimulated Activity of the p21 Promoter Fragment
Requires the C/EBP DNA-binding Site and Expression of the C/EBP
Transcription Factor--
Sequence analysis of the promoter region of
p21 revealed no canonical glucocorticoid response elements, but the
promoter does contain a putative C/EBP DNA-binding site between
nucleotides
1270 to
1256. As detailed in the preceding paper (57),
this transcription factor site is located within one of the
glucocorticoid responsive subfragments of the p21 promoter. To test
directly if the C/EBP DNA-binding site in the p21 promoter contributes to the glucocorticoid-regulated transcriptional activity, the ATCCTCTGCAATTT wild type C/EBP DNA-binding site in the
1.380- to
1.184-bp fragment of p21 promoter was mutated to
ATCCTCCCATGGTT eliminating the key nucleotides required for
transcription factor binding (27). These p21 promoter fragments,
containing either the wild type sequence or the C/EBP DNA-binding site
mutation, were linked immediately upstream of the thymidine kinase (tk) minimal promoter sequences driving the bacterial CAT gene, forming
1.380/
1.184 p21-tkCAT and C/EBPmut-1.380/
1.184 p21-tkCAT,
respectively (see diagrams in Fig.
4). The vc control cells and
C/EBP
-deficient as4 hepatoma cells were cotransfected with one of
the reporter plasmids in the presence or the absence of a C/EBP
expression vector. As shown in Fig. 4, dexamethasone failed to induce
CAT activity from either of the p21 promoter fragments in transfected as4 cells. Transient ectopic expression of C/EBP
reinstated the dexamethasone activation of p21 promoter activity only in cells transfected with the wild type p21 promoter fragment and not in cells
transfected with the reporter plasmid mutated in the C/EBP DNA site. In
vc hepatoma cells, the wild type
1.380/
1.184 p21-tkCAT reporter
plasmid was dexamethasone inducible in the presence or absence of
cotransfected C/EBP
, whereas the reporter plasmid containing the
mutated C/EBP
DNA-binding site was nonresponsive to glucocorticoids.
In all experiments, reporter gene activity in cells transfected with
the minimal promoter pTk-CAT alone was low and unaffected by
dexamethasone treatment (data not shown). Thus, the presence of an
intact C/EBP DNA-binding site and expression of a functional C/EBP
transcription factor is required for dexamethasone to confer
glucocorticoid responsiveness of the
1.380 to
1.184 p21 promoter
fragment to a heterologous promoter.
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Dexamethasone Stimulation of C/EBP and p21 Promoter Activities
and Gene Expression Are Ablated in a Glucocorticoid-resistant Hepatoma
Cell Variant--
A glucocorticoid-resistant hepatoma cell variant,
EDR1, that was selected for its inability to be growth-suppressed (2), and which produces a glucocorticoid receptor with a point mutation in
its zinc finger region,3 was
utilized to test the functional relationship between the glucocorticoid
stimulation of C/EBP
and p21 gene expression. Western blot analysis
of dexamethasone-treated and untreated hepatoma cells revealed that
under conditions in which both C/EBP
and p21 protein are stimulated
by glucocorticoids in BDS1 hepatoma cells, neither protein was induced
in the EDR1 hepatoma cell variant (Fig.
5, upper panel). BDS1 and EDR1
hepatoma cells were transiently transfected with luciferase reporter
plasmids containing either fragments of the p21 promoter (
2.4
p21-Luc) or the C/EBP
promoter (
350 C/EBP
-Luc). Determination
of relative luciferase-specific activity in both sets of transfections
revealed that dexamethasone stimulated both the p21 promoter and the
C/EBP
promoter activity to approximately the same extent in growth
suppressible BDS1 hepatoma cells (Fig. 5, lower panel). In
the EDR1 hepatoma cell line, which does not undergo a G1
cell cycle arrest (11), neither promoter was regulated by dexamethasone
(Fig. 5, lower panel). These results suggest a direct
functional connection between the glucocorticoid stimulation of
C/EBP
gene expression and that the steroid responsiveness of p21
promoter activity is a key process involved in the cell cycle control
of hepatoma cells.
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DISCUSSION |
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An intricate network of growth inhibitory and stimulatory signals transduced from the extracellular environment converge on specific sets of cell cycle components which, through their concerted action, either drive cells through critical cell cycle transitions or inhibit cell cycle progression (28-32). Our results have established that the glucocorticoid receptor-mediated signaling pathway induces a G1 cell cycle arrest of rat hepatoma cells (4) with the coordinate transcriptional control of the p21 CDK inhibitor gene. We propose that glucocorticoid receptors activate two distinct types of transcriptional cascades that together target and activate the p21 gene promoter (Fig. 6). In one branch of this pathway, the glucocorticoid receptor is proposed to target the p21 promoter through receptor-transcription factor interactions involving pre-existing promoter-bound proteins. This feature of the pathway is based on the ability of dexamethasone to stimulate p21 transcript expression in the absence of ongoing de novo protein synthesis and on the existence of at least three distinct glucocorticoid responsive subregions of the p21 promoter that contain DNA recognition sequences for members of transcription factor families known to interact with the glucocorticoid receptor (see preceding accompanying paper (57)). The p21 promoter does not contain a canonical glucocorticoid response element, suggesting that the glucocorticoid receptor does not act through direct DNA binding (16, 18, 19, 33-35). Consistent with our results, it is well established that protein-protein interactions of steroid receptors with other transcription factors and accessory factors can effectively regulate gene transcription in the absence of DNA-binding sites for the receptor (15, 17, 19, 36, 37).
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We hypothesize that there is a second branch of the glucocorticoid
signaling pathway in which the promoter of the p21 CDK inhibitor gene
is a direct downstream target of the glucocorticoid responsive C/EBP
transcription factor (Fig. 6). The proposed requirement for the
C/EBP
transcription factor in this pathway is based on our
observation that antisense ablation of C/EBP
production in as4
hepatoma cells abolished the glucocorticoid stimulation of p21 promoter
activity. Moreover, mutation of the C/EBP DNA-binding site eliminated
glucocorticoid responsiveness in wild type hepatoma cells, and ectopic
expression of C/EBP
rescued the defective glucocorticoid stimulation
of p21 promoter activity in as4 hepatoma cells. Although the antisense
C/EBP
expressing as4 cells have functional glucocorticoid receptors (11), dexamethasone was unable to induce p21 promoter activity. This
suggests that the glucocorticoid receptor alone is not sufficient to
activate the p21 promoter and functionally requires C/EBP
or an
additional factor which interacts with C/EBP
. The ability of
dexamethasone to induce p21 transcripts in the presence of cycloheximide likely reflects biological redundancy between the two
branches of the cellular cascade that target the glucocorticoid responsive region of the p21 gene promoter. The precise interactions between C/EBP
, p21 promoter elements, and promoter bound proteins may be complex because C/EBP family members contain a leucine zipper
domain and can selectively interact with a variety of other transcription factors including the glucocorticoid receptor and members
of the Ets transcription factor family (20, 38). Therefore, specific
transcriptional regulators of the p21 promoter may be utilized by both
branches of the glucocorticoid receptor transcriptional cascade,
although the molecular details of this signaling mechanism have not
been explored.
The transcriptional effects of steroid receptors on specific target
genes can be enhanced, reduced, or inhibited depending on the
availability of other transcription factors and accessory factors that
target the promoter (15, 37, 39). We propose that C/EBP represents a
tissue-specific factor that is involved in the glucocorticoid
regulation of p21 promoter activity and that its use as a
transcriptional regulator of the cell cycle is restricted to a subset
of cell types. Consistent with this possibility, our preliminary
evidence has shown that different regions of the p21 promoter are
glucocorticoid responsive in growth-arrested mammary tumor cells
compared with the hepatoma cells.2 In liver-derived cells,
C/EBP
plays a unique role in the glucocorticoid-induced cell cycle
arrest, whereas in other growth suppressible cells (9, 12, 40, 41),
such as mammary cells, osteosarcoma cells, fibroblasts, and
lymphoid-derived cells, glucocorticoid receptors may induce the
expression or interact with other tissue-specific transcription factors
to affect cell cycle control.
The activities of the cyclin-dependent kinases (CDKs),
which drive progression through the cell cycle, are regulated in part by the formation of protein complexes with appropriate cyclin and the
cyclin-dependent kinase inhibitor binding partners (30). A
key consequence of the glucocorticoid growth arrest pathway in hepatoma
cells is an elevation in the level of p21, which inhibits the ability
of the G1-acting CDKs to phosphorylate the Rb protein, and
thereby helps induce the G1 cell cycle arrest. Of the two families of CDK inhibitors, the p21 family (p21, p27, and p57) forms
complexes with a wider range of cyclins and CDKs (42, 43). The
expression of p21 appears to be important in normal liver development
because the targeted expression of this gene in hepatocytes of
transgenic mice resulted in an aberrant organization of liver tissue, a
decreased number of adult hepatocytes, reduced liver growth, and the
failure of partial hepatectomy to stimulate liver cell proliferation
(44). Mice deficient in C/EBP also have defects in the control of
hepatic growth, as well as lung development (45). We have observed that
glucocorticoids induce both C/EBP
and p21 protein production in
normal liver, suggesting a shared in vivo mechanism of
regulation. Thus, consistent with our observations in rat hepatoma
cells, which demonstrate a direct functional connection between
C/EBP
and expression of p21, these in vivo studies
suggest a potential mechanistic link between C/EBP
and p21 in the
growth and development of normal liver tissue.
Only a few studies have directly assessed the regulation of p21
promoter activity and the function of the many potential transcription factor binding sites in the p21 promoter (46-51). For example, both
vitamin D3 and retinoic acid, which act through members of the
steroid/thyroid hormone receptor family, directly stimulate p21
transcription through their cognate DNA-binding sites in the p21 gene
promoter (52-54). In addition, C/EBP has been shown to stimulate
p21 promoter activity in adipocytes (55). Our results have established
that the p21 promoter is regulated by glucocorticoids through two
distinct transcriptional mechanisms, one of which utilizes the
regulated expression of C/EBP
to induce a G1 block in
cell cycle progression of liver epithelial tumor cells (11). Because
C/EBP
plays a key role in normal liver and adipocyte function (45,
56), it is tempting to speculate that the glucocorticoid-regulated production of p21 may play a key role in the differentiated functions of normal and transformed cells. We are currently attempting to identify other targets of C/EBP
that may complement the function of
glucocorticoid responsive cell cycle components such as p21, thereby
mediating or maintaining the growth-arrested and differentiated state
of hepatic-derived tissue.
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ACKNOWLEDGEMENTS |
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We express our appreciation to Carolyn Cover, Anita Maiyar, and Paul Woo for critical reading of the manuscript. We also thank Khanh Tong, Vinh Trinh, Linda Yu, and Wei-Ming Kao for their technical assistance. We are grateful to Jerry Kapler for excellent photography.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant RPG-90-001-08-BE.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Contributed equally to this work.
§ To whom correspondence and reprint requests should be addressed: Dept. Molecular and Cell Biology, 591 LSA, University of California, Berkeley, Berkeley, CA 94720. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: glfire{at}uclink4.berkeley.edu.
1
The abbreviations used are: C/EBP,
CCAAT/enhancer binding protein-
; CDK, cyclin-dependent
kinase; Rb, retinoblastoma protein; tk, thymidine kinase; CAT,
chloramphenicol acetyltransferase; PCR, polymerase chain reaction; bp,
base pair(s); vc, vector control.
2 E. J. Cram and G. L. Firestone, unpublished results.
3 R. A. Ramos and G. L. Firestone, unpublished results.
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REFERENCES |
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