MCF-7 breast tumor cells form multicellular foci in vitro when supplemented with 17P-estradiol (E2). In the presence of E2 and the aryl hydrocarbon-receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), MCF-7 cells grow to confluence but do not form foci. To investigate the role of E2 metabolism in this antiestrogenic effect of TCDD, analyses were performed by capillary GC/MS. The results revealed that pretreatment of MCF-7 cultures with TCDD (10 nM) rapidly depletes E2. In untreated cultures supplemented with 10 nM E2, the concentration of free E2 decreased to 4 nM in the first 12 hr, followed by a slower rate of decline. After 3 days most E2 in the medium was in conjugated form(s); 1.7 nM was present as free E2, and 2.9 nM was released by treatment with glucuronidase/sulfatase. In TCDD-treated cultures, E2declined to 290 pM in 12 hr and after 2 days was not detected (<100 pM) either as free steroid or after treatment with glucuronidase/sulfatase. Intracellular E2 and estrone were likewise depleted by pretreatment with TCDD. Microsomes from TCDD-treated cells showed highly elevated aryl hydrocarbon-hydroxylase activity and catalyzed hydroxylations ofE2 at C-2, C-4, C-15a, and C-6a with a combined rate of 0.85 nmol/min per nmol of cytochrome P-450 at saturating E2.These results suggest that depletion of E2 by enhanced metabolism accounts for the antiestrogenic activity of TCDD in MCF-7 cells.The estrogen dependence of many breast tumors has been the basis of strategies for the treatment of breast cancer. Historically, ablative procedures such as ovariectomy, adrenalectomy, and hypophysectomy have been employed in treating human breast carcinomas. Currently, antiestrogens are widely used in the adjuvant treatment of the disease. Tamoxifen, which is in clinical use (1), exerts its antiestrogenic effects by antagonism of 173-estradiol (E2) binding to the estrogen receptor. Aromatase inhibitors such as 4-hydroxyandrostenedione are also used in endocrine therapy (2). Recent studies indicate that antiestrogenic activity is associated with another group of compounds. Certain aromatic hydrocarbons, chlorinated dioxins, and chlorinated dibenzofurans that bind to the aryl hydrocarbon (Ah) receptor exhibit antiestrogenic activity (3-7). The endogenous ligand for the Ah receptor is unknown, and the function of the receptor remains speculative; however, it is established that activation of the Ah locus in mice initiates transcription of several phase I and phase II enzymes, including cytochromes P-450 of the IA class, UDP glucuronosyltransferase, and glutathione transferase (8). A possible mechanism for the antiestrogenic effects of Ah-receptor agonists is that they promote increased metabolism of E2 by hepatic cytochromes P-450 (4). Alternatively, some investigators suggest that antiestrogenic effects of chlorinated dioxins and dibenzofurans are independent of cytochrome P-450 induction (3, 5, 7) and are mediated by a downregulation of the estrogen receptor (5, 7).MCF-7 is a stable cell line derived from a met...

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.