Inhibition of Retinoic Acid Receptor Function in Normal Human Mammary Epithelial Cells Results in Increased Cellular Proliferation and Inhibits the Formation of a Polarized Epitheliumin Vitro

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

236, 16–28 (1997)

EX973694

Inhibition of Retinoic Acid Receptor Function in Normal Human Mammary Epithelial Cells Results in Increased Cellular Proliferation and Inhibits the Formation of a Polarized Epithelium in Vitro Victoria L. Seewaldt,*,1 L. Elizabeth Caldwell,† Barton S. Johnson,‡ Karen Swisshelm,§ Steven J. Collins,Ø and Schickwann Tsai\ *Division of Medical Oncology and Division of Gynecologic Oncology, ‡Department of Restorative Dentistry, Division of Hospital Dentistry, and §Department of Pathology, University of Washington, Seattle, Washington 98195; †Program in Electron Microscopy, and \Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104; and ØUniversity of Washington and Division of Molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104

tion [1, 2]. Recently, retinoids have also been found to be important for the prevention of certain cancers. Retinoids can halt the progression of disease in premalignant lesions of the oral cavity, cervix, and skin, and they are effective in preventing the development of second primary tumors of the aerodigestive tract and lung [3–13]. There is also evidence that retinoids are important for the chemoprevention of breast cancer. It has been demonstrated that the risk of breast cancer is increased for women with a lower dietary intake of vitamin A and b-carotene but not for women with dietary deficiencies of vitamins C or E [14]. Phase II trials are under way to test the ability of fenretinide, a synthetic retinoid, to prevent contralateral breast cancer [15, 16]. The association between vitamin A deficiency and the development of cancer suggests that retinoid-dependent signaling pathways have a role in the suppression of carcinogenesis. Loss of expression of specific nuclear retinoid receptors may abrogate these pathways. Therefore, in order to use retinoids in a clinically rational manner it is important to define which receptors are critical to the anticancer activity of retinoids. The actions of retinoids are thought to be mediated through specific nuclear retinoic acid receptors (RAR)2 and retinoid X receptors (RXR) belonging to the steroid/ thyroid superfamily of transcription factors [17–23]. Multiple retinoic acid receptors have been identified; among these are RARa, b, and g. RARa is expressed ubiquitously in adult tissue and RARg is primarily expressed in skin. RARb is unique because it is primarily

The expression of retinoic acid receptor-b (RARb) mRNA is absent or down-regulated in a majority of breast cancers, suggesting that loss of retinoic acid receptor function may be a critical event in breast cancer carcinogenesis. We developed an in vitro system to investigate whether the loss of retinoic acid receptor (RAR) function might affect the proliferation and structural differentiation of normal cultured human mammary epithelial cells (HMECs). Utilizing a truncated retinoic acid receptor (RAR)-a construct exhibiting dominant-negative activity against retinoic acid receptor isoforms a, b, and g (DNRAR), we inhibited normal retinoic acid receptor function in HMECs. Suppression of RAR function in HMECs resulted in reduced growth inhibition mediated by all-trans-retinoic acid (ATRA). Moreover, the doubling time of HMECs expressing the DNRAR was significantly shortened, associated with a decrease in the percentage of cells in G1 and an increase in the percentage of cells in S-phase relative to controls. In addition, HMECs expressing the DNRAR cultured in prepared extracellular matrix exhibited a loss of extracellular matrix-induced growth arrest and formation of a polarized ductal epthelium. Our results suggest that ATRA and RARs may play an important role in regulating the proliferation of HMECs and in promoting differentiation. q 1997 Academic Press

INTRODUCTION

Vitamin A (retinol) and its derivatives (retinoids) are important for normal cellular growth and differentia-

2 Abbreviations used: RAR, retinoic acid receptor; RXR, retinoid X receptor; RA, retinoic acid; ATRA, all-trans-retinoic acid; HMEC, human mammary epithelial cells; RARb, retinoic acid receptor-b; RARE, retinoic acid response element; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; CAT, chloramphenicol acetyl transferase, DNRAR, dominant-negative retinoic acid receptor.

1 To whom correspondence and reprint requests should be addressed at Division of Medical Oncology, University of Washington, Box 358080, Seattle, WA 98195. Fax: (206) 667-6523. E-mail: vsee [email protected].

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0014-4827/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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expressed in epithelial cells and exhibits induced expression in response to retinoic acid, mediated by an enhancer element found in its promoter called the retinoic acid response element (RARE) [22, 23]. The retinoic acid receptor-b (RARb) is primarily expressed in normal epithelial tissues but is not expressed in many epithelial tumors or in tumor cell lines [24–30]. While normal human cultured mammary epithelial cells (HMECs) express mRNA for RARb, most breast cancer cell lines fail to express this gene [29–32]. This suggests that loss of retinoic acid receptor function may be an important event in mammary carcinogenesis. We previously developed an in vitro system to investigate how retinoic acid receptors may act to prevent malignant transformation of human mammary epithelial cells [31]. The human breast cancer cell line MDAMB-231 and our laboratory strain of MCF-7 do not normally express RARb and are resistant to all-transretinoic acid (ATRA)-mediated growth inhibition. By retrovirus-mediated gene transfer we expressed the human RARb gene in both of these breast cancer cell lines and demonstrated that MCF-7 and MDA-MB-231 cells expressing RARb readily undergo growth inhibition when treated with ATRA [31]. We next investigated how retinoids may act in HMECs in culture and observed that ATRA-treated HMECs undergo growth inhibition associated with G1 arrest, suggesting that ATRA mediates the proliferation of normal mammary epithelial cells by regulating the G1- to S-phase cell cycle transition [32]. These observations suggest that ATRA and retinoic acid receptors are important mediators of proliferation and provide a potential mechanism by which retinoids might act to prevent cancer in normal mammary epithelial cells. If the function of retinoids and RARs in normal human mammary epithelial cells is to suppress proliferation, then loss of retinoic acid receptor function might disrupt an important mechanism of maintaining normal tissue homeostasis and thereby contribute to malignant transformation. In this study we describe the use of a truncated retinoic acid receptor exhibiting dominant-negative activity (DNRAR) to inhibit retinoid receptor function in normal human mammary epithelial cells in order to investigate whether inhibition of retinoic acid receptor function results in loss of ATRA-mediated growth inhibition. Dominant-negative RARs have been recently used to unravel the physiologic function of retinoids in several cell types [33–38]. For this study we utilized a dominant-negative mutant of RARa (RARa403) driven by a retroviral LTR [34]. This RARa derivative is a negative transcriptional regulator that can simultaneously block all wild-type RAR isoforms [39]. This truncated mutant RAR retains the ability to dimerize with RXR and bind retinoic acid response elements, suggesting that the DNRAR acts by forming transcriptionally inactive heterodimers that compete for DNA binding with the natural RAR–RXR heterodimers [39]. We

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have previously inhibited retinoic acid receptor activity in hematopoietic cells by the introduction of the RARa403 DNRAR [33–35]. We now report the use of this DNRAR to inhibit retinoic acid receptor function in normal human mammary epithelial cells. Our results suggest that ATRA and retinoic acid receptors may play an important role in the expression of a normal polarized ductal epithelial structure since we observe that suppression of RAR function in results in increased proliferation and inhibits the ability of HMECs to form a polarized ductal epithelial structure. MATERIALS AND METHODS Materials. A 1 mM stock solution of all-trans-retinoic acid (Sigma, St. Louis, MO) was prepared in 100% ethanol and stored in opaque tubes at 0707C. Control cultures received equivalent volumes of the ethanol solvent. Retinoic acid stocks were used under reduced light. Cell culture and media. Normal HMEC strain AG11132 (M. Stampfer 172R/AA7) was purchased from the National Institute of Aging, Cell Culture Repository (Coriell Institute) [40]. HMEC strain AG11132 is cultivated from normal tissue obtained at reduction mammoplasty, has a limited life span in culture, and fails to divide after approximately 20 passages. AG11132 was at passage 8 at the time of receipt. Cells are grown in 4 ml/ml Mammary Epithelial Cell Basal Medium (Clonetics, San Diego, CA) supplemented with bovine pituitary extract (Clonetics CC4009), 5 mg/ml insulin (UBI, Lake Placid, NY), 10 ng/ml epidermal growth factor (UBI), 0.5 mg/ml hydrocortisone (Sigma), 1005 M isoproterenol (Sigma), 10 mM Hepes buffer (Sigma). G418 (Gibco, Grand Island, NY) containing medium was prepared by the addition of 300 mg/ml of G418 to the above standard medium. Cells were cultured at 377C in a humidified incubator with 5% CO2/95% air. We did not process our growth medium to remove endogenous retinoids. Mycoplasma testing was performed as previously reported by Russell [41]. Retroviral transduction. Construction of the LRARa403SN dominant-negative retroviral vector has been previously described [34]. AG11132 normal human mammary epithelial cells (passage 9) were plated in four T-75 tissue culture flasks (Corning) in standard medium and grown to 50% confluency. Transducing virions from either the PA317-LRARa403SN or the control PA317-LXSN (without insert) retroviral producer line were added at a multiplicity of infection at 1:1 in the presence of 4 mg/ml Polybrene (Sigma) to log-phase cells grown in T-75 flasks. The two remaining T-75 flasks were not infected with virus. After 48 h the two flasks containing transduced cells and one flask with untransduced cells were selected with standard medium containing 300 mg/ml G418. Cells were continued in G418 containing medium for 2 weeks until 100% of control untransduced cells were dead. One flask of unselected, untransduced parental control cells were passaged in parallel with the selected, transduced experimental and vector control cells. Untransduced, parental AG11132 cells are referred to in this article as AG11132-P to distinguish them from transduced HMECs. Transduced cells expressing the DNRAR construct are designated AG11132-DNRAR. Vector control clones are designated AG11132-LXSN. Northern blotting. RNA was extracted with guanidine isothiocyanate and subjected to Northern blotting in formaldehyde denaturing gels as previously described [30]. Ten micrograms of RNA was loaded per lane. Molecular probes utilized in the Northern analysis are as follows: The hRARa probe is a 1.3-kb, SmaI fragment [19]. The 36B4 probe (700-bp PstI fragment) (42) was used as a loading and transfer control probe. Transient expression assays. HMECs were plated in T-75 tissue culture flasks (Corning) in standard medium 24 h prior to transfec-

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tion and were approximately 50% confluent at the time of transfection. Cells were transfected by CellFECTIN (Gibco/BRL Life Technologies) as per the manufacturer’s recommendations for transient transfection of adherent cells. Transient transfections were performed utilizing the pRRE4-tkCAT reporter plasmid which contains four copies of the natural RA-response element (RARE) present in the promoter region of the human RARb gene [23]. Transfection control was provided by the pCMV-GH plasmid [43]. Transfection conditions were as follows: 5 ml CellFECTIN in 1.0 ml standard medium was added to 1.0 ml of standard medium containing 10 mg of pRRE4-tkCAT reporter plasmid [23] and 10 mg of pCMV-GH [43], incubated for 10 min at room temperature, and then added per T75 flask. Twenty-four hours after transfection, the cells were washed with PBS and re-fed with standard medium containing 0, 0.1, 1.0, or 10 mM RA. Control cultures received an equivalent volume of ethanol (0.01%). After 24 h, culture medium was collected for determination of growth hormone concentration using a radioimmunoassay kit (Nichols Institute, San Juan Capistrano, CA). Preparation of cell lysates and chloramphenicol acetyl transferase (CAT) assays were performed according to published methods [32]. Cell growth curves. AG11132-LXSN vector controls and AG11132DNRAR transduced cells were plated in duplicate at 1 1 104 cells per 12-well tissue culture plate (Corning) on Day 01 and allowed to adhere. On Day 0 the medium was replaced with standard medium containing 0, 0.1, 1.0, or 10 mM ATRA. Untreated controls received an equivalent volume of ethanol solvent (0.01%). Cells were trypsinized at 24-h time intervals and counted in triplicate. DNA staining of cell nuclei with propidium iodide and FACS analysis. On Day-1 5 1 105 AG11132-LXSN or AG11132-DNRAR cells were plated in T-75 flasks (Corning) and allowed to adhere. On Day 0 the medium was replaced with standard medium containing 0, 0.1, 1.0, or 10 mM ATRA. Untreated controls received an equivalent volume of ethanol solvent (0.01%). Cells were harvested at 2 days and did not exceed 70% confluency. Cells were trypsinized, and nuclei were isolated and stained with propidium iodide as previously reported [31]. Nuclei were then analyzed by FACScan [31]. Ten thousand events were collected in list mode fashion, stored, and analyzed on Multicycle AV software (Phoenix Flow Systems, San Diego, CA). HMEC culture in growth factor depleted medium. AG11132-P parental cells, AG11132-LXSN vector controls, and AG11132-DNRAR transduced cells were plated in duplicate at 1 1 104 cells per 12-well tissue culture plates (Corning) on Day 01 in standard medium and allowed to adhere. On Day 0 the medium was removed, the wells were washed three times with PBS, and then standard medium, standard medium without insulin, or standard medium without epidermal growth factor was added. Cells were trypsinized at 24-h time intervals and counted in triplicate. Final cell counts were obtained on Day 4. HMEC culture in prepared extracellular matrix. Mammary epithelial cells were grown in extracellular matrix by methods developed by Minna Bissell and others [44, 45]. One hundred microliters of prepared extracellular matrix (Matrigel, Collaborative Research, Bedford, MA) was added per well to a 48-well plate and allowed to gel at 377C for 20 min. AG11132-DNRAR cells and AG11132-LXSN vector controls were trypsinized, counted, and pelleted in a sterile microcentrifuge tube. Approximately 1 1 104 cells were resuspended in 100 ml prepared extracellular matrix on ice, gently overlaid on the initial undercoating of extracellular matrix, and allowed to gel at 377C for 20 min. Standard medium was then added and wells were inspected to ensure that there was an equal distribution of cells in each well. Cells were grown for 10–14 days in culture. Cell growth determination in extracellular matrix culture. Cell growth was determined by the following criteria. The size of growing spherical cell colonies was measured with an eyepiece equipped with a micrometer spindle. For both AG11132-LXSN vector controls and AG11132-DNRAR-transduced cells the 20 largest colonies were measured.

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Electron microscopy. AG11132-DNRAR cells and AG11132LXSN vector control cells were grown in prepared extracellular matrix as described above, fixed in half-strength Karnovsky’s fixative [46] for 6 h, rinsed in 0.1 M sodium cacodylate buffer, and postfixed in 1% collidine-buffered osmium tetroxide. Dehydration in graded ethanol and propylene oxide was followed by infiltration and embedding in Epon 812. Approximately 70- to 90-nm sections were stained using saturated aqueous uranyl acetate and lead tartrate. Photographs were taken using a Jeol 100 SX transmission electron microscope operating at 80 kV. Thick sections were obtained by cutting a 1-mm, Epon-embedded specimen and were stained with toluidine blue. The same Epon specimen block was utilized for both thick sections and electron micrographs.

RESULTS

Retroviral-Mediated Transduction of a DominantNegative Retinoic Acid Receptor into Normal Human Mammary Epithelial Cells In order to investigate how retinoids and RARs may act to regulate proliferation we utilized a dominantnegative retinoic acid receptor to inhibit retinoic acid receptor function. The LRARa403SN retroviral vector containing a DNRAR has been previously characterized [33–35]. This dominant-negative construct encodes a carboxy-terminal-truncated RARa peptide of 403 amino acids containing the amino terminus, the DNA-binding domain, and part of the hormone-binding domain of RARa. As previously described [34, 39] LRARa403SN suppresses RAR-mediated transactivation of reporter constructs harboring the natural RARE of RARb [23]. Actively dividing HMECs were infected with the retroviral vector LRARa403SN at a ratio of 1:1. Northern blots were performed on LRARa403SN-infected cells and controls to determine the levels of the DNRAR (LRARa403SN) mRNA expression. The LTRinitiated 4.8- and 3.0-kb DNRAR mRNA transcripts [33, 34] were observed in the retroviral vector-transduced cells but were not present in the parental cells or vector controls (Fig. 1). The endogenous 3.6- and 2.8kb RARa mRNA transcripts were also detected by this probe in all transduced and untransduced HMECs. Inhibition of ATRA-Mediated Trans-Activation by CAT Reporter Assays To determine whether the truncated retinoic acid receptor construct (RARa403) exhibited dominant-negative activity in HMEC strain AG11132, we performed transient expression assays utilizing the pRRE-CAT reporter plasmid which contains the natural RARE [23]. Chloramphenicol acetyl transferase reporter activity in both AG11132-P and AG11132-LXSN controls increased 2.3- and 2.4-fold, respectively, after 24 h of exposure to 0.1 mM ATRA (Fig. 2). In contrast, AG11132-DNRAR cells were resistant to ATRA-mediated trans-activation of the RARE at concentrations of 0.1 to 1.0 mM RA and demonstrated only a 1.3-fold induction at concentrations of 10 mM ATRA (Fig. 2).

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FIG. 1. Expression of a dominant-negative RAR construct LRARa403SN in HMEC strain AG11132. Northern analysis was performed on RNA extracted from AG11132-P parental cells (P), from AG11132-DNRAR cells infected with the dominant-negative construct LRARa403SN retroviral vector (DNRAR), and from AG11132LXSN vector control cells (LXSN) to determine the levels of the LRARa403SN mRNA expression. Cells were treated without (0) and with (/) 1.0 mM ATRA for 48 h. The blot was probed with a 1.3-kb (SmaI) RARa cDNA fragment [19]. The LTR-initiated 4.8- and 3.0-kb DNRAR mRNA transcripts were observed in the AG11132-DNRAR retroviral vector-transduced cells (passage 11) but were not present in the AG11132-P parental cells (passage 11) nor in AG11132-LXSN vector controls (passage 11). The endogenous 3.6- and 2.8-kb RARa mRNA transcripts were also detected by the RARa probe in all transduced and untransduced HMECs. Ten micrograms of total RNA was loaded in each lane. 36B4 is a 1.5-kb mRNA expressed uniformly in all breast cell lines and therefore serves as a loading control [42].

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AG11132-LXSN vector controls (passage 12) and AG11132-P parental cells (passage 12) exhibited a 24h doubling time (data not shown). These observations demonstrate that suppression of retinoic acid receptor function by a dominant-negative approach results in shortened doubling time in HMEC strain AG11132. Increased growth inhibition of AG11132-LXSN controls was observed with increasing concentrations of ATRA and increasing time of exposure (Fig. 3A). AG11132-LXSN controls decline in number after 2 to 3 days of treatment with 1.0 mM ATRA, suggesting that this concentration of ATRA is cytotoxic. Previous studies suggest that HMECs treated with ATRA do not undergo apoptosis [32]. AG11132-DNRAR cells were resistant to the growth inhibitory effects of ATRA relative to controls (Fig. 3B). Growth of AG11132-DNRAR cells was inhibited at 10 mM ATRA, suggesting either that the block could be overcome at higher concentrations of ATRA or that cells experienced a direct cytotoxic effect. This shifting of the dose–response curve to ATRA is evidence that the DNRAR has dominantnegative activity in HMEC strain AG11132. These data also demonstrate that ATRA inhibits the proliferation of HMECs in culture and that inhibition of retinoic acid receptor function blocks this growth inhibition. In order to further investigate the effects that reti-

This observed inhibition of ATRA-mediated trans-activation in the DNRAR-expressing HMECs relative to controls demonstrates the dominant-negative activity of our construct in HMECs and is consistent with previous observations utilizing this construct [33]. At 10 mM ATRA there was only partial suppression of ATRAmediated trans-activation, demonstrating that the dominant-negative activity of this construct could be overcome by increasing concentrations of ATRA. These data demonstrate the ability of this dominant-negative retinoic acid receptor to inhibit normal retinoic acid receptor function in HMEC strain AG11132. HMECs Expressing the Dominant-Negative Retinoic Acid Receptor Demonstrate Increased Proliferation and Resistance to ATRA-Mediated Growth Inhibition The AG11132-DNRAR-transduced cells, AG11132LXSN vector controls, and AG11132-P parental cells were grown in standard medium. Untreated AG11132DNRAR cells (passages 10 to 15) exhibited a significantly decreased doubling time relative to untreated AG11132-LXSN vector controls and AG11132-P parental cells at the same passage number. In cultures passaged in parallel we observed that AG11132-DNRAR cells (passage 12) had a 12- to 18-h doubling time while

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FIG. 2. The dominant-negative RAR construct LRARa403SN inhibits RA-mediated trans-activation of bRARE in HMECs. HMEC AG11132-P parental cells (passages 12–14), AG11132-LXSN vector controls (passages 12–14) and AG11132-DNRAR cells expressing the dominant-negative RAR (passages 12–14) were transfected with pRRE4-tkCAT plasmid and treated with 0, 0.1, 1.0, and 10 mM ATRA for 24 h, and cell lysates were assayed for CAT reporter activity. pCMV-GH was used as a transfection efficiency control. CAT counts were corrected for growth hormone activity and total protein (see Materials and Methods). Data represent an average of three independent transfections performed in duplicate.

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vector controls (passage 13–14) treated with 0, 0.1, and 1.0 mM ATRA for 2 days (Table 1). Flow cytometric analysis of isolated nuclei stained with propidium iodide demonstrated that untreated AG11132-DNRAR cells had a significant decrease in the percentage of cells in G1 and a significant increase in the percentage of cells in S-phase relative to untreated AG11132LXSN vector control cells. This is consistent with the observation that AG11132-DNRAR cells have a increased rate of proliferation relative to AG11132-LXSN controls. We also observed that cells whose normal retinoic acid receptor function was inhibited by the DNRAR were relatively resistant to the ATRA-mediated G1 block observed in vector control cells: AG11132LXSN treated with 0, 0.1, and 1.0 mM all-trans-retinoic acid had 20, 11, and 8% of cells in S-phase, respectively (Table 1). In contrast, 30, 28, and 17% of AG11132DNRAR cells treated with, respectively, 0, 0.1, and 1.0 mM ATRA were in S-phase (Table 1). These results suggest that RARs appear to be important mediators of ATRA-mediated growth inhibition and that suppression of retinoic acid receptor function in HMEC strain AG11132 results in an increased percentage of cells in S-phase relative to controls. Inhibition of Retinoic Acid Receptor Function Does Not Immortalize or Increase the Life Span of HMECs in Culture HMECs have a finite life span in culture. We investigated whether loss of RAR function in HMEC strain AG11132 resulted in immortalization or an increased life span in vitro. AG11132-P parental cells, AG11132DNRAR-transduced cells, and AG11132-LXSN vector control cells were serially passaged in culture. Cells

TABLE 1 Effects of Inhibition of Retinoic Acid Receptor Function on HMEC Cell Cycle Cells

FIG. 3. Inhibiting retinoic acid receptor function by a dominantnegative RAR blocks RA-mediated growth inhibition of HMECs. Growth curves of AG11132-LXSN vector controls (passage 12) (A) and AG11132-DNRAR cells harboring a DNRAR construct (passage 12) (B). Cells were plated on Day 01 in standard medium, in duplicate, at 1 1 104 cells per well in 12-well plates. Cells were re-fed on Day 0 with standard medium containing 0, 0.1, 1.0, or 10 mM ATRA. Cells were trypsinized at 24-h time intervals and each well was counted in triplicate.

noic acid receptors have on inhibiting the proliferation of HMECs, FACS analysis was performed on AG11132DNRAR cells (passage 13–14) and AG11132-LXSN

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AG11132-LXSN No ATRA 0.1 mM ATRA 1.0 mM ATRA AG11132-DNRAR No ATRA 0.1 mM ATRA 1.0 mM ATRA

% G0/G1

%S

% Untreated parental

60 67 70

20 11 8

100 55 40

49 49 67

30 28 17

150 140 85

Note. Adherent AG11132-LXSN vector controls or AG11132DNRAR-transduced cells were treated for 2 days with 0, 0.1, or 1.0 mM ATRA. The distribution of cells in the various phases of the cell cycle was determined by flow cytometry. These data are representative of three separate experiments. These observations demonstrate that suppression of RAR function in normal human mammary epithelial cells by a DNRAR results in an increase in the percentage of cells in S-phase relative to controls.

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harboring the dominant-negative retinoic acid receptor, as vector controls, and parental cells all underwent senescence at approximately passage 21. Thus inhibition of normal retinoic acid receptor function in HMEC strain AG11132 did not result in an increased life span or immortalization in culture. Inhibition of Retinoic Acid Receptor Function in HMECs Does Not Result in Decreased Growth Factor Requirements in Culture HMECs in culture require specific growth factors such as insulin and epidermal growth factor. In order to determine whether inhibition of retinoic acid receptor function by a dominant-negative retinoic acid receptor construct altered the requirement of HMECs for specific growth factors, we cultured AG11132-DNRAR, AG11132-LXSN vector controls, and AG11132-P parental cells in medium without added insulin or EGF. Although AG11132-DNRAR cells harboring the dominant-negative retinoic acid receptor grew at approximately twice the rate as did AG11132-LXSN controls and AG11132-P parental cells in growth factor-depleted medium, the overall percentage growth inhibition relative to cells grown in undepleted medium was similar for all three. Removal of insulin from the culture medium resulted in 44, 44, and 47% growth inhibition of AG11132-DNRAR, AG11132-LXSN, and AG11132-P parental cells, respectively, at 3 days. Similarly, EGF deprivation resulted in 35, 30, and 44% growth inhibition of AG11132-DNRAR, AG11132LXSN, and AG11132 parental cells, respectively, at 3 days. Therefore, inhibition of normal retinoid receptor function in HMECs does not result in loss of insulin or epidermal growth factor dependence. Distinctive Growth Regulation of HMECs Expressing a Dominant-Negative Retinoid Receptor vs Parental HMECs in Prepared Extracellular Matrix Interaction between basement membrane and mammary epithelial cells appears to play an important role in regulating normal mammary epithelial cell proliferation [45, 47–51]. These normal interactions may be disrupted during mammary cell tumorigenesis. It has been observed that while normal mammary epithelial cells undergo growth arrest in culture in the presence of prepared extracellular matrix, breast cancer cells and established breast cancer cell lines fail to exhibit similar growth arrest [47]. Thus it is hypothesized that interaction with basement membrane may serve to distinguish the growth patterns of normal and malignant mammary epithelial cells. AG11132-LXSN vector controls (passage 11) and AG11132-DNRAR-transduced cells (passage 11) were grown in prepared extracellular matrix as a single-cell suspension. The AG11132-LXSN vector controls grew exponentially until Day 6 and then

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FIG. 4. Inhibiting RAR receptor activity by a DNRAR enhances the proliferation of human mammary epithelial cells cultured in prepared extracellular matrix. The mean diameter of spheres formed by AG11132-LXSN vector controls (passage 10) and AG11132DNRAR cells (passage 10) whose retinoid receptor function is inhibited by a DNRAR is plotted as a function of days in culture. Cells were plated in prepared basement membrane on Day 0 and the diameter of growing spherical cell colonies was measured with an eyepiece equipped with micrometer spindle. For both AG11132-LXSN vector controls and AG11132-DNRAR transduced cells, the 20 largest colonies were measured at each time point.

growth arrested on Day 7 to 8 (Fig. 4), forming a uniform population of spherical colonies. Mean diameter of these colonies at Day 9 was 23.5 mm ({10 mm) (Fig. 4). In contrast, AG11132-DNRAR-transduced cells continued to proliferate within basement membrane matrix culture when the vector control cells had growth arrested (Fig. 4). Moreover, in contrast with control cells, AG11132-DNRAR-transduced cells formed large, dense, irregular colonies. The mean diameter of these colonies was 62 mm ({10 mm) at Day 9, which was significantly higher than that of vector control cells (Fig. 4). These data suggest that inhibition of RAR function in HMECs may result in loss of growth inhibition by extracellular matrix in this in vitro system. Loss of Retinoic Acid Receptor Function in HMECs Inhibits the Formation of a Polarized Ductal Epithelium in Vitro We observed that AG11132-DNRAR-transduced cells exhibited enhanced cell growth in the presence of prepared extracellular matrix. We next asked whether loss of retinoid receptor function could inhibit the formation of a normal epithelial ductal structure in vitro. Light micrographs of toluidine blue-stained, Epon-embedded, thick sections of AG11132-LXSN vector control

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FIG. 5. Morphologic appearance of normal human mammary epithelial cells whose retinoid receptor is suppressed by a dominantnegative retinoid receptor grown in prepared extracellular matrix. (A, B) AG11132-LXSN vector control cells grown in prepared extracellular matrix for 14 days. Cells form acinus-like spherical colonies which underwent growth arrest after 7 to 8 days in culture. Vector control cells demonstrate regular, spherical colonies surrounding a central lumen, with sharply delineated boundaries consistent with differentiated mammary glandular epithelium. (C, D) AG11132-DNRAR-transduced cells whose retinoid receptor is inhibited by a DNRAR grown in prepared extracellular matrix for 10 days. Cells form large, dense, irregularly shaped multicellular colonies with no central lumen and do not undergo growth arrest after 14 days in culture. The magnification of A and C is with a 10X objective and the magnification of B and D is with a 40X objective. Epon embedded 1-mm sections are stained with toluidine blue.

cells cultured for 10 days in prepared extracellular matrix demonstrate regular, spherical colonies surrounding a central lumen, with sharply delineated boundaries (Figs. 5A and 5B). Electron micrographs demonstrate that AG11132-LXSN control cells grow in organized, acinus-like structures consisting of a single layer of epithelial cells, connected by numerous desmosomes, organized around a central lumen. A fundamental property of normal epithelial cells is their ability to

organize into polarized structures manifested by the characteristic location of intracellular organelles. When AG11132-LXSN control cells are cultured in prepared extracellular matrix and examined by electron microscopy, the resulting acinus-like structure exhibits cellular polarity typical of polarized normal mammary epithelium: (i) there are microvilli primarily distributed on the luminal and lateral surfaces but not on the basal surface, (ii) secretory vacuoles are present on the

FIG. 6. Phenotypic changes observed by electron microscopy in normal human mammary epithelial cells expressing a dominant-negative retinoid receptor suggest that RARs play an important role in the formation of a polarized ductal epithelium. (A, B) Electron micrographs of AG11132-LXSN vector control cells grown in prepared extracellular matrix for 10 days (magnification of A and B is 15001 and 25001, respectively). AG11132-LXSN control cells form acini-like structures which demonstrate a central lumen (L) surrounded by correctly polarized luminal cells consistent with differentiated mammary glandular epithelium: (i) microvilli (Mv) are primarily distributed on the luminal and lateral surfaces, (ii) secretory vacuoles (V) are present on the luminal surface and not on the basal surface, and (iii) mitochondria (M) are primarily located at the basal surface. (C, D) AG11132-DNRAR-transduced cells whose RAR function is inhibited by a DNRAR grown in prepared extracellular matrix (magnification of C and D is 10001 and 25001, respectively). Cells grow in large, disorganized, multilayered, irregularly shaped colonies, with no lumen formation and a loss of normal cellular polarity. Evidence of lack of polarity includes the following: (i) cells are not organized into spherical, single-layer structures, (ii) a lumen is not present, (iii) mitochondria (M) are present throughout the cell, (iv) secretory vacuoles (V) are randomly distributed throughout the cytoplasm. These morphologic changes observed in AG11132-DNRAR cells suggest that loss of retinoid receptor function in HMECs inhibits the formation of a polarized ductal epithelium in vitro.

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luminal surface and not on the basal surface, and (iii) mitochondria are primarily located at the basal surface (Figs. 6A and 6B). In contrast, light micrographs of AG11132-DNRAR-transduced cells grown in extracellular matrix for 10 days exhibit large, dense, irregularly shaped multilayered clusters of cells, with no central lumen (Figs. 5C and 5D). Unlike normal control cells, the surface of these cell clusters projects irregularly into the surrounding extracellular matrix. Electron micrographs demonstrate disorganized clusters of cells with an absence of normal epithelial polarity (Figs. 6A and 6B). Evidence of lack of polarity includes the following: (i) cells are not organized into spherical, single-layered structures; (ii) lumens are not present; (iii) microvilli are present on all cell surfaces; and (iv) secretory vaculoles and mitochondria are randomly distributed throughout the cytoplasm. These data suggest that loss of retinoic acid receptor function in normal mammary epithelial cells inhibits the formation of a polarized ductal epithelium in vitro. DISCUSSION

We utilized a dominant-negative approach to study the effects of inhibiting retinoic acid receptor function in human mammary epithelial cells. A dominant-negative retinoic acid receptor has the advantage of being able to simultaneously interfere with all retinoic acid receptor isoforms with a single construct in a specific human cell type. Previous studies utilizing trangenic mouse lines resulting from targeted disruption of specific retinoic acid receptors by homologous recombination have been complicated because the loss of a specific isoform has in some cases resulted in embryonic lethality (RXRa) [52], or in other cases, no phenotypic alteration (RARb2) [53]. In the latter case it is postulated that other members of the RAR family or other RAR isoforms can compensate for loss of a specific retinoic acid receptor isoform and therefore no phenotypic alteration is observed. This suggests that multiple receptors must be knocked out to completely disable the retinoic acid receptor pathway. Dominant-negative RARs have been recently used to unravel the physiologic function of retinoids in several cell types [33–38]. Studies utilizing the targeted expression of a DNRAR in transgenic mice suggest that retinoic acid promotes normal epidermal differentiation [37, 38]. We also previously utilized a dominant-negative approach to provide evidence that RARs are critical for hematopoietic development [33– 35]. We now report that inhibition of RAR function by a dominant-negative approach in HMECs results in dysregulated growth and inhibits the formation of a polarized ductal epithelium, suggesting that ATRA and RARs also play an important role in regulating proliferation and differentiation in mammary cells. Retinoids are important for the normal growth and development of epithelial cells [1, 2]. Selective lack of

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expression of specific RARs has been observed in ductal carcinoma in situ and in a majority of breast cancers, suggesting that loss of RAR function may be an important early event in breast cancer development [54]. The molecular mechanism by which the loss of RAR function might promote mammary carcinogenesis, however, is poorly understood. We previously observed that ATRA mediates G1/S arrest in HMECs [32]. We show here that inhibition of RAR function may enhance cell proliferation and inhibits the ability of mammary epithelial cells to form a polarized epithelial structure in vitro. First, HMECs whose RAR function is inhibited by a DNRAR construct exhibit enhanced proliferation (Fig. 3) associated with an increase in the percentage of cells in S-phase and a decrease in the percentage of cells in G1 (Table 1). Second, HMECs whose RAR function is suppressed by a DNRAR are resistant to the growth suppressive effects of extracellular matrix (Fig. 4). Third, suppression of RAR function in HMECs inhibits the formation of a polarized ductal epithelium in our in vitro system (Figs. 5 and 6). These observations provide a potential model for elucidating how loss of retinoid receptor function may promote breast cancer carcinogenesis. We previously reported that ATRA mediates G1/S arrest in HMECs [32]. Moreover, we have also observed that breast cancer cell lines which have lost RARb function and are resistant to ATRA-mediated growth inhibition undergo ATRA-mediated G1/S arrest when transduced with a functional RARb receptor [31]. These observations suggest that ATRA and RARs mediate proliferation by regulating the G1/S cell cycle transition. We now show that if RAR function is inhibited by a dominant-negative retinoid receptor, HMECs increase their rate of proliferation (Fig. 3), associated with a decrease in the percentage of cells in G1 (Fig. 4). This provides further evidence that retinoids and RARs are critical regulators of the G1 to S-phase cell cycle transition and that loss of RAR receptor function may result in altered cell cycle control. Epithelial cell differentiation is typically characterized by decreased cellular proliferation, increased cellular adhesion, and the expression of cell-specific proteins. Retinoic acid receptors are transcription factors, which, when activated by ligand binding, can modulate the expression of a wide variety of genes. Many of these target genes are likely to be important for promoting differentiation. Retinoids have been shown to regulate the expression of genes important for the expression of a differentiated phenotype including extracellular matrix glycoproteins, proteases such as collagenase and stromelysin, intermediate filaments such as keratin, and gap junction proteins [for a review, see ref. 2]. These gene products play an important role in normal cellular architecture, cellular adhesion, and cell–cell communication. The relative importance of these RAregulated proteins in mediating differentiation of

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HMECs is unknown. It is likely, however, that changes in the expression of a large number of these RA-regulated proteins would alter the ability of cells to regulate normal cellular homeostasis. We have suppressed RAR function in HMECs and observe that these cells exhibit dysregulated growth (Table 1, Figs. 3 and 4) and inhibition of the formation of a polarized ductal epithelium (Figs. 5 and 6). We speculate that this may be in part the result of altered expression of RA-regulated proteins critical for the promotion of growth and differentiation. Further studies will be necessary to investigate the mechanism by which the DNRAR acts to regulate proliferation in HMECs. The retinoid signaling pathway is very complex and the DNRAR utilized in this study may be acting via pathways which are not anticipated by current models. We hypothesize that the enhanced proliferation (Table 1, Fig. 3) observed when AG11132DNRAR cells are cultured in the absence of added ATRA is the result of inhibition of the growth regulatory signal exerted by the low levels of endogenous retinoids present in our standard medium. The mechanism, however, by which the DNRAR exhibits dominant-negative activity is not fully understood and it is possible that the increased rate of proliferation that we observe is a direct effect of the DNRAR. It is unclear whether estrogen receptor expression is important for ATRA-mediated growth regulation of HMECs in vitro. In general, estrogen receptor-positive breast cancer cell lines are sensitive to the growth inhibitory effects of retinoids, whereas estrogen receptornegative breast cancer cell lines are resistant [54]. HMEC strain AG11132 is a heterogeneous population of normal epithelial cells, approximately 10% of which stain positively, by immunocytochemistry, for the estrogen receptor (data not shown). The remainder of this population exhibits low levels of estrogen receptor expression. Further experiments will be required to define the role of estrogen receptor expression in HMECs in ATRA-mediated growth inhibition and the formation of a polarized epithelium in vitro. Breast cancer development involves multiple alterations in the normal mammary epithelial cell phenotype including dysregulated growth, loss of cellular polarity, and invasion through a basement membrane. This transition from a normal mammary architecture to an invasive cancer is felt to reflect a multistep deregulation of many cellular pathways involving growth regulation and cell–cell signaling. The relative contributions of oncogenes, chemical mutagens, and genetic predisposition in the development of breast cancer are unclear. We show that inhibition of RAR function in HMECs in this in vitro model results in loss of normal cellular polarity and of growth regulation by extracellular matrix. We do not observe growth factor independence or immortalization in HMECs expressing the DNRAR. This suggests that inhibition of RAR function

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in HMECs results in loss of growth regulation but does not result in immortalization. Evidence suggests that loss of RAR function may be an early event in solid tumor carcinogenesis. Dysplastic lesions of the lung and oral cavity demonstrate loss of retinoid receptor expression, demonstrating that loss of RAR function can occur before the expression of a fully malignant phenotype [55, 56]. Our observations suggest how loss of RAR expression might contribute to the development of breast cancer. If the normal function of ATRA and RARs involves inducing growth arrest and promoting the formation of a normal polarized ductal epithelium by mammary epithelial cells, then loss of this function might disrupt this normal cycle of breast epithelial cell proliferation and differentiation, leading to an expanded abnormal epithelial cell population. Further mutations of cellular oncogenes or tumor suppressor genes in this expanded population of cells might lead to the development of overt breast cancer. This model predicts the loss of RAR function could play an early role in the pathogenesis of breast cancer. The authors acknowledge the excellent technical assistance of LeMoyne Mueller. This work was supported by a Pilot and Feasibility Award from the Clinical Research Unit, NIH NIDDK, 2P30 DK 35816-11 (V.L.S.). Victoria L. Seewaldt was supported by NIH KO8 Clinical Investigator Award CA 68210-01, a New Investigator Award from the Clinical Nutrition Research Unit NIH NIDDK, 2P30DK 35816-11, and a Susan G. Komen Breast Cancer Fellowship. Schickwann Tsai was supported by NIH DK-48622.

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Received March 24, 1997 Revised version received June 26, 1997

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