[CANCER RESEARCH 46, 2271-2275, May 1986] Control of Cell Proliferation: Evidence for Negative Control on Estrogen-sensitive T47D Human Breast Cancer Cells1 Ana M. Soto,2 James T. Murai, Peniti K. Siiteri, and Carlos Sonnenschein Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 021 ¡l[A. M. S., C. S.J, and Department of Obstetrics and Gynecology, University of California School of Medicine, San Francisco, California 94143 [J. T. M., P. K. SJ ABSTRACT The human breast tumor cloned cell lines 1471)-AX and All are estrogen dependent for cell proliferation in the nude mouse model. In contrast, these cells multiplied at similar rates when grown in serum-free cultures, regardless of the presence of 170-estradiol (3 x 10 "to3x IK"" M estradiol). Addition of 10% charcoal-dextran stripped human female serum to the culture medium resulted in a marked inhibition of cell proliferation. The addition of 3 x IO " M estradili! overcame the inhibitory effect of serum. Similar results were obtained with the human breast tumor C7MCF7 cell line. Both cell lines contain similar estrophilin levels. The A,, of the estrophilin-estradiol complex was 0.39 x 10 '" M for C7MCF7 cells and 4.4 x IO"10 M for T47D-A11 cells. Maximal cell yields were achieved at 5 X III"12M free estradici levels in 10% charcoal- dextran stripped serum supplemented medium. These data are compatible with the following interpretation: (a) estradiol-sensitive cells are inhibited from proliferating by a serum-borne factor; and (b) estraditi! neutralizes this inhibitory effect. This mechanism seems not to be mediated by estradiol binding to the cellular estrophilins because (a) the free estradici levels needed for maximal response are significantly lower than the estrophilin A'ds. and (t>) maximal proliferation rates occur at similar estradiol concentrations for these three cell lines, regardless of the binding properties of their estrophilins. INTRODUCTION The mechanism of estrogen action on cell proliferation is poorly understood (1-5). Experimentalists in this field are currently following three distinct working hypotheses: (a) the direct-positive hypothesis, according to which 17/3-estradiol by itself triggers the multiplication of its target cells (3, 4, 6, 7); (b) the indirect-positive hypothesis, whereby estradiol triggers the synthesis and/or release of a "growth factor" or estromedin, that in turn causes proliferation of the estrogen-sensitive cells; this factor could be secreted in the endocrine (2), paracrine (8), or autocrine manner (9); and (c) an indirect-negative hypothesis which postulates that (a) estrogen blocks the synthesis and/or release of a specific inhibitor of estradiol-sensitive cells secreted by an intermediary organ (10, 11), and/or (/>)estrogen in the circulation may neutralize or inactivate the action of a putative inhibitor (12, 13). We explored this fundamental issue of control of cell multi plication under the direct and indirect positive hypotheses; the data collected suggested that the evidence was not entirely compatible with either hypothesis (5, 10, 14, 15). We therefore explored and subsequently adopted the indirect-negative hy pothesis based on what we considered increasingly compelling empirical evidence in its favor (16-20). Recently, we have developed evidence for the presence of a blood-borne, specific inhibitor of the proliferation of the human breast tumor MCF7 in culture and in nude mice. Estrogens reverse this inhibition; neither androgens, corticoids, nor growth factors cancel the inhibitory effect of this blood-borne factor(s). These results not only are compatible with the indirect-negative hypothesis, but they challenge the direct and indirect-positive working hy potheses (12, 13). This paper describes the proliferative behavior of the human breast tumor cell line T47D in an "in animal-in culture" model. We provide data regarding parameters considered by many as being directly implicated in the proliferative response attributed to estradiol, namely, the presence of intracellular estrophilins and the levels of free hormone in the culture medium. MATERIALS AND METHODS Cell Lines and Culture Conditions. T47D-A11 and T47D-A8 cells were graciously supplied by Dr. E. Medrano and Dr. A. Baldi from the Instituto de Biologia and Medicina Experimental, Buenos Aires, Ar gentina, and T47D-26 and 82 cells were purchased from the American Type Culture Collection, Rockville, MD. The results reported below did not show significant differences when cells from both sources were compared. Al 1 and AS refer to clonal populations from the uncloned T47D cells originally described by Keydar et al. (21). C7MCF7-173 cells are derived from a single-cell clone. An original sample was generously provided by Dr. Charles McGrath from the Michigan Cancer Foundation, Detroit, MI (22). The partial character ization of this cloned cell line was reported elsewhere (23). Cells were routinely grown in 5% (v/v) FBS3 (Lots 100345 and 100405 from Sterile Systems, Inc., Logan, UT) in DME medium in an atmosphere of 5% CO2-95% air under saturating concentrations of humidity at 37°C. To determine estradiol sensitivity in nude mice and cellular estrophilin properties, large volumes of the cells were grown in plastic roller bottles in a Bélico rolling apparatus placed in a Bélico incubator: CO: was introduced directly into the bottles to balance the pH in the culture medium when the culture was initiated. Cell Proliferation Experiments in Culture. T47D-A11, T47D-A8, and C7MCF7-173 cells were plated into Costar 3512 multiplates at initial concentrations of about IO4cells. At optimal proliferation rates, these cells would accrue a minimum of 4 to 6 doublings during an observation period of 10 to 14 days. The cells were allowed to attach for 24 h, and then the seeding medium (5% FBS in DME) was removed and replaced by the experimental one. Serum-supplemented medium was not changed during the length of the proliferative period. When serumless medium was used, an adequate supply of nutrients was provided by changing the medium every 48 h. Blood Drawing and Charcoal-Dextran Treatment of Sera. Venous blood was drawn from 20 healthy young women during the first day of their menstrual cycle; these sera will be called Day-01 FHS. Heart blood was drawn from CD of mouse retired breeders (Charles River Animal Laboratories, Wilmington, MA). Blood was allowed to clot in sterile plastic tubes for 4 h; sera were clarified by centrifugation (3000 rpm for 10 min in a Model PR6 centrifuge, IEC; Damon Corp., Needham Heights, MA). Sera were then filtered through a 0.45->im pore size Nalgene filter unit or with Millex filters (Millipore Corp., Bedford, MA), used immediately, or aliquoted and stored in a freezer at —20°C for future use. Sera from individual donors were thawed and heat inactivated for 30 min at 56°Cbefore use. This procedure is designed to denature heat-sensitive estrogen binders (24). To strip the sera of steroids, a 0.5% charcoal (Norit A, acid washed; Sigma)-0.05% Dextran T70 (Pharmacia, Piscataway, NJ) suspension Received 4/16/85; revised 8/28/85, 12/10/85; accepted 1/21/86. 'The abbreviations used are: FBS, fetal bovine serum; DME, Dulbecco's 1This work was supported in part by USPHS Grant CA 13410 awarded by modification of Eagle's medium; FHS, female human serum; CD, charcoal- the National Cancer Institute. dextran stripping treatment; IET, 100 ng insulin, 100 ng epidermal growth factor, 1To whom requests for reprints should be addressed. and 2 ¿ig transferrin per ml Dulbecco's modification of Eagle's medium. 2271 Research. on January 11, 2016. © 1986 American Association for Cancer cancerres.aacrjournals.org Downloaded from