A Role for CCAAT/Enhancer Binding Protein b-Liver-enriched ......[CANCER RESEARCH 61, 261–269, January 1, 2001] A Role for CCAAT/Enhancer Binding Protein b-Liver-enriched Inhibitory

[CANCER RESEARCH 61, 261–269, January 1, 2001]

A Role for CCAAT/Enhancer Binding Protein b-Liver-enriched Inhibitory Proteinin Mammary Epithelial Cell Proliferation 1

Cynthia A. Zahnow,2 Robert D. Cardiff, Rodolfo Laucirica, Daniel Medina, and Jeffrey M. RosenDepartments of Cell Biology [C. A. Z., D. M., J. M. R.] and Pathology [R. L.] and The Methodist Hospital [R. L.], Baylor College of Medicine, Houston, Texas 77030, and Centerfor Comparative Medicine, University of California at Davis, Davis, California 95616 [R. D. C.]

ABSTRACT

The transcription factor, CCAAT/enhancer binding protein b(C/EBPb), regulates the expression of genes involved in proliferation andterminal differentiation. Dimerization of the dominant-negative C/EBPb-liver-enriched inhibitory protein (LIP) isoform with the C/EBP b-liver-enriched activating protein (LAP) isoform inhibits the transcriptionalactivation of genes involved in differentiation. Consequently, an increasein LIP levels may inhibit terminal differentiation and lead to proliferation.C/EBPb-LIP and LAP are crucial for mammary gland development(G. W. Robinsonet al.,Genes Dev.,12: 1907–1916, 1998; T. N. Seagroveset al., Genes Dev.,12: 1917–1928, 1998) and are also overexpressed inbreast cancer (B. Raughtet al., Cancer Res.,56: 4382–4386. 1996; C. A.Zahnow et al.,J. Natl. Cancer Inst., 89: 1887–1891, 1997); however, littleis known about how these isoforms differentially regulate cell cycle pro-gression. To address this question, C/EBPb-LIP was overexpressed inboth the mammary glands of transgenic mice and in cultured TM3mammary epithelial cells. Here we report that the involuted mammaryglands from transgenic mice overexpressing C/EBPb-LIP contain bothfocal and diffuse alveolar hyperplasia and, less frequently, contain mam-mary intraepithelial neoplasias (high grade) and invasive and noninvasivecarcinomas. Likewise, cultured TM3 cells, stably expressing C/EBPb-LIP,showed an increase in proliferation and foci formation attributable to areentry into S-phase during cellular confluence. These results demonstratethat C/EBPb-LIP can induce epithelial proliferation and the formation ofmammary hyperplasias and suggest that a C/EBPb-LIP-initiated growthcascade may be susceptible to additional oncogenic hits, which could resultin the initiation and progression of neoplasia.

INTRODUCTION

Although a majority of breast cancer research has focused onstudies of advanced tumors and metastases, the molecular mecha-nisms responsible for the regulation of normal mammary gland de-velopment and the initiation of premalignant disease are still not wellunderstood. Breast cancer originates primarily in the normal mam-mary epithelium of the terminal ducts and has been hypothesized toinvolve the clonal expansion of an initiated cell into an epithelialhyperplasia prior to local invasion of the mammary stroma. Themolecular changes that occur during this progression include theamplification and/or overexpression of transcription factors, growthfactors, and growth factor receptors or the silencing of tumor sup-pressor genes, which can then act to disrupt the delicate balancebetween cellular proliferation, terminal differentiation, and pro-grammed cell death. C/EBP3b is one such transcription factor, which

has been implicated in cell cycle regulation and plays an importantrole in mammary gland proliferation and differentiation.

C/EBPb is a member of the C/EBP family of transcription factorsthat bind to specific DNA sequences as homo- and heterodimers andaffect the transcription of target genes involved in proliferation anddifferentiation. Six C/EBP genes have thus far been identified(C/EBPa, C/EBPb, C/EBPg, C/EBPd, C/EBPe, andC/EBPz), and allof the genes are intronless except forC/EBPe andC/EBPz. Transcrip-tion of C/EBPb results in a single mRNA that can be translated intofour isoforms: LAP (full-length LAP-Mr 38,000 and LAP-Mr 35,000);LIP (LIP-Mr 20,000); and a smallerMr 16,000 isoform. The predom-inant isoforms expressed in the mouse mammary gland are theMr35,000 andMr 20,000 family members. Several different mechanismshave been described to account for the differential expression of theC/EBPb isoforms: (a) a leaky ribosome scanning mechanism (1); (b)the interaction of a CUG repeat binding protein (CUGBP1) with the59 region of C/EBPb mRNA (2); (c) a mechanism involving theevolutionary conserved upstream open reading frame of the 59 regionof C/EBPb mRNA and the eukaryotic translation initiation factorseIF-2a and eIF-4E (3); and (d) specific proteolytic cleavage in hema-topoietic progenitor cells present in mouse liver (4). All C/EBPbfamily members share a strong homology in the COOH-terminal,leucine-rich dimerization domain (bZIP) and the DNA-binding basicregion. The truncated C/EBPb-LIP isoform, translated from the thirdAUG, lacks most of thetrans-activation domain and can, therefore,dimerize and bind to DNA but is unable to activate gene transcription.Because of an increased DNA affinity of the C/EBPb-LIP isoform,this inhibition of transcriptional activity can occur even at substoi-chiometric ratios of LIP:C/EBP, thereby suggesting a dominant-negative function for C/EBPb-LIP (1). Thus, the LAP:LIP ratio,rather than their absolute amounts, may be an important indicator oftranscriptional activity by C/EBPb. Dimerization of bZIP proteins canoccur in the absence of DNA but is a prerequisite for DNA binding(5). Additionally, dimers of bZIP proteins are usually unstable whennot bound to DNA and will rapidly dissociate back to monomers (6).

C/EBPb is vital for development of the mouse mammary gland (7,8). As demonstrated in the C/EBPb knockout mouse, mammaryglands contain enlarged, undeveloped ducts that have a low prolifer-ative rate and decreased tertiary branching. C/EBPb-LAP expressionis detectable throughout murine mammary gland development and isin contrast to C/EBPb-LIP expression levels, which are highest duringpregnancy (proliferative state) and reduced in the virgin (mice,4months of age) gland and lactating gland (8, 9). TheC/EBPa andC/EBPd genes are also expressed in the murine mammary gland.Although C/EBPa mRNA is expressed throughout mammary devel-opment, C/EBPa is not essential for mouse mammary gland devel-opment (8). Additionally, knockout mice have been generated forC/EBPd (10), but a mammary gland phenotype has not been reported.Nevertheless, the C/EBPd transcript is overexpressed during involu-tion of the mouse mammary gland (11, 12), and cell culture studies

Received 6/23/00; accepted 10/26/00.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This research was supported by Grant CA 16303 from the National Cancer Institute(to J. M. R.), Grant JB-0014 from the State of California Breast Cancer Research Program(to R. D. C.), and Contract DAMD17-96-1-6086, a postdoctoral fellowship from theDepartment of Defense (to C. A. Z.).

2 To whom requests for reprints should be addressed, at Johns Hopkins ComprehensiveCancer Center, Room 542, 1650 Orleans Street, Baltimore, MD 21231.

3 The abbreviations used are: C/EBP, CCAAT/enhancer binding protein; WAP, wheyacidic protein; LAP, liver-enriched activating protein; LIP, liver-enriched inhibitoryprotein; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium; BrdUrd, bromodeoxyuridine; FACS, fluorescence-activated cell sorter;

MIN, mammary intra-epithelial neoplasia; HAN, hyperplastic alveolar nodule; CMV,cytomegalovirus; Rb, retinoblastoma; HOG, hyperplastic outgrowth.

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have determined that its predominant role in mammary gland devel-opment is in growth arrest of mammary epithelial cells (13, 14).

Consequently, the differential expression pattern of the C/EBPbisoforms suggests a dual and opposing role in mammary gland devel-opment and the importance of the LAP:LIP ratio as a cell cycleswitch, resulting either in cellular differentiation or proliferation.Although C/EPBb-LIP is also overexpressed in breast cancer and isassociated with biological predictors of poor survival, such as loss ofestrogen and progesterone receptor expression, increased cellular pro-liferation, aneuploidy, and poor histological and nuclear grades (15),its role in tumorigenesis is unknown. Taken together, these observa-tions have led to the hypothesis that overexpression of the C/EBPb-LIP isoform in the mammary gland can result in epithelial cellproliferation that may render the mammary gland more susceptible toadditional oncogenic hits, resulting in the initiation and progression ofneoplasia. Persistent, aberrant expression of the C/EBPb-LIP isoformin these neoplasms may contribute to an increased growth rate andresult in a more proliferative or aggressive tumor. To test this hypoth-esis, complementary approaches have been used to study the overex-pression of C/EBPb-LIP in both transgenic mice and mammaryepithelial cell lines. Our studies have demonstrated that C/EBPb-LIPoverexpression is associated with increased epithelial proliferation,resulting in mammary hyperplasias and the stochastic development ofinfrequent carcinomas.

MATERIALS AND METHODS

Transfection and Maintenance of TM3 Cells. TM3 cells were grown andmaintained using HEPES buffered DMEM/F-12 growth medium containing2% fetal bovine serum, 10mg/ml insulin, L-glutamine, 5 ng/ml epidermalgrowth factor, and 5mg/ml gentamicin sulfate (16). At 20–40% confluence,cells were stably transfected with pCIneo-LIP or pCIneo (as control) usingSuperfect (Qiagen). Stably transfected cells were cloned using cloning cylin-ders (PGC Scientifics) and maintained with 0.2 mg of G418 per ml growthmedia.

MTS Cell Proliferation Assay. Five independent LIP clones and fiveindependent neomycin control clones were plated in quadruplicate into a96-well tissue culture format at a density of 23 105 cells/well. The number ofviable or proliferative cells was determined for days 1, 3, 5, 7, 9, and 12 ofculture using the CellTiter 96 Aqueous Non-Radioactive Cell ProliferationAssay from Promega, according to the manufacturer’s instructions. Afterapplication of the MTS reagent, the cells were incubated for 2–3 h at 37°C, andabsorbance at 490 nm was measured using a Dynex Technologies ELISA platereader. Data were plotted as fold change in growth rate.

BrdUrd Staining for FACS Analysis. Two independent LIP clones andtwo independent neomycin control clones were plated into 100-mm tissueculture dishes at a density of 13 106 cells/plate. At days 3, 7, 10, and 15 ofculture, the cells were pulse labeled for 15 min with 10mM BrdUrd (AmershamLife Science), washed with Hanks’ medium, and removed from the plate usingthe enzyme Dispase II (Boehringer Mannheim). The pellet was resuspended in200 ml of Hanks’ medium, and the cells were fixed by the addition of 70%ethanol while vortexing to avoid cell clumping. Cells were stored at 4°C in70% ethanol until collection of the last time point. Approximately 43 106

cells were removed from the initial pellet and incubated for 10 min with 3 mlof pepsin (0.04% in 0.10N HCl) on a rocker at room temperature. Aftercentrifugation (1200 rpm for 5 min), the pepsin supernatant was aspirated, 3 mlof 2 N HCl were added to a vortexed pellet, and the mixture was incubated for20 min at 37°C. After incubation, 6 ml of 0.1M sodium borate were addedwhile vortexing, and the cells were pelleted. After aspiration of the superna-tant, 6 ml of PBST-B (PBS with 0.5% Tween 20, 0.5% BSA) were added whilevortexing, the cells were pelleted, the supernatant was aspirated, and 1 ml ofPBS containing 1 unit of DNase-free RNase was added and incubated for 30min at 37°C. The nuclei were again pelleted, the supernatant was removed, and20 ml of anti-BrdUrd FITC and 100ml of PBST were added. The nuclei wereincubated for 1 h in the dark at room temperature, and 3 ml of PBST-B wereadded while vortexing, the nuclei were pelleted, the supernatant was aspirated,

and propidium iodide (Sigma) was added for a final concentration of 5 or 10mg/ml in PBST-B. Nuclei were stored at 4°C overnight and examined 1 daylater by FACS analysis.

Active Caspase-3 Determination.Active-caspase-3 levels were deter-mined both by a fluorogenic assay and FACS analysis. TM3 cells stablyexpressing either PCI-neo-LIP or PCI-neo as control were plated at an equaldensity of 1.53 106/100-mm plate and cultured for 3, 7, 10, and 15 days. Ateach time point, cells were harvested and processed by two methods: scrapingand freezing of the cell pellet for the fluorogenic assay; or digestion withDispase (Boehringer Mannheim), followed by fixation with 4% paraformal-dehyde for FACS analysis in the Flow Cytometry Core Lab (Baylor College ofMedicine, Houston, TX). Active caspase-3 was determined using either Ac-DEVD-AMC Caspase-3 (CPP32) fluorogenic substrate or phycoerythrin-con-jugated polyclonal rabbit anti-active caspase-3 according to the manufacturer’sinstructions (PharMingen).

Plasmid Construction: WAP-LIP-WAP. The first step in the generationof this construct was theEcoRI linearization and Klenow fill-in of a pBlue-script SKII(1) plasmid containing 843 bp of rat WAP 39sequence, with aportion of the third exon, the third intron, all of the fourth exon, and 70 bp of39 flanking DNA. The second step included the removal of;865 bp of anNcoI/XhoI cDNA fragment (LIP) from the COOH-terminal region of ratC/EBPb (MSV/C/EBPb, kindly provided by Dr. S. McKnight, University ofTexas Southwestern, Dallas, TX). This cDNA insert contains only the thirdtranslation initiation Met codon and encodes a full-length protein for LIP andnot the LAP isoforms. After fill-in with Klenow, the LIP cDNA fragment wasligated to a position immediately 59to the 39 WAP sequence in pSCPTSKII(1). In the third step, the LIP-WAP 39construct was excised using bothKpnI andSpeI, filled-in with Klenow, andXbaI linkers were attached. ThisLIP-WAP39 fragment was then ligated to anXbaI-linearized WAP 59frag-ment, which consists of 982 bp of a rat WAP 59 promoter fragment (2949 to11) and WAP 59untranslated region (from11 to 133). The integrity of theWAP-LIP-WAP construct was confirmed by sequencing the WAP-LIP bound-aries. The WAP-LIP-WAP construct was removed from pSCPT SKII(1) bydigestion withBstXI andKpnI producing a vector (2.9 kb) and insert fragment(2.75 kb) that were similar in size. Further digestion usingPvuI, which cutsonly the vector, allowed complete size fractionation and separation usingagarose gel electrophoresis. The DNA was further purified and concentrated ona silica matrix (Glassmilk; Geneclean). Transgenic mice (FVB inbred) weregenerated by the transgenic core facility at Baylor College of Medicine.

Plasmid Construction: PCI-neo-LIP. To construct PCI-neo-LIP, an865-bp cDNA, which codes only LIP, was excised from the WAP-LIP-WAPconstruct usingXbaI andEcoRV and directionally cloned into the PCI-neoplasmid (Promega) at theXbaI andSmaI restriction sites using T4 DNA ligase.

Analysis of Tail DNA. Hot-start PCR reactions (25ml) were performedusing a bottom and top mix initially separated by a wax barrier. The bottommix, containing 1mg of genomic tail DNA, 1 mM MgCl, 0.2 mM deoxynucle-otide triphosphates, 10% DMSO, and 103Promega thermocycle buffer in afinal volume of 14.5ml, was heated to 90°C for 10 min to denature the DNAand melt the wax pellet and then cooled to 4°C. The top mix, containing 12.5pmol of each primer, 103Promega thermocycle buffer, and 2.5 units of Taqpolymerase (Promega) in a final volume of 10.5ml, was added to the top of thehardened wax barrier and allowed to mix with the bottom reagents by heatingto 94°C. The reaction profile consisted of 30 cycles of 1 min at 94°C, 2 minat 60°C, and 3 min at 72°C. After the final cycle, the samples were incubatedat 72°C for an additional 5 min. Reactions were performed in a DNA thermo-cycler (Perkin-Elmer). The PCR products were resolved on a 1.5% agarose gel.The sequences of the synthetic oligonucleotides used in the PCR reactionswere as follows (59to 39): rWAP11 (F), ATCAGTCATCACTTGCCTGC-CGCCG; and LIP 1574 (R), GTGTGTTGCGTCAGTCCCGTGTCCA.

Protein Extraction and Western Blot Analysis. Tissue and/or cells weredisrupted in RIPA buffer [50 mM Tris-Cl (pH 7.4), 1% NP40, 0.25% desoxy-cholate, 150 mM NaCl, 1 mM EGTA, and 0.2% SDS] containing the followingkinase, phosphatase, and protease inhibitors: 1 mM NaVO3, 1 mM NaF, 1 mMNa2MoO4, 10 nM okadaic acid, and 1mg/ml each of benzamidine, aprotinin,soybean trypsin inhibitor, and antipain. Aliquots of these lysates containing100 mg of protein were electrophoresed on denaturing SDS 12%-polyacryl-amide minigels and then transferred to polyvinylidene difluoride membranes(Millipore, Bedford, MA) overnight at 75 mA. Blots were blocked 90 min inTBST [20 mM Tris (pH 7.5), 150 mM NaCl, and 0.5% Tween 20] containing

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3% nonfat dry milk (Carnation, Glendale, CA) and then incubated for 90 minin this solution containing antibodies (0.5 ng/ml; Santa Cruz) prepared againstC/EBPb. Blots were washed with TBST (without milk) three times for 5–10min each, with agitation. Blots were then incubated for 60 min in blockingsolution containing 200 ng/ml biotinylated donkey antirabbit immunoglobulin(Amersham, Little Chalfont, England) and washed. Lastly, blots were incu-bated for 30 min in blocking solution containing 40 ng/ml streptavidin-horseradish peroxidase (Oncogene Science, Uniondale, NY) and washed asbefore. Enhanced chemiluminescence (Hyperfilm; Amersham) and chemifluo-rescence reagents (Storm Fluoroimager; Molecular Dynamics) were used forvisualization per the manufacturer’s instructions.

Tissue. Approximately 43 glands from lactating mice (day 10 of lactation)were examined. Lactating mothers were separated from pups 2 h prior toexcision of the mammary glands to reduce the variability associated withsuckling and milk stasis. Inguinal glands were fixed in 10% neutral bufferedformalin for ;6 h, embedded in paraffin, sectioned at 5mm, deparaffinizedthrough a graded series of xylenes and alcohols, rehydrated in water, andstained with H&E. Involuted, inguinal, and thoracic mammary glands wereremoved and examined from 22 transgenic LIP mice and 14 control mice(nontransgenic siblings and wild-type FVBs), 6–32 months of age. All micewere multiparous, and most had undergone involution.3 months beforebiopsy; however, a few mice were permitted to involute for a shorter intervalof 14 days prior to biopsy. Tissues were processed in a manner identical to thatfor lactating tissue. Whole-mount analysis was performed as described bySeagroveset al. (8).

RESULTS

Overexpression of C/EBPb-LIP in Transgenic Mice. Sequencesin the rat WAP promoter and 39 untranslated region were used tocreate a transgenic construct, WAP-LIP-WAP (Fig. 1A), which pref-erentially targets high levels of C/EBPb-LIP expression to the mam-mary gland in mice, starting at about days 7–10 of pregnancy andextending throughout lactation (17). Seven WAP-LIP-WAP foundermice were generated. One female founder did not produce live off-spring, and the other three female founders were mosaic and did notexpress theLIP transgene in their mammary glands. The six remain-ing founders were bred further, and transgene expression was detectedin lactating glands of the F1 generation from three of the six founderlines (6067, 6074, and 6070) by Western blot analysis (Fig. 1B) andby reverse transcription-PCR (data not shown). Unfortunately, be-cause of limitations of the currently available C/EBPb antibodies,transgenic LIP expression could not be detected via immunocyto-chemistry for two reasons: (a) the antibody recognizes the COOHterminus and cannot distinguish between the C/EBPb-LIP and LAPisoforms; and (b) the antibody cannot discriminate between endoge-

nous mouse C/EBPb-LIP and transgenic rat C/EBPb-LIP because theproteins are.98% similar in amino acid identity. The level oftransgene expression was relatively constant in subsequent genera-tions, as evidenced by the similar levels of transgene expressionobserved in the F1 as well as in the F5 generation (data not shown).Although the construct is not epitope-tagged, the transgenic C/EBPb-LIP protein is developmentally distinguishable from endogenousC/EBPb-LIP during lactation, because the native LIP isoform is notexpressed during lactation and is primarily expressed during preg-nancy (Fig. 1B, FVB lane). Consequently, it was hypothesized thatany phenotypic effects resulting from the overexpression of C/EBPb-LIP would, therefore, be most readily detected during lactation andsubsequent involution.

The mammary glands from;22 C/EBPb-LIP transgenic mice and21 control mice (nontransgenic siblings and wild-type FVBs) corre-sponding to days 1–18 of lactation were examined at both the grossand microscopic levels. C/EBPb-LIP transgenic mice did not experi-ence any difficulties in nursing their pups, and no histological abnor-malities were observed in the mammary glands of lactating mice.Next, involuted glands from 22 transgenic and 14 control mice (non-transgenic siblings and wild-type FVBs), 6–32 months of age, wereexamined for abnormalities. Mammary gland neoplasia was observedin 9% (2 of 22) of transgenic mice and included two invasive carci-nomas (Fig. 2A) and three MINs (high grade; Fig. 2B) from one27-month-old mouse. MINs comprise a variety of intraluminal epi-thelial proliferations with atypical cytology, includingin situ carci-nomas (18). Additionally, the gland from a 20-month-old mousecontained a highly proliferative, poorly differentiated carcinoma (Fig.2D), and the gland contralateral to the tumor contained diffuse alve-olar hyperplasia (Fig. 2C). A more thorough, blind examination (bytwo independent researchers, R. D. C. and D. M.) of a subset of the 22involuted glands revealed that 30–40% (3 of 10 or 4 of 10) oftransgenic mice contained two distinct forms of mammary hyperpla-sias known as focal hyperplastic alveoli or HAN (Fig. 3,A andB) anddiffuse alveolar hyperplasia (Fig. 3,C andD). The epithelial cells inthese hyperplasias are characterized by their large shape and activenuclei with open chromatin, large nucleoli, and a high rate of mitosis,as evidenced by an abundance of mitotic figures. In contrast to normalepithelial cells that have undergone a delayed involution, hyperplasticcells contain very little lipid and are not actively secreting. Thedescribed neoplasias and hyperplasias were observed in both the 6067and 6074 lines, and no tumors or hyperplasias were observed inage-matched, nontransgenic siblings or wild-type FVB mice.

Fig. 1. Structure of the WAP transgenic construct and detection ofexpression by Western blot analysis.A, the transgene was constructedusing 949 bp of rat WAP 59noncoding sequence and the first 33 bp ofthe open reading frame, followed by 865 bp of a C/EBPb rat cDNAfragment that codes only for the C/EBPb-LIP isoform. An additional843 bp of rat WAP sequence containing part of exon III, intron C, andexon IV plus 70 bp of 39flanking DNA was positioned immediately 39to the cDNA (see “Materials and Methods” for further details).B,representative Western blot of mammary gland extracts prepared from(10-day) lactating, F1 generation, female mice from the followingtransgenic founder lines: 6067, 6074, 6070, 6060, and 6065. Thetransgenic (LIP) construct was detected in three lines (6067, 6074, and6070), and the endogenous (LAP) isoform was detectable in all mice.A nontransgenic (FVB) mouse was included in the Western blot todemonstrate that endogenous C/EBPb-LIP protein levels are not de-tectable during lactation. Cross-reactive material (CRM), which isobserved in mammary gland extracts from C/EBPb knockout mice (8),serves as an internal loading standard.

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Overexpression of C/EBPb-LIP in Cultured Mammary Epithe-lial Cells. To investigate the molecular mechanisms responsible forthe proliferation and hyperplasias associated with C/EBPb-LIP over-expression, cell cycle studies were initiated in cultured mammaryepithelial cells. Two considerations helped to determine which mam-mary epithelial cell line was used for testing the effects of overex-pression of C/EBPb-LIP on cell growth and tumorigenicity: (a) thatthe endogenous levels of C/EBPb-LIP were low; and (b) that the cellline should exhibit normal, nontumorigenic growth patterns. The TMcell lines were established from hyperplastic alveolar outgrowths,which resulted from thein vivo transplantation of FSK cell lines (16).These lines are maintained both in culture (in vitro) and as mammarytransplants, which grow (in vivo) either as hyperplastic outgrowths ortumors. TM3 (HOG) is a slow-growing, hyperplastic alveolar out-growth that is ovarian hormone dependent and infrequently progressesinto tumors when maintained beyond transplant generation 16 (19). Incontrast, (low passage,,10) TM3 in vitro cultures do not producesuccessful outgrowths after transplantation into cleared mammary fat

pads, have low endogenous C/EBPb levels as compared with a moreneoplastic TM line (TM6; Ref. 20), exhibit a marked dependence onepidermal growth factor for growth (21), and contain a mutant p53(Ser

233 ins; Refs. 22 and 23), which has been associated with a higher

rate (4–7%) of apoptosis (19). Consequently, both the TM3 out-growths and the TM3 cell line fit the necessary criteria for containingrelatively low levels of endogenous C/EBPb and exhibiting normal orweakly tumorigenic growth patterns.

The TM3 cell line was stably transfected with either CMV-drivenPCIneo-LIP or PCIneo (the vector without LIP cDNA insert, ascontrol), and stable cellular clones were generated. In the parental,nontransfected cells, endogenous C/EBPb protein levels were ob-served to change with growth. The expression of the native LIPisoform was consistently observed to be higher when the cells wereexponentially growing than when the cells were contact inhibited orconfluent (Fig. 4,left panel). Consequently, CMV-driven expressionof C/EBPb-LIP from the PCIneo-LIP construct was easily detectedduring confluence (Fig. 4,middle panel). Passage number had little

Fig. 2. The mammary glands from transgenic C/EBPb-LIP mice develop invasive carcinoma and high-grade MIN.A, representative photomicrograph (320) of one of two invasivecarcinomas from mouse (7869, case 1). These H&E-stained lesions contained extensive fibrosis, infiltrated by small cords of atypical epithelial cells (seearrow) with large pleomorphicnuclei and scattered mitoses.B, the same gland from mouse (7869, case 1) also contained three high-grade MINs. The lesion represented inB (320) consists of several expanded alveoli(large arrow) filled with hyperchromatic, atypical nuclei with prominent nucleoli and abnormal mitotic figures. The oval profiles typically form a cribriform-like pattern (small arrows),as is often observed in ductal carcinomain situ,and a dense lymphocytic infiltrate (w) is present in the upper portions of the micrograph. (CandD, 320). Mouse (case 35) developeda mammary carcinoma with some foci of squamous metaplasia.C, the gland contralateral to the tumor and depicts a profile of incomplete involution or diffuse hyperplasia. The contoursof the residual alveoli are described as rounded (arrows), as opposed to the angulated acini present in a normally regressed gland (see Fig. 3,C andD). Duct ectasia was also commonlyobserved in involuted glands. The large carcinoma (D) is composed of nests (short arrow) and cords (long arrow) of very hyperchromatic cells in a dense connective tissue stroma.The sizes of the cords vary. The tumor cells have large, pleomorphic nuclei with prominent and multiple nucleoli but with delicate chromatin. The cytoplasm is amphophilic, and themitotic rate is very high.

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effect on loss or gain of endogenous C/EBPb expression, as shown bycomparison of the first panel containing the earlier passage parentalTM3 line and the last panel (Fig. 4), which represents a late-passageTM3 clonal line expressing only neomycin. Endogenous C/EBPb-LAP levels were more variable during confluence, but endogenousC/EBPb-LIP levels were usually low and were never observed toexceed the expression levels for C/EBPb-LAP during confluence inTM3 cells.

TM3 clones stably expressing CMV-driven LIP and vector-only,neomycin controls were randomly chosen and tested for proliferativepotential using an MTS cell proliferation kit. The clones displayeddifferent growth rates, but the five clonal lines expressing LIP were,on average, twice as proliferative as the five control (Neo) clonal lines(Fig. 5). The fold change in growth was determined by dividing thenumber of proliferating, viable cells (as measured by the amount ofabsorbance at 490 nm) at days 12, 9, 7, 5, and 3 by the value for

proliferation at day 1 for each clone. To determine whether thisincrease in cell number or growth was attributable to an increase in thenumber of cells entering S phase, cells were pulse labeled withBrdUrd at 3, 7, 10, and 15 days of culture and analyzed by flowcytometric (FACS) analysis (Fig. 6B). The data indicate that expres-sion of LIP in TM3 cells facilitates entry into S-phase and DNAsynthesis. Both the LIP-expressing cells and the control cells exhib-ited similar levels of BrdUrd incorporation during exponential growth(day 3) and early confluence (day 7); however, the LIP-expressingcells did not remain contact inhibited, and by day 15 of culture, atleast 10% of the cells had re-entered the cell cycle, were proliferating,and formed foci as compared with the neomycin control cells, whichremained a monolayer (Fig. 6,A andB). Interestingly, CMV-drivenLIP expression does not coincide, temporally, with the renewedgrowth and reentry of the LIP clones into the cell cycle (Fig. 6C).Nuclear LIP expression, as well as the LIP:LAP ratio, was higher in

Fig. 3. Involution in the mammary glands of transgenic CEBPb-LIP mice is incomplete and characterized by diffuse and focal hyperplasia.A, whole-mount analysis of the involutedmammary gland from mouse 8/98#2, case 15, showing both focal and diffuse hyperplasia. The focal hyperplasia or HAN is evident as a grape-like cluster on theright sideof the photo(arrow). H&E analysis of the HAN (arrow) is presented inB (320). The diffuse hyperplasia resembles a delayed or incomplete regression. The residual alveoli are characterized byan abnormally round appearance as opposed to the more normal, collapsed, and angulated acini observed after a normal regression.C (320) shows a normal pattern of mammary glandregression (mouse 2160, case 16) with collapsed and angulated acini, often containing lipid droplets (arrow;inset,340). D (320), a diffuse alveolar hyperplasia (mouse 6074 invT,case 19). The rounded alveoli do not contain lipid (arrow;inset,340) and are either filled with cells or contain multilayers of epithelial cells possessing large, active nuclei with anopen chromatin and large nucleolus (not visible in this magnification).

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the LIP-expressing clones during the first week of culture than duringthe second week (Fig. 6C). It is highly unlikely that the proliferationobserved in these cells can be attributed to clonal variation, becausefoci formation was observed in at least four clonal lines stablyexpressing C/EBPb-LIP and was never observed in the control cells.In addition, the increase in cellular proliferation was not accompaniedby a decrease in apoptosis. The TM3 clones were assayed for changesin caspase-3 activity using two independent methods (see “Methodsand Materials”), and the levels of active caspase-3 were found not tobe significantly decreased in the proliferating LIP-expressing cells ascompared with the control (Neo) cells (data not shown).

Transplantation of LIP-overexpressing TM 3 Cells into the FatPads of BALB/c Mice. To determine whether the TM3 cells main-tained their proliferative growth potentialin vivo, stably expressingLIP and vector control (Neo) cells (13 106 cells) were transplantedinto the right and left inguinal mammary fat pads, respectively, ofvirgin, syngeneic BALB/c mice. An inherent difficulty in cell line/transplantation experiments is that many nontransfected, high-passagemammary epithelial cell lines will spontaneously form tumors aftertransplantation into a cleared mammary fat pad.4 This may be attrib-utable to the fact that during immortalization, these cells have lostexpression of p16, p53, or other cell cycle regulators. Thus, thisexperiment was not designed to examine the oncogenic capacity ofC/EBPb-LIP but rather to test the reproducibility of LIP-overexpress-ing cells to proliferate in the mammary fat padin vivo as well as onplastic. Because clonal selection of the TM3 cells resulted in higherpassage lines, it was expected that the CMV-driven LIP expressionmight generate larger more proliferative tumors with a decreasedlatency. Accordingly, palpable tumors were detectable in the LIP butnot the Neo transplants 6 weeks after transplantation. Although notpalpable, the Neo cells also formed some histologically identifiablesmall tumors. Examination of the transplanted fat pads, via H&Estaining of paraffin-embedded sections, revealed that the LIP-express-ing transplants either grew out into large, undifferentiated tumors thatcompletely filled the fat pad (four of seven) or did not grow out at all(three of seven). In contrast, the vector control (Neo) transplants grewas small palpable tumors (three of seven) or undifferentiated cellmasses that did not fill the fat pad but with additional time couldgenerate palpable tumors (four of seven). Analysis of the transplantsdemonstrated that the LIP tumors were approximately four timeslarger, as determined by tumor volume (mm3) and wet weight (g, datanot shown) than the vector control (Neo) outgrowths and tumors (Fig.7C). The larger size is suggestive of a more proliferative tumor. Thiswas confirmed by the detection of 10-fold more mitotic figures in the

LIP than in the control tumors or outgrowths (Fig. 7,A andB). Thus,evidence from bothin vitro tissue culture andin vivo transplantationstudies demonstrate that overexpression of C/EBPb-LIP in mammaryepithelial cells results in increased proliferation.

DISCUSSION

These results have demonstrated that overexpression of C/EBPb-LIP in the mammary glands of transgenic mice as well as in mammaryepithelial cells cultured on plastic results in increased epithelial cellproliferation. These mammary hyperplasias may, therefore, be inher-ently more susceptible to additional oncogenic “hits” resulting in thestochastic formation of infrequent tumors, as was observed in 9% ofthe C/EBPb-LIP transgenic mice. Expression of C/EBPb-LIP hasbeen observed in many rodent mammary tumors and some humanbreast cancers and may increase the number of proliferative cells,potentially resulting in more highly proliferative and aggressive tu-mors. Consequently, overexpression of C/EBPb-LIP may be an im-portant indicator for breast epithelium at risk for hyperplasia andcancer.

The incidence of hyperplastic and neoplastic lesions in our WAP-LIP-WAP mice are in agreement with several other published reportsof genetically engineered mice bearing WAP-driven transgenes. Forexample, in WAP-stromelysin transgenic mice, 6–24 months of age,20% of mice contained atypical proliferative lesions and 7.4% devel-oped mammary carcinomas (24). Transgenes driven by the WAPpromoter are preferentially expressed in alveolar epithelial cells and,to a lesser extent, in ductal epithelial cells in the mammary gland (17,25, 26). The rat WAP promoter used in our transgenic study isminimally active during each estrous cycle in virgin as well as inmultiparous females but is maximally expressed starting at day 10 ofpregnancy and extending throughout lactation (17, 26, 27). Conse-quently, the induction of hyperplasias by WAP-LIP-WAP probablyoccurs during pregnancy and/or lactation but may be detectable onlyafter involution, following the regression of the surrounding normalalveolar epithelium. Attempts to detect an early, LIP-induced, prolif-erative response during pregnancy were unsuccessful because it wasdifficult to distinguish small increases in proliferation from the highlyproliferative background during pregnancy (data not shown). WAP-driven C/EBPb expression after involution was not detectable byWestern blots and could not be localized by immunohistochemistry asdiscussed previously. Thus, the hyperplasias observed in the involuted

4 D. Medina, personal communication.

Fig. 4. C/EBPb-LIP and LAP levels are elevated in exponentially growing TM3 cellsbut are decreased as the cells become contact inhibited during confluence. Western blotanalysis of whole-cell protein extracts from TM3 cells, which were either exponentiallygrowing (exp) or confluent and contact inhibited (conf), is shown. The parental TM3 cellsrepresent the early-passage cells from which the LIP and Neo clones were derived. LIPand Neo clones refer to clonal TM3 lines that stably express either C/EBPb-LIP orneomycin (Neo) as the control.

Fig. 5. Overexpression of LIP causes TM3 cell proliferation. TM3 clones stablyexpressing LIP (n5 5; L1, L3, LL5, L6, and L9) and vector-only controls (n5 5; N2,N3, N5, N6, and N10) were randomly chosen, plated at equal density, and tested forproliferative potential using an MTS cell proliferation kit (Promega). The various clonesdisplayed different growth rates, but the clones overexpressing LIP were, on average,twice as proliferative as the cells without exogenous LIP. The fold change in growth wasdetermined by dividing the number of proliferating viable cells (as measured by theamount of absorbance at 490 nm) at days 12, 9, 7, 5, and 3 by the value for proliferationat day 1 for each clone. Bars, SE.

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tissue are either no longer dependent on LIP expression or are main-tained by the expression of LIP in a small subset of cells, possibly asa result of limited transgene expression that may occur during eachestrous cycle. Likewise, in the cell culture studies, CMV-driven LIPexpression was higher during exponential growth (day 3) and earlyconfluence (day 7) than during days 10 and 15 of confluence, whenLIP-induced proliferation is evident. Although these TM3 cells havebeen isolated as subclones that stably express C/EBPb-LIP, it ispossible that the subset of cells that forms foci and proliferate duringlate confluence has higher levels of C/EBPb-LIP expression than theadjacent cells, which are less proliferative.

Targeted dominant-negative constructs are especially difficult tooverexpress in transgenic mice, because the transgene has the poten-

tial to negatively regulate its own promoter. In fact, only one othertransgenic study thus far has successfully overexpressed a dominant-negative, C/EBP-related protein (28). Consequently, WAP regulatorysequences were chosen to target transgene expression to the mammarygland in this study because these sequences did not contain anyknown, functional C/EBP consensus sites. However, subsequent anal-ysis of milk protein gene expression from the mammary glands ofC/EBPb knockout mice has demonstrated that loss of C/EBPb candramatically reduce the levels of both WAP mRNA and protein (7, 8).Similarly, when our WAP-LIP-WAP mice were crossed withC/EBPb-knockout mice, expression of theLIP transgene was reducedor was nondetectable (data not shown). Taken together, these datademonstrate that C/EBPb is indeed important in the regulation ofWAP, and that autoregulatory effects of LIP may account for themoderate levels of transgene expression and subtle phenotype ob-served in these mice. Additionally, variegated or sectored localizationof gene expression in transgenic mice can also account for variationsin the level of transgene expression (29). Several different transgenicmouse studies, including our studies with WAP-driven transgenes,have demonstrated that cells expressing the transgene often appear asscattered clusters, leading to a variegated pattern of gene expression

Fig. 6. Overexpression of C/EBPb-LIP in TM3 mammary epithelial cells results inincreased growth and foci formation.A, micrographs (34) of confluent TM3 monolayersgrown on plastic at days 10 and 15 of culture. Note the presence of foci in theLIP-expressing clonal line (upper panels) and the absence of foci in the control lines (Neo,neomycin-expressing only;lower panels). The LIP-expressing cells are also smaller andmore crowded in appearance than the control cells.B, LIP and Neo clonal lines (n5 2,each) were plated at equal density, and at days 3, 7, 10, and 15 of culture, the cells werepulse labeled for 15 min with 10mM BrdUrd, harvested, and analyzed by FACS analysisfor percentage of BrdUrd incorporation. Although foci formation was also observed in atleast four other LIP-expressing clones, the BrdUrd analysis was conducted with only twoof these clones, and consequently SEs could not be determined.C, Western blot analysisof cytoplasmic (Lanes C) and nuclear (Lanes N) extracts from LIP expressing TM3 clone(LIP1) and control clone (Neo 10) at 7 and 15 days of culture. TM6 cells serve as apositive control (1) for C/EBPb-LIP and LAP, and cross-reactive material (CRM) isindicated on the blot.

Fig. 7. Transplantation of TM3 cells stably overexpressing C/EBPb-LIP result in moreproliferative tumors in vivo. Approximately 1 3 106 TM3 cells stably expressingC/EBPb-LIP were transplanted into the right, cleared inguinal fat pad of BALB/c mice,and an equal number of non-LIP-expressing control cells (Neo/control) were transplantedinto the contralateral gland. Mammary glands were harvested 6 weeks after transplanta-tion, fixed overnight in 10% neutral buffered formalin, and processed via standardmethods for paraffin sectioning.A, H&E micrograph (340) of a C/EBPb-LIP-expressingtransplant that grew out as a poorly differentiated tumor. Numerous mitotic figures(boxed) are visible within this one high-powered field. The tumors derived from C/EBPb-LIP-expressing cells were approximately four times larger, as determined by tumorvolume or wet weight (C), and contained 10 times more mitotic figures/10 high-poweredfields than the outgrowths and smaller tumors derived from the control cells (B). The totalnumber of mitotic figures/10 high-powered fields (HPF) was determined by a pathologist(R. L.) as a blind comparative study.Bars,SE.

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(30, 31). This may also account, in part, for the focalversusdiffusepattern of hyperplasia observed in the WAP-LIP-WAP mice. Further-more, the timing of transgene expression may also be an importantfactor because WAP-driven transgenes are not expressed until pu-berty. If it were possible to selectively target C/EBPb-LIP expressionto the mammary gland either during early ductal development or invirgin transgenic mice, one might expect to observe a very different ormore severe phenotype.

It is generally accepted that breast cancer originates in the terminalduct lobular unit (32). Although the mouse mammary gland does notcontain a terminal duct lobular unit, an equivalent structure would bethe tertiary branches that give rise to the alveoli. HANs can form inthis region, as was observed in the WAP-LIP-WAP transgenic mice.HAN is a low-grade, focal alveolar hyperplasia that persists in theinvoluted mammary gland and has been experimentally proven viatransplantation experiments to be a precancerous, clonal lesion withhigh malignant potential (33–35). Squamous metaplasia, inflamma-tion, or lymphocytic infiltration, also frequently present in the invo-luted glands of WAP-LIP-WAP mice, has been proposed to be anormal repair response of the mammary gland to the hormonal chal-lenges and damage caused by multiple pregnancies (18).

Numerous reports in tissues other than the mammary gland supportthe observation that C/EBPb-LIP plays a proliferative role in cellcycle control. In adipocytes, C/EBPb and C/EBPd have been shownto induce C/EBPa expression, which arrests the ongoing proliferationand facilitates terminal cell differentiation (36, 37). Moreover, over-expression of C/EBPb-LIP results in continued proliferation and isable to inhibit the adipocyte conversion into the differentiated pheno-type (38). Similarly, a recent study has demonstrated that the intro-duction of C/EBPb-LIP via retroviral gene transfer into 3T3-L1 cellsresults in proliferation, foci formation, and a loss of contact inhibition(3). Although C/EBPa is primarily responsible for regulating terminaldifferentiation in hepatocytes (39), cellular proliferation in Hep G2hepatoma cells is not blocked by C/EBPa expression but is abrogatedby C/EBPb-LAP (40). In adult hepatocytes, differentiation and pro-liferation are mutually exclusive (40), and during rat postnatal devel-opment, the levels of LAP in liver nuclei are elevated much more thanthose of LIP (1). This is suggestive that the LAP:LIP ratio is importantfor differential regulation of gene expression and differentiation in theadult liver. In contrast, during hepatocyte proliferation after partialhepatectomy, C/EBPa levels decline, but both C/EBPd and C/EBPblevels increase. In fact, C/EBPa:C/EBPb heterodimers are replacedwith C/EBPb homodimers during the early G1 period after partialhepatectomy (41, 42).

C/EBP family members have been historically described as DNA-binding proteins; however, the C/EBPs are also capable of protein-protein interactions with cell cycle proteins such as Rb and p21. TheC/EBPb-LIP and LAP isoforms can directly interact with the SV40Tantigen domain of hypophosphorylated Rb (43). This transient butdirect interaction with Rb increases DNA binding and transactivationpotential of the C/EBPb isoforms, and depending on the ratio ofLIP:LAP, may inhibit the transactivation potential of LAP to tran-scribe genes involved in cellular differentiation (43). Additionally, anin vivo analysis in the liver of C/EBPa knockout mice showed thatC/EBPa and p21 interact via protein-protein interactions to stabilizep21 levels (44). At the transcriptional level, studies in rat hepatomacells have demonstrated that C/EBPa can bind to the canonical C/EBPDNA binding site in the p21 cyclin-dependent kinase inhibitor gene,resulting in the elevation of p21 expression, the inhibition of cyclin-dependent kinase-dependent Rb phosphorylation, and the induction ofcell cycle arrest at G1 (45–47). Similarly, in human colorectal cancercell lines, C/EBPb has been shown to increase p21 transcription, butit was not determined whether the C/EBPb isoforms have opposing

effects on p21 regulation (48). However, in primary cultures ofkeratinocytes, the deletion of theC/EBPb gene did not alter expres-sion of p21 (49). Consequently, the regulation of p21 by C/EBPb maybe a tissue-specific process. Further investigation of p21 regulation byLIP in mammary epithelial cells is clearly warranted. These observa-tions are important, because the canonical C/EBP DNA binding site inthep21gene promoter should be capable of binding all of the C/EBPs,including C/EBPb. The C/EBPs have identical binding specificities,and the hierarchy of DNA binding affinities for the C/EBP consensussequence is C/EBPb . C/EBPa . C/EBPd (50). If C/EBPb-LIP wereto dimerize with C/EBPa or form homodimers with itself, the tran-scriptional regulation of p21 might be inhibited, resulting in phospho-rylation of Rb and progression through the G1-S transition. Thisprovides a potential mechanism by which C/EBPb-LIP might induceentry into S-phase.

Alternatively, alterations in p21 or other cyclin-dependent kinaseinhibitors may result in changes in apoptosis. Although no decreasesin apoptosis were observed in the clonally selected TM3 cells, mod-ulation of the LIP:LAP ratio may result in increased apoptosis inmammary epithelial cells5 or a rescue from apoptosis by matrixdetachment in intestinal epithelial cells.6 Effects on apoptosis may betissue specific and dependent on the amount of LIP present in thecells. Failure to obtain TM3 clones that highly express LIP, may bethe result of induction of apoptoin in induction of apoptosis in theseclones during selection. Thus, LIP expression may potentially regulatecell proliferation and/or apoptosis, depending on the cell type andcell-substratum interactions.

In conclusion, these studies indicate that the overexpression ofC/EBPb-LIP in mammary epithelial cells promotes proliferation andthe development of hyperplasias. The data also support the hypothesisthat LIP overexpression may stimulate a growth cascade, which maybe susceptible to additional oncogenic hits and result in the stochasticformation of tumors. The elucidation of the molecular mechanisms bywhich C/EBPb-LIP regulates cell cycle progression may, therefore,be critical for defining protein targets associated with premalignancyand neoplastic progression.

ACKNOWLEDGMENTS

We thank Liz Hopkins for assistance with histology, Jeff Scott for help withFACS analysis, Jason Gay for assistance with surgical techniques, ShirleySmall for mouse husbandry, and Frances Kittrell for helpful discussions abouttechniques related to mammary epithelial cell lines and mice.

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2001;61:261-269. Cancer Res Cynthia A. Zahnow, Robert D. Cardiff, Rodolfo Laucirica, et al. Inhibitory Protein in Mammary Epithelial Cell Proliferation

-Liver-enrichedβA Role for CCAAT/Enhancer Binding Protein

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A Role for CCAAT/Enhancer Binding Protein b-Liver-enriched ......[CANCER RESEARCH 61, 261–269, January 1, 2001] A Role for CCAAT/Enhancer Binding Protein b-Liver-enriched Inhibitory