17β-Estradiol-mediated growth inhibition of MDA-MB-468 cells stably transfected with the estrogen receptor: Cell cycle effects

Molecular and Cellular Endocrinology 133 (1997) 49 – 62

17b-Estradiol-mediated growth inhibition of MDA-MB-468 cells stably transfected with the estrogen receptor: Cell cycle effects Weili Wang a, Roger Smith III b, Robert Burghardt c, Stephen H. Safe a,* a

Departments of Veterinary Physiology and Pharmacology and Biochemistry and Biophysics, Texas A&M Uni6ersity, College Station, TX 77843 -4466, USA b Department of Veterinary Pathobiology, Texas A&M Uni6ersity, College Station, TX 77843 -4466, USA c Department of Veterinary Anatomy and Public Health, Texas A&M Uni6ersity, College Station, TX 77843 -4466, USA Received 21 May 1997; accepted 23 July 1997

Abstract Estrogen receptor (ER)-negative MDA-MB-468 human breast cancer cells were stably transfected with wild-type human ER and utilized as a model for investigating estrogen- and aryl hydrocarbon (Ah)-responsiveness. Treatment of the stably transfected cells with 10 nM 17b-estradiol (E2) resulted in a significant inhibition ( \ 60%) of cell proliferation and DNA synthesis, which was blocked by 10 − 7 M ICI 182 780. Analysis by flow cytometry indicated that treatment with E2 increased the percentage of cells in G0/G1 (from 68.8 to 89.4) and decreased cells in S (from 18.4 to 3.4) and G2/M (from 12.8 to 7.2) phases of the cell cycle. The effects of E2 on the major cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, retinoblastoma protein (RB), E2F-1, and cyclin-dependent kinase activities were also investigated in the stably transfected MDA-MB-468 cells. The results demonstrated that the growth inhibitory effects of 10 − 8 M E2 in ER stably transfected MDA-MB-468 cells were associated with modulation of several factors required for cell cycle progression and DNA synthesis, including significant induction of the cyclin-dependent kinase inhibitor p21cip-1 (\4-fold increase after 12 h) and decreased E2F1 and PCNA protein levels. These results show that the growth-inhibitory effects of E2 in the stably transfected cells were due to multiple factors which result in growth arrest in G0/G1 and inhibition of DNA synthesis. © 1997 Elsevier Science Ireland Ltd. Keywords: Estradiol; Growth inhibition; MDA-MB-468 cells

1. Introduction Breast cancer is one of the leading causes of premature death in women in North America and approximately one in nine women will develop this disease during their lifetime (Kelsey and Berkowitz, 1988). Increased lifetime exposure to estrogens is an established risk factor for development of breast cancer and a high percentage of early stage mammary tumors are estrogen receptor (ER)-positive and many of these * Corresponding author. Tel.: +1 409 8455988; fax: + 1 409 8624929; e-mail: [email protected]

patients respond to antiestrogen or endocrine therapy (Muss, 1992; Santen et al., 1990; Lerner and Jordan, 1990; Jordan, 1993). Later stage breast cancers are more aggressive and refractory to most forms of antiestrogen therapy and this correlates with the ER-negative phenotype of these tumors (Clarke et al., 1994; Morrow and Jordan, 1993; Ethier, 1995; Dickson and Lippman, 1995; King, 1992). Factors which regulate hormone-dependent mammary tumor growth, development of hormone resistance and loss of estrogen-responsiveness have been extensively investigated in both ER-positive and ER-negative breast cancer cell lines. For example, estrogen-responsive MCF-7 cells have

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been utilized to determine the role of hormone- and growth factor-induced gene expression on proliferation in cell culture and in athymic nude mice bearing MCF7 cell xenografts. These cells have also been used as models to understand the mechanism of cancer cell/tumor growth inhibition by antiestrogens (Brunner et al., 1987). Long term growth of MCF-7 cells under selective growth conditions including high concentrations of antiestrogens has provided several clonal populations which exhibit altered phenotypic and genotypic characteristics including partial or complete loss of hormoneresponsiveness (Brunner et al., 1993a,b; Katzenellenbogen et al., 1987; Wolf and Jordan, 1994; Lykkesfeldt et al., 1994; Pink et al., 1995; Herman and Katzenellenbogen, 1994). The importance of altered estrogen-responsiveness and ER expression has also been studied in drug resistant ER-negative breast cancer cells, normal mammary epithelial cells and ER-independent cell lines stably transfected with wild-type ER (Levenson and Jordan, 1994; Jiang and Jordan, 1992; Catherino et al., 1995; Garcia et al., 1992; Zajchowski et al., 1993; Zajchowski and Sager, 1991; Lundholt et al., 1996; Watts et al., 1989; Gaben and Mester, 1991; Watts and King, 1994; Webb et al., 1992; Maminta et al., 1991; Kushner et al., 1990; Touitou et al., 1990). The effects of 17b-estradiol (E2) and antiestrogens on ER stably transfected cell lines varies markedly with the cell-type and specific clone. For example, Zajchowski and Sager (1991) showed that E2 induced TGF-a and cathepsin D in ER negative cells and in non-tumorigenic immortal ER-negative epithelial cells stably transfected with wild-type ER; in contrast, pS2 was induced only in stably transfected tumor cell lines. The variability of E2-induced gene expression in these ER stably transfected cell lines contrasts with their uniform response to E2 on cell proliferation in which the hormone paradoxically inhibits cell growth. Thus activation of some estrogen-regulated genes in ER-stably transfected human breast cancer cell lines does not correlate with hormone-induced growth inhibition and the mechanism of the latter response is unknown. This study reports the isolation of MDA-MB-468 human breast cancer cells stably transfected with wildtype ER. Proliferation of these cells is also inhibited by E2 and this was comparable to effects observed in other cell lines stably transfected with the ER. Therefore these stably transfected cells were utilized as models for investigating the role of cell cycle and DNA synthesis enzymes as mediators of E2-induced growth inhibition. The results indicated that treatment with E2 resulted in an increase in the cyclin-dependent kinase inhibitor p21 (p21cip-1), a decrease in cdk4-dependent kinase activity and lower levels of E2F1 and proliferating cell nuclear antigen (PCNA) proteins.

2. Experimental procedures

2.1. Chemicals, oligonucleotides, plasmids and antibodies E2, acetyl CoA and histone (type III-SS) were purchased from Sigma (St. Louis, MO). ICI 182 780 was a kind gift from Dr Alan Wakeling, ICI Pharmaceuticals (Alderley Park, UK). Geneticin was purchased from GIBCO BRL, Life Technologies (Grand Island, NY). p21 Antibody was purchased from Oncogene Science, (Uniondale, NY). Cyclin D1, cyclin A, cyclin E, cdk2, cdk4, retinoblastoma (RB), E2F1 and PCNA antibodies, protein A/G-agarose beads and GST-RB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). [14C]Chloramphenicol (58.4 mCi/mmol) and [g 32P]ATP (3000 Ci/mmol) were obtained from New England Nuclear (Boston, MA). pBLCAT2 contains the regulatory region of the human thymidine kinase gene fused to the bacterial CAT reporter gene and was obtained from the American Type Culture Collection (ATCC). TATA-CAT plasmid was derived from pBLCAT2. The p21-Luc plasmid was kindly provided by Dr Bert Vogelstein, Johns Hopkins University, and contains a 2.4 kb fragment of the p21 promoter. The Vit-CAT plasmid contains the −821/− 87 5%-flanking region from the frog vitellogenin A2 gene linked to a thymidine kinase promoter and a bacterial chloramphenicol acetyl transferase (CAT) gene and was kindly provided by Drs Klein-Hitpass and Ryffel, Institute for Cell Biology (Essen, Germany). A cDNA for Northern blot analysis of the ER was prepared from an hER expression plasmid kindly provided by Dr Ming Jer Tsai, Baylor College of Medicine (Houston, TX). Oligonucleotide primers for c-myc, c-fos, c-jun and bactin for RT-PCR have been previously described (Irving et al., 1992; Do¨hr et al., 1995). Primers sequences for p21 and p27 were obtained from GeneBank (p21: Sense 5%-CCCAGTGGACAGCGAGCAGC-3%; Antisense 5%-ACTGCAGGCTTCCTGTGGGC-3%; p27: Sense 5%-TAACGGGAGCCCTAGCCTGG-3%; Antisense 5%-AACTCGGGCAAGCTGCCCTT-3%. The sizes of PCR products for p21 and p27 were 449 and 240 bp, respectively). Primers were synthesized by the DNA Technologies Laboratory, Texas A&M University (College Station, TX). All other chemicals and biochemicals were purchased from commercial sources and were either reagent or molecular grade.

2.2. Cell culture maintenance MDA-MB-468 and MCF-7 human breast cancer cells were obtained from the ATCC and maintained in DME/F12 and MEM media with or without phenol red and supplemented with 5% fetal bovine serum plus 10 ml antibiotic/antimycotic solution at 37°C, respectively.

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2.3. Stable transfection of the estrogen receptor A full length hER expression plasmid, 10 mg (1.8 kb EcoRI-digested wild type ER cDNA was cloned into pcDNA3 vector) or vector pcDNA3 (as a control) were transfected into ER-negative MDA-MB-468 cells. After 2 days, cells were treated with selective medium containing 400 mg/ml of geneticin for the first week, then 800 mg/ml of geneticin until drug-resistant colonies were formed. Cloning cylinders were used to isolate colonies which were then propagated in selection medium. Approximately 20 resistant colonies were isolated, and the effects of E2 on cell growth were determined for ten different stable transfectants. The growth of four of these cell lines was inhibited by E2 as previously reported for other cell lines stably transfected with the ER (Levenson and Jordan, 1994). The cell line described in this study was among the most sensitive to the growth inhibitory effects of E2 and was utilized as a model for studying the mechanism of growth inhibition by E2.

2.4. Cell proliferation and morphology change Cells were seeded at 6.0 – 7.5 ×104 cells/well in 6-well plates in DME-F12 media plus 5% dextran-coated charcoal-stripped fetal bovine serum (DCC-FBS). For cell proliferation, cells were treated with appropriate chemicals for specific times as indicated. The medium was changed and cells were redosed every 48 h. Cells were harvested and counted using Coulter Z1 cell counter after appropriate treatments. For morphology studies, cells were treated with DMSO (0.1% v/v as control) and 10 nM E2 for 5 days. Medium was changed, cells were redosed every 48 h, and morphological alterations of cells were examined by phase contrast microscopy.

2.5. Transient transfection assays Cells were seeded in 100 mm Petri dishes and grown in DME/F12 plus 5% DCC-FBS until 70% confluence; 25 mg of pBLCAT2 (TK-CAT) or TATACAT, 5 mg Vit-CAT or 7 mg p21-Luc plasmids and 20 mg polybrene/ml were used for the assays. After incubation for 6 h, cells were shocked using 25% DMSO for 4 min followed by treatment with DMSO (0.1% total volume) or E2 (10 nM) in DMSO for 48 h in DME/F12 medium supplemented with 5% stripped FBS. Cells were then washed with phosphate buffered saline (PBS) and scraped from the plates. Cell lysates were prepared in 0.18 ml of 0.25 M Tris – HCl, pH 7.5 by three freezethaw-sonication cycles (3 min/each). CAT activity was determined using 0.2 mCi D-threo-[dichloroacetyl-114 C]chloramphenicol and 4 mM acetyl-CoA as substrates. Protein concentrations were determined using BSA as a standard. Following TLC, acetylated prod-

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ucts were visualized and acetylated band intensities were quantitated using a Betascope 603 Blot analyzer. Luciferase activities in cell lysates were determined using the Luciferase Assay System with Reporter Lysis Buffer from Promega. The intensity of light emission from assays of cell extracts containing 20 mg of total protein was determined using a Packard 1600 liquid scintillation counter.

2.6. Preparation of whole cell extracts Cells were seeded in 100 mm plates and grown in DME/F12 media plus 5% DCC-FBS. After cells reached 70% confluence, they were treated with DMSO (0.1% total volume as a control) and 10 nM E2 for 12, 24 and 48 h, respectively. Cell monolayers were then washed once in ice-cold PBS and scraped into lysis buffer [50 mM HEPES (pH 7.5), 150 mM sodium chloride, 10% (v/v) glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 1 mM phenylmethylsulfonylfluoride (PMSF), 200 mM sodium orthovanadate, 10 mM pyrophosphate, and 100 mM NaF (Watts et al., 1995). Cells were incubated for 30 min, then centrifuged at 10 000×g for 5 min. Supernatants were precleaned by addition of 20 ml of protein A-agarose beads for 30 min followed by centrifugation for 5 min at 10 000× g. The lysates used for both Western blot and kinase assays were stored at − 80°C until required. All procedures were carried out at 4°C.

2.7. Western immunoblot analysis Cell lysates, prepared as described above, were loaded on SDS-page polyacrylamide gel. After electrophoresis, proteins were transferred to nitrocellulose membrane using an electroblotting apparatus overnight at 4°C. Membranes were blocked with TBS (10 mM Tris–HCl pH, 8.0; 150 mM sodium chloride) plus 5% milk (blotto buffer) for 1 h and then incubated in primary antibody at 0.1–1.0 mg/ml in the blotto buffer for 1–2 h at 20°C or overnight at 4°C. Membranes were rinsed in water and washed 5 min (2× ) in TBS. The secondary anti-mouse or rabbit/HRP (1:1000–2000 dilution) was added in blotto buffer and incubated for 1 h at 20°C. Membranes were then rinsed with water and washed 5 min (2× ) in TBS buffer. Membranes were incubated in Amersham ECL reagents for 1 min, excess ECL reagent was removed by dabbing with a Kimwipe, and the membrane was sealed in plastic wrap. Membranes were then exposed to ECL hyperfilm for visualization of immunoreactive bands. ER antibodies were purchased from Santa Cruz Biotechnology, (Santa Cruz, CA) and the assay was carried out as described in the treatment protocols provided by the company.

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2.8. Kinase assays

2.11. Semi-quantitati6e RT-PCR

Cell lysates (400 mg), prepared as described above, were incubated with individual antibodies (500 ng/each) for 3 to 14 h at 4°C, then immunoprecipitated by protein A/G-agarose beads for 3 h at 4°C. Beads were washed with lysis buffer (3×5 min) and with kinase buffer (3 ×5 min) (50 mM Tris – HEPES, pH 7.5, 10 mM MgCl2). The kinase reaction was carried out in 30 ml of kinase buffer supplemented with 400 mg/ml histones (Sigma type III-SS) for cdk2 kinase assay or 0.3 mg GST-RB (sc-4112, Santa Cruz, CA) for cdk4 kinase assay, 10 mM ATP, 0.5 mM dithiothreitol, 0.5 mM EGTA and 5 mCi [g 32P]ATP for 30 to 40 min at 30°C. Assays were stopped with 30 ml of 2 ×SDS-PAGE sample buffer, boiled for 5 min. Samples were loaded and separated on 12% SDS-PAGE gel. A major 30 kDa pattern of phosphorylated histone products was observed and visualized by autoradiography using Kodak film; bands were quantitated using a densitometer (JX330, Sharp, Japan) (Watts et al., 1995).

Cells were seeded in 100 mm plates and synchronized in serum-free medium for 2 days. Cells were then treated with DMSO (0.1% v/v as control) and 10 nM E2 in DME/F12 medium supplemented with 5% DCCFBS for 1, 3, 7, 12, 24, 32 and 48 h, respectively. Total RNA was extracted as described above. RNA (0.2 mg) from each sample was reverse-transcribed for 25 min at 42°C, then heated for 5 min at 99°C and cooled for 5 min at 4°C. Sense primers were labeled using [g32 P]ATP. The amplification conditions were denaturation at 95°C for 45 s, annealing at 63°C for 45 s for c-myc, c-fos, c-jun, p21 and p27 or at 58°C for 1 min for b-actin, and extension at 72°C for 1 min (25 cycles) in a total volume of 50 ml. After PCR reaction, samples were incubated at 72°C for an additional 10 min. Each PCR reaction mixture (20 ml) was subjected to 6% polyacrylamide gel electrophoresis. The gel was dried, exposed to X-ray film and quantitated using the densitometer. b-Actin mRNA transcript was used as an internal control to standardize quantitation of various mRNAs.

2.9. Thymidine incorporation Cells were seeded in 6-well plates at 2× 105 cells/well (DME/F12 plus 5% DCC-FBS) and then treated with DMSO (0.1% v/v as a control) and 10 nM E2 for 12, 24 and 48 h, respectively. Methyl-[3H]Thymidine (2.0 uCi/ well) was added for an additional 2 h. Cells were then washed once with ice-cold PBS and scraped into 15 ml tubes containing 1 ml of 10% trichloroacetic acid. Cells were then incubated for 2 h on ice and centrifuged at 10 000× g for 10 min at 4°C. Cell pellets were washed repeatedly with ethanol to remove all radioactivity; pellets were then dissolved in 100 ml of 0.25 N sodium hydroxide solution and neutralized with equimolar hydrochloric acid followed by counting using a liquid scintillation counter.

2.10. Northern blot analysis RNA was extracted using an RNA extraction kit from TEL TEST (Friendswood, TX). Of the total RNA obtained from each treatment group 25 mg was separated by electrophoresis on 1.2% agarose gel, transferred onto a nylon membrane, bound to the membrane by UV crosslinking, and baked at 80°C for 2 h. The membrane was then prehybridized in a solution containing 0.1% BSA, 0.1% Ficoll, 0.1% polyvinyl pyrolidone, 10% dextran sulfate, 1% SDS and 5× SSPE (0.75 M sodium chloride, 50 mM NaH2PO4, 5 mM EDTA) for 18 to 24 h at 60°C and hybridized in the same buffer for 24 h with the 32P-labeled DNA probe (106 cpm/ml). The cDNA probes were labeled with [a-32P]dCTP.

2.12. Flow cytometry analysis Cells were seeded in 6-well plates and treated with DMSO (0.1% v/v) and 10 nM E2 at indicated times. Cells were then washed once with PBS and harvested. Their nuclei were stained using the hypotonic lysis procedure (Tate et al., 1983). The staining solution was 4 mM sodium citrate, 0.1% Triton X-100, 30 U/ml RNase A (boiled for 30 min to inactivate DNase), and 50 mg/ml propidium iodide. Washed cells were incubated in 1 ml of the staining solution for 10 min at 37°C followed by addition of 100 ml of 1.5 M sodium chloride. Cells were then analyzed on a FACS Calibur instrument (Becton–Dickinson, San Jose, CA). Data were analyzed using ModFit LT software (Verity Software House, Topsham, ME).

2.13. Statistics Results are expressed as means9SE for at least three independent (replicate) experiments for each treatment group. Statistical significance was determined by ANOVA and Student’s t-test and the levels of probability are noted.

3. Results The results illustrated in Fig. 1 show that in MDAMB-468 cells stably transfected with wild-type ER only the 18 S ER mRNA transcript is detected (Fig. 1A, lane 3). In contrast, the higher molecular weight (28 S)

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Fig. 1. Analysis of ER mRNA and protein levels in MCF-7, wild-type and ER stably transfected MDA-MB-468 cells, and estrogen-responsiveness in the MDA-MB-468 cells. (A) Northern blot analysis. RNA (25 mg/each) was used for each assay. Lanes 1 – 3 represent RNA samples from MCF-7, wild-type and ER stably transfected MDA-MB-468 cells, respectively. MCF-7 cells express a full length ER mRNA transcript whereas ER stably transfected MDA-MB-468 cells express a 1.8 kb ER cDNA-related mRNA transcript. ER mRNA was not detected in wild-type MDA-MB-468 cells. (B) Western blot analysis. Whole cell lysates from MCF-7 (50 mg) and from wild-type and stably transfected MDA-MB-468 cells (100 mg) (lanes 1 – 3, respectively) were used for the assay as described in Section 2. The 65 kDa ER protein was observed in all cell lines; however, only trace levels of the immunoactive ER band was detected in wild-type MDA-MB-468 cells after longer exposure times. The relative ratios of ER protein levels in MCF-7, wild-type MDA-MB-468 and stably transfected MDA-MB-468 cells were 90 9 9.8:1:16 94.3. (Results are expressed as means 9SE for three determinations, relative to levels in wildtype MDA-MB-468 cells). (C) Vit-CAT activity. CAT activity in cells transiently transfected with the Vit-CAT plasmid were determined as described in Section 2. Relative levels of acetylated product in lanes 1–4 were 1.0, 1.3 9 0.5, 0.7 90.2, and 2.4 9 0.1, respectively (means 9 SE for three separate determinations). Ten nM E2 did not significantly induce CAT activity in wild-type cells (lanes 1 and 2), whereas a 3.4-fold induction was observed in ER stably transfected cells (lanes 3 and 4).

transcript is expressed in MCF-7 cells (lane 1) whereas the ER mRNA transcript was not detected in wild-type MDA-MB-468 cells (lane 2). Immunoreactive wild-type human ER protein was detected in MCF-7, wild-type and stably transfected MDA-MB468 cells at ratio of 90:1:16, respectively (Fig. 1B, lanes 1 – 3, respectively) [note: the equivalent amount of protein used for the wild-type and stably transfected MDA-MB-468 cells was 2× higher than used for MCF-7 cells (lane 1)]. These results are consistent with previous studies with ER-negative cell lines stably transfected with wild-type ER (Levenson and Jordan, 1994). Although low levels of immunoreactive protein were expressed in wild-type MDA-MB-468 cells, evidence for E2 responsiveness was not observed (Wang et al., 1997). Moreover, ligand binding was not observed in nuclear extracts from cells treated with 1 nM [3H]E2 (data not shown). In transient transfection studies with an estrogen-responsive construct derived from the vitellogenin A2 gene promoter, 10 nM E2 induced reporter gene activity (3.4fold) in the ER stably transfected cells whereas no induction response was observed in wild-type cells (Wang et al., 1997) (Fig. 1C). In parallel studies using MCF-7 cells, the induction response was comparable to that observed for the stably transfected cell line (data not shown). Treatment of stably transfected MDA-MB-468 cells with 10 nM E2 for 6 days resulted in 53% inhibition of cell growth whereas E2 did not affect growth of wildtype cells or MDA-MB-468 cells stably transfected with control vector pcDNA3 (Fig. 2A). The antiestrogen ICI 182 780 (0.1 mM) did not inhibit proliferation of the ER stably transfected cells (Fig. 2B); however, 0.1 mM ICI

182 780 inhibited the antiproliferative activity of E2. E2 also significantly inhibited [3H]thymidine uptake in ERstably transfected cells whereas this response was not observed in wild-type MDA-MB-468 cells (Fig. 2C). The effects of E2 on the morphology of wild-type, ER and pcDNA3-(vector) stably transfected MDA-MB-468 cells is illustrated in Fig. 3. E2 treatment of ER-stably transfected cells resulted in enlarged and flattened cells whereas no significant morphological changes were observed in the wild-type and pcDNA3 stably transfected MDA-MB-468 cells. These results indicate that cells stably transfected with pcDNA3 (vector) did not affect the wild-type phenotype with respect to cell growth or morphology suggesting that the stably transfected ER was responsible for hormone-induced growth inhibition and altered morphology. The potential role of several genes on the growth inhibitory effects of E2 were also investigated in stably transfected MDA-MB-468 cells. Previous studies in this laboratory and others have shown that TGF-a and EGF inhibit growth of wild-type cells (Wang et al., 1997; Armstrong et al., 1994, 1994; Ennis et al., 1989; Filmus et al., 1985) and other antiproliferative agents such as TPA and TCDD affect EGF receptor gene expression or binding (Wang et al., 1997; Bjorge et al., 1989). Although TGF-a and EGF also inhibited growth of the stably transfected cells, E2 did not affect EGF, TGF-a or EGF receptor gene expression in stably transfected MDA-MB-468 cells (data not shown). The results in Fig. 4 demonstrate that E2 did not significantly modulate c-fos, c-myc or c-jun expression in stably transfected MDA-MB-468 cells whereas these ‘early-induced genes’ are generally up-regulated by E2

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Fig. 2. Effects of E2 on cell proliferation and DNA synthesis in wild-type, vector pcDNA3 and ER stably transfected MDA-MB468 cells. (A) Time-dependent effects of 10 nM E2 on cell proliferation in wild-type ( ), pcDNA3 () and ER stably transfected (“) MDA-MB-468 cells. Cells were seeded in 6-well plates and treated with DMSO (as control) and 10 nM E2 for specific times as indicated. Cells were then harvested and analyzed as described in Section 2. Relative cell growth is standardized against solvent-treated control. (B) Effects of ICI 182 780 on E2 induced growth inhibition for ER stably transfected MDA-MB-468 cells. Cells were treated with appropriate compounds for 6 days and redosed every 48 h. (C) Effects of E2 on DNA synthesis in wild-type (“) and ER stably transfected () MDA-MB-468 cells. Cells were seeded in 6-well plates and treated with DMSO (control) or 10 nM E2 for 12, 24, and 48 h. Cells were then treated with [3H]thymidine for an additional 2 h. Cells were harvested and DNA synthesis was measured as described in Section 2.

in ER-positive breast cancer cells (Weisz and Bresciani, 1993). The effects of E2 on cell cycle phase distribution of stably transfected MDA-MB-468 cells (Table 1) shows that there was a significant 9.2–27.5% increase of cells in G0/G1 phase, an 11.6–1.0% decrease in S phase, and a 5.9–8.2% decrease in G2/M phase 1–6 days after treatment. These results showed a significant initial arrest of cells in G0/G1; however, after 6 days, the number of cells in G0/G1 decreased and this was accompanied by a comparable increase of cells in S phase. The most persistent effect of E2 was a permanent decrease in cells entering G2/M (Table 1). Based on these data, the effects of E2 on enzymes/genes which regulate the cell cycle and DNA synthesis were investigated. The results in Fig. 5 show that treatment of stably transfected MDA-MB-468 cells with E2 did not significantly alter cyclin D1, cyclin A, cdk2 or cdk4 immunoreactive protein levels whereas there was a time-dependent increase of cyclin E protein and a maximal 3.7-fold induction after 48 h. The results illustrated in Fig. 6 show that cyclin E- and cdk2-dependent kinase activities are initially decreased in stably transfected MDA-MB-468 cells 12 to 24 h after treatment with 10 nM E2 whereas after 48 h both kinase activities are slightly higher than observed in untreated cells. In contrast, cdk4-mediated phosphorylation of RB protein was decreased by over 40% after treatment of stably transfected MDA-MB-468 cells with 10 nM E2 for 48 h (data not shown). Cyclin and cdk activities are also regulated by cdk inhibitors and initial studies examined the effects of E2 on p27 and p21 gene expression in stably transfected MDA-MB-468 cells. Although p27 mRNA levels were unchanged (data not shown), E2 caused a 2.5- to 3-fold induction of p21 mRNA levels within 6 to 12 h, and after 48 h, no significant induction was observed (Fig. 7C). E2 induced a 9-fold increase in immunoreactive p21 protein levels in stably transfected MDA-MB-468 cells after 48 h whereas in wild-type cells low basal levels of p21 protein are unchanged during the same time period (Fig. 7A and 7B). The cell specific induction of p21 was further investigated in wild-type and stably transfected MDA-MB-468 cells transiently transfected with p21-Luc which contains a 2.4 kb region of the p21 gene promoter (El-Deiry et al., 1993). The results show that in wild-type cells, 10 nM E2 did not induce luciferase activity, whereas in the stably transfected cell line, there was 7.8-fold increase in luciferase activity after treatment with 10 nM E2. This induced response was inhibited by the antiestrogen ICI 182 780 (Fig. 7D). The results summarized in Fig. 8A show that immunoreactive E2F1 and PCNA protein levels were

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Fig. 3. Effects of E2 on cell morphology in wild-type, pcDNA3 and ER stably transfected MDA-MB-468 cells. Each cell line was treated with DMSO (0.1% v/v) and 10 nM E2 for 5 days. Medium was changed and cells were redosed every 48 h. The morphological alterations of cells were examined by phase contrast microscopy.

decreased by 58.0% and 43.8%, respectively, in stably transfected MDA-MB-468 cells treated with 10 nM E2 for 48 h. In parallel studies E2 also decreased CAT

activity in stably transfected MDA-MB-468 cells transiently transfected with a TK-CAT plasmid whereas no significant effects were observed using a TATA-CAT

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plasmid (Fig. 8B). These data were consistent with decreased E2F1 protein in these cells after treatment with E2 since this transcription factor enhances TK expression.

Table 1 Cell cycle distribution of ER stably transfected MDA-MB-468 human breast cancer cells after treatment with 17b-estradiol (E2)a Cell cycle phase (%)

G0/G1 S G2/M

Control

70.0 91.5 15.9 9 6.4 14.1 96.3

Time after treatment (days)

1

3

6

87.5 92.8b 4.3 92.4b 8.2 9 3.3b

85.79 6.8b 6.4 93.3b 7.9 94.7b

79.29 7.5 14.99 6.8 5.9 95.7b

a Cells were seeded in 6-well plates at 7×104 cells/well. Cells were treated with DMSO (0.1% v/v as control) and 10 nM E2 in DME/ F12 medium supplemented with 5% DCC-FBS for different time periods. Cells were then washed once with the medium and the assay was carried out as described in Section 2. The cell cycle phase distribution in DMSO-treated (control) cells was determined after 6 days. In a parallel study using wild-type MDA-MB-468 cells, E2 had no significant effect on % of cells in G0/G1, S or G2/M phases. Experiments were performed in triplicate and results are expressed as means9 SE. b Significantly different (PB0.05) than control cells (day 0).

4. Discussion

Fig. 4. Effects of E2 on c-fos, c-myc and c-jun protooncogene expression in ER stably transfected MDA-MB-468 cells. Cells were seeded in 100 mm plates and synchronized in serum-free DME/F12 medium for 48 h. Cells were treated with DMSO (0.1% v/v) and 10 nM E2 for various times, then harvested. RNA was extracted and RT-PCR was carried out as described in Section 2 for (A) c-fos, (B) c-myc and (C) c-jun mRNAs. The experiments were repeated at least three times and the values presented in the figure are means of these experiments. SE values were 510% for all time points. EGF, TGF-a and EGFR mRNA levels were not significantly affected by treatment with E2 (data not shown). (), Control; (“), 10 nM E2.

In estrogen-responsive MCF-7 human breast cancer cells E2 stimulates cell proliferation and DNA synthesis and this is accompanied by decreased cells in G1 phase and an increased percentage of cells in S and G2/M phase (Foster and Wimalasena, 1996; Musgrove et al., 1993; Thomas and Thomas, 1994; Sutherland et al., 1983; Osborne et al., 1984; Wilcken et al., 1996). The antiestrogen ICI 182 780 alone did not change the cell cycle status of growth arrested MCF-7 cells; however, in cells co-treated with E2 plus ICI 182 780, the changes in cell cycle status and growth induced by E2 were inhibited (Foster and Wimalasena, 1996). Although stably transfected MDA-MB-468 cells expressed wildtype ER protein, E2 inhibited cell growth and DNA synthesis in stably transfected MDA-MB-468 cells (Figs. 2–4). ICI 182 780 inhibited the E2-induced response suggesting that the paradoxical antimitogenic activity of E2 in stably transfected MDA-MB-468 cells was mediated through the ER. The growth inhibitory effects of E2 have also been observed in other cell lines stably transfected with the ER (Levenson and Jordan, 1994; Jiang and Jordan, 1992; Catherino et al., 1995; Garcia et al., 1992; Zajchowski et al., 1993; Zajchowski and Sager, 1991; Lundholt et al., 1996; Watts et al., 1989; Gaben and Mester, 1991; Watts and King, 1994; Webb et al., 1992; Maminta et al., 1991; Kushner et al., 1990; Touitou et al., 1990), and therefore stably transfected MDA-MB-468 cells were used as a model to determine which pathways associated with the cell cycle and DNA synthesis mediated hormone-induced growth inhibition. Initial studies showed that genes such as c-fos, c-jun and c-myc which are rapidly induced in ER-positive

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breast cancer cells (Weisz and Bresciani, 1993) were not affected after treatment of stably transfected MDAMB-468 cells with E2 (Fig. 4), and induction of TGF-a and EGF receptor was also not observed (data not shown). The failure to observe induction of these genes by E2 in the ER stably transfected MDA-MB-468 cells demonstrates that expression of the ER is not sufficient to fully restore E2-responsiveness. This is consistent with studies showing that other factors such as coactivators are also important for cell-specific gene expres-

Fig. 5. Western blot analysis of cyclin D1, cyclin E, cyclin A, cdk2 and cdk4 protein levels in ER stably transfected MDA-MB-468 cells treated with E2. Cells were seeded in 100 mm plates and treated with 10 nM E2. Whole cell lysates were extracted and 100 mg of protein from each sample was loaded and separated on 12% SDS-PAGE gel as described in Section 2. Lane 1: untreated; lanes 2–4: 10 nM E2 for 12, 24 and 48 h, respectively. The Western blot experiments were repeated at least three times and no time-dependent changes in immunoreactive cyclin D1, cyclin A, cdk2 or cdk4 were observed in any of the experiments. Only cyclin E protein levels were increased. The relative intensities of bands in lanes 1 – 4 were 1, 1.99 0.5, 3.39 1.1 and 3.79 1.3, respectively (means 9 SE for three separate determinations). Cyclin E protein levels were not significantly affected by E2 in wild-type MDA-MB-468 cells (data not shown).

Fig. 6. Effects of E2 on cyclin E-, cdk2- and cdk4-dependent kinase activities in ER stably transfected MDA-MB-468 cells. Immunoprecipitates were obtained using anti-cyclin E, anti-cdk2 and anti-cdk4 antibodies and whole cell lysates (400 mg/each) from ER stably transfected MDA-MB-468 cells. Cyclin E-dependent and cdk2 activities were determined using histone (Sigma type III-SS) as a substrate. Cdk4 activity was determined using GST-RB as a substrate. Kinase activities were analyzed by SDS-PAGE gel and band intensities were measured by a densitometer as described in Section 2. (A) Cyclin E-dependent kinase activity. The relative intensities of bands in lanes 1 – 4 were 100, 45.79 6.5, 67.49 5.1 and 128.79 15.8, respectively. (B) Cdk2-dependent kinase activity. The relative intensities of bands in lanes 1 – 4 were 100, 61.4910.8, 44.49 9.0 and 108.89 20.8, respectively. (C) Cdk4-dependent kinase activity. The relative intensities of bands in lanes 1 – 4 were 100, 99.89 33.1, 91.0 94.3 and 64.9 96.3, respectively. Lane 1: untreated (0.1% v/v of DMSO); lanes 2 – 4: 10 nM E2 for 12, 24 or 48 h, respectively. Cdk4-dependent kinase activity was decreased by over 40% (PB 0.05) after treatment of ER stably transfected MDA-MB-468 cells with 10 nM E2 for 48 h. All data are means 9 SE for three separate determinations.

sion and hormone inducibility (Horwitz et al., 1996). EGF and TGF-a inhibit growth of wild-type MDAMB-468 cells (Wang et al., 1997; Armstrong et al., 1994; Ennis et al., 1989; Filmus et al., 1985), and similar results were observed in the stably transfected

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Fig. 7. Effects of E2 on p21 mRNA, protein levels, and promoter activity in wild-type and ER stably transfected MDA-MB-468 cells. (A) Western blot analysis of p21 protein levels in ER stably transfected MDA-MB-468 cells treated with 10 nM E2 for 6, 9, 12, 24, 48 and 60 h, respectively. Whole cell lysates (100 mg/each) were used for the assay which was carried out as described in Section 2. p21 Immunoreactive protein was increased 9 91.1-fold (PB0.001) after 48 h of treatment with E2. This experiment was carried out in triplicate. (B) Western blot analysis of p21 protein levels in wild-type MDA-MB-468 cells. The assay was carried out as described above. p21 Protein levels in wild-type cells were not significantly affected by treatment with E2 compared to that observed in ER stably transfected MDA-MB-468 cells. (C) RT-PCR analysis of p21 mRNA levels in ER stably transfected MDA-MB468 cells treated with 10 nM E2. Cells were seeded at 100 mm plates and synchronized in serum-free medium for 48 h. Cells were treated with DMSO (0.1% v/v) and 10 nM E2 for various times. RNA was extracted and RT-PCR was carried out as described in Section 2. p21 mRNA levels were increased approximately 3-fold (PB 0.05) after treatment with E2 for 9 h. (open circle), Control; (filled circle), 10 nM E2. This experiment was carried out in triplicate and the results are expressed as means; SE values were 510% for all time points. (D) Wild-type (diagonally striped box) and ER stably transfected (filled box) MDA-MB-468 cells were transiently transfected with p21 Luc, treated with various chemicals, and luciferase activity was determined as described in Section 2. Luciferase activities relative to control (DMSO) cells were: (a) wild-type cells, DMSO (100 915%), 10 nM E2 (72919%); (b) ER stably transfected cells, DMSO (100%), 10 nM E2 (775 9 132%), 10 nM E2 plus 1 mM ICI 182 780 (225 913%), and 1 mM ICI 182 780 (225 912%). In the stably transfected cells, E2 significantly induced luciferase activity (PB 0.05) and ICI 182 780 significantly inhibited (PB 0.05) this response (results are expressed as means9SE for three separate determinations).

cells (data not shown). This suggested that the sites of E2 action may be more closely linked to proteins directly involved in regulating cell cycle kinetics and DNA synthesis, and this was confirmed by showing that after treatment of the stably transfected cells with E2, there was an increased accumulation of cells in

G0/G1 and a decrease in cells in S and G2/M phases (Table 1). Previous studies with ER-positive breast cancer cells have shown that after growth arrest, treatment with E2 resulted in an increase in cyclin D1 and E proteins whereas only minimal changes were observed in other

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Fig. 8. Effects of E2 on E2F1, PCNA and TK-CAT expression in ER stably transfected MDA-MB-468 cells. (A) Western blot analysis of E2F1 and PCNA protein levels after treatment with E2. Whole cell extracts (see Fig. 5) were utilized for the assay which was carried out as described in Section 2. E2F1 and PCNA protein levels were decreased 589 5.7% and 43.8 9 7.6% (P B 0.05), respectively. E2 had no effect on E2F1 and PCNA protein levels in wild-type MDA-MB468 cells (data not shown). (B) Effect of E2 on TK-CAT activity in ER stably transfected MDA-MB-468 cells. Cells were seeded in 100 mm plates. After cells reached 70% confluence, 25 mg of TK-CAT or TATA-CAT (as control) plasmids were transfected into the cells followed by treatment with DMSO and 10 nM E2 for 48 h in DME/F12 medium supplemented with 5% stripped fetal bovine serum. The assay was carried out as described in Section 2. TK-CAT activity was decreased 3695.4% (P B0.05) after treatment with E2 whereas TATA-CAT activity was not affected. E2 did not alter the TK-CAT activity in wild-type MDA-MB-468 cells (data not shown). Results are means 9SE for three separate determinations.

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cyclins and cdks (Foster and Wimalasena, 1996). The results in Fig. 5 show that cyclin A, cyclin D1, cdk2 and cdk4 levels were unchanged in stably transfected MDA-MB-468 cells after treatment with E2 whereas cyclin E protein was increased 3.7-fold after 48 h (Fig. 5). Despite the increased cyclin E protein levels, there was not a comparable increase in cyclin E-dependent kinase activities (Fig. 6). The critical E2-induced responses in MCF-7 cells associated with cell-cycle progression were induction of cdk4- and cdk2-dependent kinase activities; hypophosphorylation of RB and decreased expression of the cdk inhibitor p27 (Foster and Wimalasena, 1996). The results of this study show that the growth-inhibitory activity of E2 and modulation of cell cycle enzymes and DNA synthesis does not bear an inverse relationship to the effects of E2 in ER-positive cell lines (e.g. MCF-7). In stably transfected MDA-MB-468 cells, E2 treatment resulted in a temporal decrease (0 to 24 h) in both cdk2and cyclin E-dependent kinase activities; however, after 48 h these activities were at or slightly above those observed in untreated cells (Fig. 6). Treatment of these cells with E2 also resulted in a marked 40% decrease in cdk4-dependent kinase activity (Fig. 6), and this was associated with increased p21 gene expression and immunoreactive protein levels (Fig. 7). The E2-treated cells were also significantly enlarged with a flattened appearance (Fig. 3), and this may be associated with induction of p21 since MCF-7 and T47D cells transfected with p21 exhibited similar morphological characteristics (Sheikh et al., 1995). Thus, one of the major differences between wild-type and ER stably transfected MDA-MB-468 cells is the induction of p21 in the latter cell line by E2. E2 decreases p27 but not p21 protein levels in MCF7 cells (Foster and Wimalasena, 1996); however, the antiestrogen ICI 182 780 induces both p27 and p21 immunoreactive proteins in MCF-7 cells (Wilcken et al., 1996). Thus, the growth inhibitory effects of E2 in the stably transfected MDA-MB-468 cells (Fig. 2) resembled, in part, the effects of ICI 182 780 in MCF-7 cells (Wilcken et al., 1996). In some cells, there is an association between increased expression of p53 tumor suppressor gene and increased p21 (El-Deiry et al., 1993, 1994; Macleod et al., 1995); however, since p53 is mutated in MDA-MB-468 cells (Nigro et al., 1989), the upregulation of p21 appears to be a p53-independent response (Jiang et al., 1994; Parker et al., 1995). The results illustrated in Fig. 7C confirm the dramatic cellspecific differences in hormonal regulation of p21 expression in wild-type (diagonally striped box) and ER stably transfected (filled square) MDA-MB-468 cells. E2 does not induce luciferase activity in wild-type cells transiently transfected with the p21-Luc construct (ElDeiry et al., 1993), whereas a 7.8-fold interaction was observed in stably transfected cells. Moreover, E2-induced luciferase activity was inhibited by the antiestro-

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gen ICI 182 780 (Fig. 7C). These results suggest that wild-type MDA-MB-468 cells express factors which suppress induction of p21 by E2, whereas in MDAMB-468 cells stably transfected with ER, hormone-responsiveness is observed. Some of these cell-specific factors could include coactivator and corepressor proteins (Horwitz et al., 1996) which may be differentially regulated in the wild-type and stably transfected cells. Current studies are focused on identifying nuclear proteins and cis-genomic sequences within the p21 gene promoter which are associated with the cell-specific hormonal regulation of this gene. ICI 164 384 and retinoids inhibit ER-positive T47D breast cancer cell growth by different mechanisms; however both compounds inhibit RB phosphorylation (Wilcken et al., 1996). PGA2 also inhibited growth of MCF-7 cells and modulated several other responses including decreased cyclin D1 and cdk4 mRNA levels and cdk2/cdk4 activities (Gorospe et al., 1996), whereas only decreased cdk4 activity was observed in stably transfected MDA-MB-468 cells (Fig. 6). The direct effects of PGA2 on E2F1 in MCF-7 were not reported; however, PGA2 decreased expression of E2F1-regulated c-myc and cyclin D1 (Gorospe et al., 1996). In contrast, E2 did not inhibit expression of either c-myc or cyclin D1 gene expression in stably transfected MDA-MB-468 cells (Figs. 4 and 5); however, both E2F1 and PCNA protein levels and TK-CAT activity were significantly decreased in the stably transfected cell line (Fig. 8). In summary, the results of this study demonstrate that growth inhibition of stably transfected MDA-MB468 cells by E2 is associated with modulation of several responses which decrease both cell cycle progression and DNA synthesis, namely increased p21 levels, decreased cdk4 activity, E2F1 and PCNA protein levels. The results of this study also demonstrate that stable transfection of the ER did not result in restoration of an estrogen-responsive phenotype with respect to E2-induced growth, cell cycle progression or DNA synthesis, suggesting that other factors in addition to the ER are required for estrogen-induced cell proliferation. Thus, as previously pointed out by Levenson and Jordan (1994), breast cancer cells stably transfected with ER can serve as a model for understanding the multiple factors which determine an ER-responsive phenotype with respect to both induced gene expression and cell growth.

Acknowledgements The financial assistance of the National Institutes of Health (CA-64081) and the Texas Agricultural Experiment Station is gratefully acknowledged.

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