Serum Regulation of the Estrogen Responsiveness of the Human Breast Cancer Cell Line MCF-71
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[CANCER RESEARCH 43. 1244-1250. March 1983] 0008-5472/83/0043-OOOOS02.00 Serum Regulation of the Estrogen Responsiveness of the Human Breast Cancer Cell Line MCF-71 Martin J. Page, John K. Field, Nie P. Everett, and Chris D. Green2 Department of Biochemistry. The University of Liverpool, P. O. Box 147. Liverpool L69 3BX. United Kingdom ABSTRACT England, and tamoxifen was a gift from S. R. Slater, I.C.I. Pharmaceu ticals Division, Alderloy Edge, England. Initially after receiving MCF-7 cells, we were able to confirm Maintenance of Stock Cultures. MCF-7 human breast cancer cells their estrogen responsiveness. We observed significant in were kindly provided by Dr. M. Rich, Michigan Cancer Foundation, creases in thymidine incorporation, in thymidine kinase activity, Detroit, Mich., and Dr. M. Lippman, National Institute of Health, Be- and in cell numbers in response to 10~8 M estradiol. Subse thesda, Md. The cells were grown as monolayer cultures in glass bottles at 37° in an atmosphere of 5% CO? in air. Growth medium quently, however, the cells failed to show a response to estra consisted of Dulbecco's minimal essential medium (Gibco, Paisley, diol. A growth response to estradiol could be restored by Scotland), supplemented with 4 mw L-glutamine (BDH, Liverpool, Eng increasing the serum concentration in the medium. Cells grown land) penicillin:streptomycin (250 units/ml; Gibco), and 10% (v/v) in 15% serum (calf or human) responded to estradiol with NBCS3 (Gibco or Flow, Irvine, Ayrshire, Scotland). Cells were harvested increased rates of growth and thymidine incorporation and by rinsing the monolayer with PBS, followed by a 15-min incubation increased activities of thymidine kinase and DNA polymerase. with PBS containing EDTA (0.1%). Cultures were tested for Myco- We suggest that there is present in serum a "factor" which plasma contamination using the Hoechst fluorescent staining method can influence the expression of a growth response to estradiol. (3). Preparation of Steroid-depleted Serum. Serum was stirred over night at 4° with 0.25% activated charcoal (Sigma) and 0.0025% INTRODUCTION dextran (Pharmacia, Hounslow, England). The serum was then centri- MCF-7 is a human breast cancer cell line derived from the fuged at 10,000 x g to remove the charcoal and passed through a 0.22-nm Millipore filter. Removal of steroids was monitored by the pleural effusion of a patient with metastatic breast adenocar- addition of a trace of [3H]estradiol at the beginning of the procedure. cinoma (21). These cells have been characterized extensively Greater than 97% of the tritiated steroid was removed. and have been shown to be of human and breast origin. [3H]Thymidine Incorporation Experiments. Cells were uniformly Because growth and macromolecular synthesis of MCF-7 cells plated in 35-mm plastic dishes using medium containing 5% serum. have been reported to be stimulated by physiological concen After the cells were left overnight to allow attachments, the medium trations of estradiol (13, 14), they have been proposed as an was aspirated and replaced by medium containing 0.5% steroid-de in vitro model system for the study of estrogen-responsive pleted serum (unless otherwise stated) and left for 24 hr. The medium was then aspirated and replaced by fresh medium containing hormone breast cancer (15). additions in ethanol. The cells were labeled with [3H]thymidine for the However, reports from other laboratories and our own ex last 2 hr of the next 48-hr incubation period. The dishes were then perience with these cells have shown that the degree of re thoroughly washed with ice-cold PBS, and the cells were harvested sponsiveness of MCF-7 cells to estradiol is rather variable. and spun at 800 x g for 5 min. To the cell pellets was added 1.0 ml Early reports from Lippman ef al. showed that estradiol more distilled water, and the cell suspension was sonicated (MSE Sonicator). than doubled rates of macromolecular synthesis (13) and could To 600 ut of this homogenate was added an equal volume of ice-cold double cell number yield (15). However, more recently, the 10% trichloroacetic acid, and the mixture was left at 4°for 30 min. same group has observed an abrupt decrease in the response The resulting precipitate was collected on a GF/C filter (Whatman, of their MCF-7 cells to estradiol (22). Similarly, other workers Maidstone, Kent, England) and washed with ice-cold 5% trichloroacetic have recently shown that estradiol failed to stimulate growth acid (10 ml). The filters were then placed in scintillation vials and dried (5, 8) and produced only a minimal stimulation of [3H]thymidine at 100° for 15 min, and the radioactivity was determined using Triton:toluene scintillation fluid (700 ml toluene, 300 ml Triton X-100, incorporation (10). Other recent reports would suggest that estradiol stimulation of MCF-7 cell growth can still be demon 5 g PPO, and 0.3 g POPOP). The remaining 400 ¡i\ofeach homogenate were used for protein determinations (16). strated (1, 17). Cell Number Determinations. Cells were harvested in PBS:EDTA, In this paper, we describe the characteristics of MCF-7 cells pelleted at 800 x g (5 min), resuspended in culture medium, and which had become apparently unresponsive to estradiol, and counted using a hemocytometer. we also report new conditions under which estrogen respon Thymidine Kinase Assay. Cells were cultured in a manner identical siveness can be regained. to that described for [3H]thymidine incorporation except that the step involving the addition of [3H]thymidine was omitted. Cells were har vested with a rubber policeman in 200 fi\ of 0.05 M Tris-HCI:1 mM MATERIALS AND METHODS dithiothreitol:0.25 M sucrose buffer, pH 7.8, and sonicated. The ho Materials. [3H]Thymidine (48 Ci/mmol) and 17/J-[3H]estradiol (108 mogenate was spun at 20,000 x g for 10 min, and the supernatant Ci/mmol) were obtained from the Radiochemical Centre, Amersham, was transferred to a Sarstedt tube. Blue dextran (10 /il of a 10% England. 17/3-Estradiol was obtained from Sigma Chemical Co., Poole, solution) was added, and the mixture was loaded onto a Sephadex G- 25 column (2 ml) equilibrated with the Tris:dithiothreitol:sucrose buffer ' Supported by grants from the Medical Research Council and the North West at 4°. The column was eluted with Tris:dithiothreitol:sucrose buffer, Cancer Research Fund. 2 To whom requests for reprints should be addressed. 3The abbreviations used are NBCS, newborn calf serum; PBS, phosphate- Received May 26, 1982; accepted November 23, 1982. buffered saline. 1244 CANCER RESEARCH VOL. 43 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1983 American Association for Cancer Research. Estrogen-responsive MCF-7 Cells and the blue fraction (excluded volume containing all molecules with Table 1 molecular weight greater than 5000) was collected and assayed for Effects of estradiol and tamoxifen on [3H]thymidine incorporation in ' 'unresponsive MCF- 7 celts thymidine kinase activity as described by Green and Martin (7). The final assay mix (100 fil) contained 4.4 /¿molTris-HCI (pH 7 to 8), 0.44 Cells were plated with 5% NBCS medium (2 ml) in 35-mm-diameter culture dishes. After 24 hr, the medium was replaced with 2 ml of fresh medium /¿molMgCI2,0.089 /unol NaF, 17.8 /¿molunlabeled thymidine, 1.0 /¿mol containing 0.5% steroid-depleted serum. After a further 24-hr, the medium was [3H]thymidine, and 50 /¿Iofenzyme extract. replaced with 2 ml of fresh medium containing 0.5% steroid-depleted serum together with estradiol (10 8 M) and/or tamoxifen (10~6 M). The cells were DMA Polymerase Assay. Cells were cultured and harvested as for the thymidine kinase assay. DMA polymerase activity was measured as incubated in these media for 48 hr, and 0.5 iiCi of pHJthymidine was added for the final 2 hr. Thymidine incorporation rates were determined as described in incorporation of [3H]dTTP into trichloroacetic acid-insoluble product "Materials and Methods." using the basic method of McLennan and Keir (12) except that acti vated calf thymus DNA was used as the template primer. Assay mixture (75 til) contained 10 mM potassium phosphate (pH 7.4), 4 mM MgSO4, x 10~Vmg pro- 1 mM mercaptoethanol, 15 iig activated DMA, 50 /¿MdATP, 50 /¿M TreatmentControl tein/hr%17.3 dCTP, 50 /IM dGTP, 0.1 iiCi [3H]dTTP, and enzyme extract. After 1 hr ±1.6a 120° at 37°,the reaction was stopped by adding 50 /¿Iof50% trichloroacetic Estradiol 20.7 ±1.8 acid and cooling the mixture on ice. The acid-insoluble product was Tamoxifen 6.7 ±0.7 39 Tamoxifen + estradiolpHJThymidmeincorporationdpm16.6 ±1.2100 98C collected on GF/C filters. Mean ±S.E. of triplicate determinations. Estradici Receptor Assays. MCF-7 cytosol and nuclear extracts " Stimulation by estradiol not significant by ( test ( p = 0.05). were prepared and assayed using the single-dose protamine sulfate c Stimulation by estradiol significant by f test (p = 0.01 ). method of Zava and McGuire (28), except that the assay for total nuclear receptors was carried out at 30°for 3 hr. Table 2 Karyotyping. Logarithmically growing cells were treated with col- Cytoplasmic and nuclear estrogen receptor contents of control and estradiol- chicine at a final concentration of 0.0004% for 4 to 5 hr and then treated unresponsive MCF-7 cells swollen with hypotonie solution (0.075 M KCI) and fixed with metha- MCF-7 cells were grown for 5 days in glass roller bottles containing 5% nol:acetic acid (3:1, v/v).