CCAAT/Enhancer-binding Protein delta  Regulates Mammary Epithelial Cell G0 Growth Arrest and Apoptosis*

John P. O'RourkeDagger §, Garret C. NewboundDagger , Julie A. HuttDagger , and Jim DeWilleDagger §parallel

From the Dagger  Department of Veterinary Biosciences, the § Ohio State Biochemistry Program, and the  Division of Molecular Biology and Cancer Genetics, Ohio State Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CCAAT/enhancer-binding proteins (C/EBPs) are a highly conserved family of DNA-binding proteins that regulate cell-specific growth, differentiation, and apoptosis. Here, we show that induction of C/EBPdelta gene expression during G0 growth arrest is a general property of mammary-derived cell lines. C/EBPdelta is not induced during G0 growth arrest in 3T3 or IEC18 cells. C/EBPdelta induction is G0-specific in mouse mammary epithelial cells; C/EBPdelta gene expression is not induced by growth arrest in the G1, S, or G2 phase of the cell cycle. C/EBPdelta antisense-expressing cells (AS1 cells) maintain elevated cyclin D1 and phosphorylated retinoblastoma protein levels and exhibit delayed G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. Conversely, C/EBPdelta -overexpressing cells exhibited a rapid decline in cyclin D1 and phosphorylated retinoblastoma protein levels, a rapid increase in the cyclin-dependent kinase inhibitor p27, and accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal. When C/EBPdelta levels were rescued in AS1 cells by transfection with a C/EBPdelta "sense" construct, normal G0 growth arrest and apoptosis were restored. These results demonstrate that C/EBPdelta plays a key role in the regulation of G0 growth arrest and apoptosis in mammary epithelial cells.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (alpha , beta , delta , gamma , epsilon , 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-kappa 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/EBPalpha and C/EBPbeta play major roles in the regulation of proliferation and differentiation of adipocytes and hepatocytes (2, 5, 17-19). C/EBPbeta 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/EBPalpha and C/EBPbeta , C/EBPdelta is also associated with adipocyte differentiation (2). In addition to adipocyte differentiation, C/EBPdelta also functions in the differentiation of lung epithelial cells (20) and myelomonocytic cells (21). C/EBPdelta also plays an important role in the hepatic acute phase response (22, 23). C/EBPepsilon , 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/EBPbeta in mammary gland biology using C/EBPbeta knockout mice (18, 19). In both reports, mammary epithelial cell proliferation and differentiation were dramatically reduced in female C/EBPbeta knockout mice. C/EBPdelta also functions in mammary gland biology; however, instead of promoting mammary gland proliferation and differentiation, C/EBPdelta is associated with mammary epithelial cell G0 growth arrest and programmed cell death (24-26). Most reports indicate that C/EBPalpha 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/EBPdelta 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/EBPdelta is induced in G0 growth-arrested COMMA D mouse mammary epithelial cells (24). C/EBPdelta 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/EBPdelta in multiple mammary-derived cell lines. In addition, the induction of C/EBPdelta in mammary epithelial cells is G0-specific. Altering mammary epithelial cell C/EBPdelta content by transfection with C/EBPdelta 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/EBPdelta in the regulation of major cell fate determining pathways in mammary epithelial cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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/EBPdelta RNA antisense plasmid was produced by digesting MSV/EBPdelta with EcoRI-PstI, generating an ~300-base pair 5' C/EBPdelta cDNA fragment containing the ribosomal binding site. To directionally clone the C/EBPdelta 5' fragment in the antisense orientation in the PcDNA 3 expression vector, the C/EBPdelta 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/EBPdelta overexpression plasmid was produced by ligating an EcoRI-HindIII restriction fragment containing the full-length C/EBPdelta 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/EBPdelta 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 eta 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/EBPdelta , C/EBPbeta , 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/EBPdelta , 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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C/EBPdelta mRNA and Protein Is Induced in Serum-deprived Mammary Epithelial Cells-- We previously described C/EBPdelta induction in the G0 growth-arrested COMMA D mammary epithelial cell line (24). To investigate whether G0 induction of C/EBPdelta was a general characteristic of mammary cells, we investigated C/EBPdelta 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/EBPdelta mRNA levels were extremely low in the growing mammary-derived cells (Fig. 1A, G). After 48 h in growth arrest medium (GAM) C/EBPdelta 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/EBPdelta mRNA levels were constitutively elevated in growing and 48 h growth-arrested NIH 3T3 cells. C/EBPdelta mRNA was barely detectable in growing and 48 h growth-arrested IEC 18 cells. Unlike C/EBPdelta , relatively high levels of C/EBPbeta mRNA were detected in all mammary-derived cell lines, regardless of growth status. C/EBPbeta 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/EBPdelta 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/EBPdelta antibody and detected with a horseradish peroxidase-conjugated anti-rabbit secondary antibody. Blots were visualized with the ECL system.

Western blots of nuclear and cytoplasmic proteins were used to correlate C/EBPdelta protein content with mRNA data and to investigate C/EBPdelta subcellular localization. Consistent with the mRNA data (Fig. 1A), C/EBPdelta protein was barely detectable in growing mammary-derived cells but markedly induced in 48 h growth-arrested cultures (Fig. 1B). In contrast, C/EBPdelta protein levels were elevated and unchanged in growing and growth-arrested NIH 3T3 cells. In all cell lines, the majority of C/EBPdelta protein was localized to the nucleus. The northern and Western blot data support a novel mammary-specific induction of C/EBPdelta during G0 growth arrest.

C/EBPdelta mRNA Is Specifically Induced during G0-mediated Growth Arrest-- To investigate cell cycle-dependent induction of C/EBPdelta 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/EBPdelta mRNA was induced 7-fold during G0 growth arrest. C/EBPdelta 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/EBPdelta , 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/EBPdelta mRNA in mammary epithelial cells, suggestive of a role for C/EBPdelta in the initiation and/or maintenance of G0 growth arrest.


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Fig. 2.   G0-specific induction of C/EBPdelta 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/EBPdelta , 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.

Construction of HC 11 Antisense and Overexpression Cell Lines-- To further investigate the role of C/EBPdelta in HC 11 mammary epithelial cells, we generated C/EBPdelta antisense and overexpression cell lines. Antisense 1 cell line (AS1), which had the greatest reduction in C/EBPdelta protein, was chosen for further analysis. AS1 C/EBPdelta protein levels were reduced by approximately 90% compared with control transfected cells (Fig. 3A). This level of C/EBPdelta 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/EBPdelta 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/EBPdelta construct (TnT) were separated by 12.5% SDS-polyacrylamide gel electrophoresis. Blots were probed with C/EBPdelta antisera. B, nuclear proteins from growing control transfected cells (control) or the OV cell line. C, cytoplasmic; N, nuclear.

Despite multiple attempts, only one C/EBPdelta -overexpressing colony survived drug selection, and therefore, only a single cell line (OV) was available for investigation. C/EBPdelta protein levels and subcellular localization was analyzed by immunoblotting of the OV cell line during normal growth. C/EBPdelta protein levels were increased 2.3-fold compared with growing control cells (Fig. 3B). The constitutively expressed C/EBPdelta protein, like endogenously expressed C/EBPdelta , was localized in the nucleus.

C/EBPdelta 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/EBPdelta 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/EBPdelta as a "conditional" cell cycle brake. Reducing C/EBPdelta levels (AS1 cells) increases proliferation under suboptimal growth conditions, whereas increasing C/EBPdelta levels (OV cells) decreases proliferation under suboptimal growth conditions.


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Fig. 4.   Growth curves of HC11 control, C/EBPdelta antisense, and C/EBPdelta 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.

C/EBPdelta 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/EBPdelta antisense (AS1), and C/EBPdelta 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.

We next investigated the influence of C/EBPdelta 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/EBPdelta antisense (AS1), and C/EBPdelta 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.

C/EBPdelta 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/EBPdelta antisense (AS1), and C/EBPdelta 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/EBPdelta antisense (AS1) and C/EBPdelta 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.

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/EBPdelta Sense RNA-- AS1 cells were stably transfected with a plasmid containing the same 300-base pair fragment of C/EBPdelta as the antisense plasmid but in the sense orientation. C/EBPdelta 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/EBPdelta 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/EBPdelta expression in the C/EBPdelta antisense cell line AS1 corrects defects in G0 growth arrest and cell survival. This demonstrates that C/EBPdelta 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/EBPdelta antisense AS1 and C/EBPdelta 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/EBPdelta 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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study investigated the growth regulatory role of C/EBPdelta in mouse mammary epithelial cells in vitro. In a previous report, we showed that C/EBPdelta 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/EBPdelta is induced during G0 growth arrest is a general property of mammary epithelial-derived cell lines. C/EBPdelta expression is unrelated to growth status in 3T3 cells, which express constitutively high levels of C/EBPdelta , and the IEC18 rat intestinal epithelial cell line, which expresses relatively low levels of C/EBPdelta . This indicates that C/EBPdelta functions in a mammary epithelial cell-specific G0 growth control in vitro. We and others (25, 26) have shown that C/EBPdelta 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/EBPdelta 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/EBPdelta in mammary epithelial cells have not yet been identified. Factors that induce C/EBPdelta gene expression in other tissues and cell types do not appear to induce C/EBPdelta expression in cultured HC 11 mammary epithelial cells. For example, glucocorticoids induce C/EBPdelta expression in intestinal epithelial cells, lung epithelial cells and in adipocytes (20, 33, 34) but have no effect on C/EBPdelta mRNA levels in HC 11 cells (data not shown). Insulin induces C/EBPdelta mRNA in adipocytes (35); however, C/EBPdelta levels are low in mammary epithelial cells cultured in insulin-containing complete growth medium (24). Interleukin-6 and cAMP induce C/EBPdelta 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/EBPdelta mRNA (data not shown). Other candidate molecules that may act as inducers of C/EBPdelta 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/EBPdelta 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/EBPdelta -negative (AS1 cells express C/EBPdelta 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/EBPdelta restored appropriate cycle exit/G0 entry growth arrest and apoptosis in AS1 cells. These results suggest that C/EBPdelta , like VHL, functions in the regulation of cell cycle exit.

The difficulty we encountered in the generation of a C/EBPdelta overexpressing mammary epithelial cell line supports a growth inhibitory role for C/EBPdelta . Similar difficulties were not encountered in producing C/EBPdelta antisense cell lines or other C/EBP isoform expression cell lines. Once produced, however, the presence of elevated levels of C/EBPdelta (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/EBPdelta , or C/EBPdelta alone is insufficient to induce growth arrest. Additional factors, such as subcellular compartmentation and/or posttranscriptional modification of C/EBPdelta may be required for full function.

C/EBPdelta is regulated by subcellular localization in hepatocytes (39). In cultured mammary epithelial cells and the mammary gland in vivo, C/EBPdelta protein is primarily localized to the nucleus, regardless of growth or differentiation status (24). C/EBPdelta 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/EBPdelta regulation in mammary epithelial cells. The inability of C/EBPdelta to inhibit OV cell growth in complete growth medium could be due to a lack of phosphorylation in growing cells. Phosphorylation of C/EBPdelta 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/EBPdelta 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/EBPdelta overexpression in OV cells did not induce cell cycle exit/G0 growth arrest in optimal growth medium, C/EBPdelta 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/EBPdelta in the nucleus (OV cells) rapidly exit the cell cycle in response to growth arrest conditions. This suggests that C/EBPdelta 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/EBPdelta levels, p27, and G0 growth arrest in vitro. This association may extend to the mammary gland in vivo, as C/EBPdelta and p27 are both induced in the mammary gland during involution.2

C/EBPdelta 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/EBPalpha and C/EBPbeta both transactivate the cyclin-dependent kinase inhibitor p21waf1 promoter (49), and C/EBPalpha 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/EBPdelta levels were reduced (AS1 cells), cyclin D1 levels remained elevated and cell cycle progression continued in low serum medium. When C/EBPdelta levels were increased (OV cells), cyclin D1 levels rapidly declined and cell cycle progression stopped in low serum medium. These data indicate that C/EBPdelta and cyclin D1 are induced under opposing growth conditions. C/EBPdelta may influence cyclin D1 levels by acting as a transcriptional repressor of cyclin D1 in G0 growth-arrested mammary epithelial cells. C/EBPdelta 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/EBPdelta antisense-expressing COMMA D mouse mammary epithelial cells (24). In both studies, reducing endogenous C/EBPdelta levels consistently delayed cell cycle exit. In this report, however, we have extended the analysis of the C/EBPdelta antisense-expressing HC11 cells (AS1) to include cell cycle regulatory proteins and apoptosis. The results indicate that C/EBPdelta plays an important role in regulating cell cycle exit. When this role is compromised by reducing endogenous C/EBPdelta 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/EBPdelta 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/EBPdelta overexpressing OV cells and percentage of cells undergoing apoptosis was reduced in C/EBPdelta antisense AS1 cells. Apoptosis in both cell lines, however, was similar in response to staurosporine (data not shown). This suggests that C/EBPdelta 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/EBPdelta 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/EBPdelta 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/EBPalpha 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/EBPbeta is uncertain. C/EBPbeta knockout mice exhibit defective mammary epithelial cell proliferation and differentiation (18, 19); however, overexpression of C/EBPbeta , or LIP, the dominant negative inhibitor of C/EBPbeta , 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/EBPdelta is similar to that described for the VHL gene product (29). In addition, C/EBPdelta 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/EBPdelta gene transcription in mammary epithelial cells.

    Acknowlegements

We thank Drs. Peter Johnson and Esta Sterneck (ABL-Basic Research Program, NCI, National Institutes of Health, Frederick, MD), for valuable reagents, helpful discussions, and support and specific comments on the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant RR00136CA (to J. A. H.) and NCI, National Institutes of Health Grants CA 57607 (to J. D.) and P30CA16058.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.

parallel To whom correspondence should be addressed: Dept. of Veterinary Biosciences, The Ohio State University, Columbus, OH 43210. Tel.: 614-292-4261; Fax: 614-292-6473; E-mail: dewille.1{at}osu.edu.

2 L. Dearth, and J. DeWille, manuscript in preparation.

    ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; CGM, complete growth medium; GAM, growth arrest medium; Rb, retinoblastoma protein; FITC, fluorescein isothiocyanate; OV, overexpression; FBS, fetal bovine serum; CHOP, C/EBP homologous protein; VHL, von Hippel-Lindau.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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