Estrogen receptor in a human cell line (MCF-7) from breast carcinoma.

Abstract A stable cell line (MCF-7) derived by pleural effusion from a breast cancer patient has been demonstrated to contain significant amounts of 17β-estradiol receptor. This binding protein is specific for 17β-estradiol and has a Kd equal to 2.5 nm, a sedimentation constant of 4.0 S (and 9.2 S), and a mechanism capable of transporting the 17β-estradiol into the nucleus.

Michigan 48901 SUMMARY A stable cell line (MCF- 7) derived by pleural effusion from a breast cancer patient has been demonstrated to contain significant amounts of 17fi-estradiol receptor. This binding protein is specific for 17@-estradiol and has a K, equal to 2.5 nM, a sedimentation constant of 4.0 S (and 9.2 S), and a mechanism capable of transporting the 17P-estradiol into the nucleus.
Considerable work, carried out in recent years, has culminated in elucidation of the receptor mechanism for the stimulation of "target" tissues by steroid hormones (l-4). This knowledge recently has given investigators greater insight into the molecular events involved in hormonal control of normal and neoplastic target tissues (5-7).
Present understanding regarding the estrogen receptor has been acquired through in vivo studies and by short term in vitro incubations of tissue slices or cell fractions. JIYc would like to report herein the first demonstration of 17/S estradiol binding protein in a stable cell line.
The primary culture of human breast carcinoma cells was obtained originally by pleural effusion from a female patient with metastatic disease. A stable epithelioid cell line, MCF-7, was derived from free-floating passages and had been maintained through 71 weekly subcultivations.
The cells were cultured in Eagle's minimal essential medium supplemented with nonessential amino acids and 20 pg per ml of insulin prepared in Hanks' salt solution.
All media contained 250 units of penicillin and 250 pg of streptomycin per ml and were made 10% with respect to calf serum. Details of culturing and cell morphology will be published elsewhere (8).
For the experiments described herein, cells were inoculated into closed plastic containers (Falcon T-75) and allowed to grow into a conlluent monolayer (approximately 20 X lo6 cells per bott'le, 15 to 21 days). Cells from passages 71 through 87 were used in these investigations.
The microsome-free supernatant fraction prepared from these cells contained a significant number of 17@-estradiol binding sites (6.3 X 1OW pmole per mg of protein, Fig. 1 receptor. The absence of estrogen binding protein in these cells from a tumor of a nontarget tissue indicates that prolonged maintenance in cell culture did not generate receptor. This was true in spite of constant exposure to the plasma hormones found in calf serum (12).
The 17fi-estradiol binding protein was somewhat labile to extended storage of cells at -70".
For example, storage for 36 days reduced the number of binding sites in the experiment described in Fig. 2 to approximately onc-third (2.7 x 10W2 pmole of 17,Kestradiol per mg of protein) that of an aliquot of the same cells stored for 14 days and utilized in the studies described in Fig. 1. Although this decrease in picomoles of 17/3estradiol bound per mg of protein occurred, the binding constant of the remaining receptor would be expected to be unchanged (5). The Scatchard plot which resulted from analysis of nine aliquots of the microsome-free supernatant fraction from frozen cells is shown in Fig. 2 binding of 17P-estradiol by Scphadex G-25 column material (9). The lco was found to be 2.5 nM which is comparable to the dissociation constants determined by others for estrogen receptor in human breast tumors (5). 1 he specificity of the binding for 1 Tfi-estradiol is shown in Table I. Preincubation of cytosol from cells with a 103-fold excess of progesterone did not depress the binding by tritiated 17P-estradiol (1.8 nM). However, prior exposure to unlabeled 17fi-estradiol at lo2 and lo3 times the concentration of tritiated estrogen significantly decreased radioactivity in the receptor complex.
1 his inhibition was observed also in experiments with high concentration of a specific estrogen blocking agent, UllJOOA (13). While sediment.ation constants reported for the complex vary according to conditions of the experiment, most of the values obtained for tissues homogenized in Tris buffer with high or low KC1 concentrations are near 4 and 9, respectively (14). The density gradient pattern of cytoplasmic l'ifi-estradiol binding protein derived from the incubation of MCF-7 cells in Krebs-Ringer bicarbonate buffer with 20 no tritiated 17P-estradiol showed peaks at 4.0 and 9.2 S (Fig. 3). Although incubated in bicarbonate buffer salts, these cells were homogenized and centrifuged through a gradient in Tris-EDTA buffer containing no KCl.
The two sedimentation constants found for the estrogen   ever, many of the experiments carried out to demonstrate uptake of the receptor complex by nuclei are presently in doubt due to evidence of posthomogenization binding of extracellular tritiated l'ifi-estradiol by released cytoplasmic receptor (17). In Table  II experiments are presented which utilized a wash with a 102-fold  excess of nonlabeled 17,B-estradiol prior to homogenization of the tissue, cells, or nuclei.
The results clearly show migration of cytoplasmic 17P-estradiol receptor complex into the nuclei during incubation at 37". Both porcine uterine nuclei and nuclei from RICF-7 cells exhibited appreciable nuclear uptake after 1 hour at 0"; a similar observation has been reported in the recent publication of Williams and Gorski (17).
These experiments demonstrated the presence of significant amounts of 17fi-estradiol binding protein in a stable cell line derived from a human breast tumor.
As previously reported for the receptor in human tumors, the estrogen binding protein from MCF-7 has a Ku equal to 2.5 no, a sedimentation constant of 4.0 S (and 9.2 S), and a mechanism capable of transporting the 17P-estradiol complex into the nucleus.
Utilizing in viva experimentation and short term incubations, it previously has not been possible to investigate the induction of steroid receptor protein with strictly controlled environmental exposure to hormones and nutrients.
With cell culture techniques these investigations are now feasible; and, in addition, a stable cell line will permit experiments to be carried out which will add to the present knowledge regarding intracellular binding constants, transport mechanisms, and the mode of nuclear uptake.