[CANCER RESEARCH 39, 3319-3327, September 1979] 0008-5472/79/0039-0000$02.00 Prolonged Retention of Estradiol by Human Breast Cancer Cells in Tissue Culture1 Jeannine S. Strobl and Marc E. Lippman2 Department of Pharmacology, The George Washington University Medical Center, Washington, 0. C. 20037 (J. 5. S.J, and Medicine Branch, National Cancer Institute, NIH, Bethesda, Maryland 20014 (M. E. LI. containing both cytosol and nuclear receptors for estrogen and exhibiting an increased rate of incorporation of radiolabeled thymidine, leucine, and uridine in response to physiological concentrations of estradiol (9, 10, 12). To examine estrogenic responses more closely requires that cell functions prior to the addition of exogenous estradiol be independent of estrogen. The MCF-7 cells under our standard culture conditions in Richter's medium supplemented with 10% fetal calf serum serum may be influenced by estrogen since estrogen concen trations in medium containing 10% fetal calf serum may often be as high as 0.5 nM (5, 7, 8, 10). When estrogen receptor is assayed in MCF-7 cells grown in media containing 10% fetal calf serum, only 11% of the estrogen receptors are located in the cytosol. The remainder are found in the nucleus with over one-half in the form of an estrogen receptor complex as dem onstrated by assays performed under exchange or nonex change conditions (20). The presence of occupied nuclear estrogen receptors is associated with cells grown in medium containing estrogen (21). We report here on the ability of serum-free medium washes to remove cell-bound [3H]estradiol following a 3-hr incubation with 3 to 5 n@ [3H]estradiol. We observe a prolonged retention of specifically and nonspecifi cally bound estradiol and discuss the implications of our results with respect to estrogen action in tissue culture systems. MATERIALS AND METHODS Materials. Unlabeled 17/3-estradiolwas obtainedfrom Ster aloids (Pawling, N. Y.), and prepared in absolute ethanol. Tamoxifen (ICI 46474), the generous gift of Imperial Chemical Industries, U. S. (Wilmington, Del.), was prepared as the free base in absolute ethanol at 1O@ M. These unlabeled stock solutions of estradiol and tamoxifen were stored at — 20°for Upto 2 months and diluted i :1000 in medium just prior to use. The final concentration of ethanol in the culture media was 0.1 % which does not affect MCF-7 viability or response to hormones. [3HjEstradiol (87 to 91 Ci/mmol) from Amersham/ Searle (Arlington Heights, Ill) was purified by Sephadex LH-20 (Pharmacia Fine Chemicals, Piscataway, N. J.) chromatogra phy using a benzene:chloroform:methanol, 6:3:i solvent sys tern. The purity was assessed as greater than 98% by thin layer chromatography on silica gel (Baker-flex; J. T. Baker, Phillipsburg, N. J.) using either chloroform:ethanol (90:1 0) or benzene:ethanol (80:20) as the solvent system. The purified estradiol was dried under nitrogen and stored for up to 5 days at 0.1 nM in benzene:ethanol (90:iO) at 4°with no loss of purity. This solution was dried under nitrogen, then redissolved in absolute ethanol to a final concentration of approximately 5 x 1O-@M. A 1:1000 dilution into Richter's medium to a final concentration of 5 nM was used for the washout experiments. [3H]Estrone sulfate (40 to 60 Ci/mmol) was obtained from New ABSTRACT Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 flM) and then were washed with 3 successive exchanges of medium 3, i 7, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture: (a) the concentration of [3H]estradiol and [3H]estradiol metab olites in the media washes; (b) the intracellular concentration Of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidmne incorporation. The (3H]estradiol concentration in the final me dium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H)estradiol was initially i 8 flM, and after 78 hr of wash, it was 2.8 n@.i. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 flM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed through out the 78 hr monitored. When 10° M tamoxifen or i O'@M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estra diol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem. INTRODUCTION The MCF-7 cell line is a human breast cancer cell line , From a dissertation to be presented to the Department of Pharmacology. The Graduate School of Arts and Sciences, The George Washington University, In partial fulfillment of the requirements for the Ph.D. Supported by a Predoctoral Fellowship from the National Science Foundation. A preliminary report of this work has been presented (16). 2 To whom requests for reprints should be addressed. Received July 17. 1978; accepted May 16, 1979. SEPTEMBER 1979 3319 Research. on December 3, 2021. © 1979 American Association for Cancer cancerres.aacrjournals.org Downloaded from