Breast Cancer Research and Treatment

, Volume 5, Issue 3, pp 231–243 | Cite as

Antiestrogen action in breast cancer cells: Modulation of proliferation and protein synthesis, and interaction with estrogen receptors and additional antiestrogen binding sites

  • Benita S. Katzenellenbogen
  • Margaret Ann Miller
  • Alaka Mullick
  • Yhun Yhong Sheen
Seventh Annual San Antonio Breast Cancer Symposium


Antiestrogens have proven to be effective in controlling the growth of hormone-responsive breast cancers. At the concentrations of antiestrogens achieved in the blood of breast cancer patients taking antiestrogens (up to 2 × 10−6 M), antiestrogens selectively inhibit the proliferation of estrogen receptor-containing breast cancer cells, and this inhibition is reversible by estradiol. Antiestrogens also inhibit estrogen-stimulation of several specific protein synthetic activities in breast cancer cells, including increases in plasminogen activator activity, progesterone receptor levels and production of several secreted glycoproteins and intracellular proteins.

Antiestrogens bind with high affinity to the estrogen receptor and to additional microsomal binding sites to which estrogens do not bind. These latter sites, called antiestrogen binding sites (AEBS), are present in equal concentrations in estrogen receptor-positive and -negative breast cancer cells and are present in a wide variety of tissues, with highest concentrations being found in the liver. The antiestrogenic and growth suppressive potencies of a variety of antiestrogens correlate best with their affinity for estrogen receptor and not with affinity for AEBS.

Antiestrogens undergo bioactivation and metabolismin vivo and hydroxylated forms of the antiestrogen have markedly enhanced affinities for the estrogen receptor. Detailed studies with high affinity radiolabelled antiestrogens indicate that antiestrogens induce important conformational changes in receptor that are reflected in the enhanced maintenance of a 5 S form of the estrogen receptor complex; reduced interaction with DNA; and altered activation and dissociation kinetics of the antiestrogen-estrogen receptor complex. These conformational changes effected by antiestrogens likely result in different interactions with chromatin, causing altered cell proliferation and protein synthesis.

Analyses of the rates of synthesis and turnover of the estrogen receptor through pulse-chase experiments utilizing the covalently attaching antiestrogen, tamoxifen aziridine, and studies employing dense amino acid labeling of estrogen receptor reveal that the antiestrogen-occupied receptor is degraded at a rate (t 1/2 = 4 h) similar to that of the control unoccupied receptor. Hence, antiestrogens do not prevent estrogen receptor synthesis and they do not either accelerate or block estrogen receptor degradation.

Our findings raise serious doubts about the role of the AEBS in mediating directly the growth suppressive actions of antiestrogens, and suggest that interaction with the estrogen receptor is most likely the mechanism underlying the growth-inhibitory effects of antiestrogens. At present, the role of the AEBS in the actions of antiestrogens or in possible antiestrogen metabolism remains unclear.


antiestrogens antiestrogen binding sites breast cancer estrogen receptors growth inhibition mechanism of antiestrogen action tamoxifen 


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  1. 1.
    Bloom HJG, Boesen E: Antioestrogens in treatment of breast cancer: value of nafoxidine in 52 advanced cases. Br Med J 2: 7–10, 1974Google Scholar
  2. 2.
    Heuson JC, Englesman E, Blonk-Van der Wijst J, Maass H, Drochmans A, Michel J, Nowakowski H, Gorins A: Comparative trial of nafoxidine and ethinyloestradiol in advanced breast cancer: a European Organization for Research on Treatment of Cancer study. Br Med J 2: 711–713, 1975PubMedGoogle Scholar
  3. 3.
    Horwitz KB, McGuire WL: Antiestrogens: mechanism of action and effects in breast cancer. In: McGuire WL (ed): Breast Cancer Advances in Research and Treatment, vol. 2. Plenum Press, New York, pp 155–204, 1978Google Scholar
  4. 4.
    McGuire WL: Steroid receptor sites in cancer therapy. Adv Intern Med 24: 127–140, 1979PubMedGoogle Scholar
  5. 5.
    Tormey DC, Simon RM, Lippman ME, Bull JM, Meyers CE: Evaluation of tamoxifen dose in advanced breast cancer: a progress report. Cancer Treat Rep 60: 1451–1459, 1976PubMedGoogle Scholar
  6. 6.
    Coezy E, Borgna J-L, Rochefort H: Tamoxifen and metabolites in MCF-7 cells: correlation between binding to estrogen receptor and inhibition of cell growth. Cancer Res 42: 317–323, 1982PubMedGoogle Scholar
  7. 7.
    Eckert RL, Katzenellenbogen BS: Physical properties of estrogen receptor complexes in MCF-7 human breast cancer cells: differences with antiestrogen and estrogen. J Biol Chem 257: 8840–8846, 1982PubMedGoogle Scholar
  8. 8.
    Eckert RL, Katzenellenbogen BS: Effects of estrogens and antiestrogens on estrogen receptor dynamics and the induction of progesterone receptor in MCF-7 human breast cancer cells. Cancer Res 42: 139–144, 1982PubMedGoogle Scholar
  9. 9.
    Katzenellenbogen BS, Bhakoo HS, Ferguson ER, Lan NC, Tatee T, Tsai TL, Katzenellenbogen JA: Estrogen and antiestrogen action in reproductive tissues and tumors. Recent Prog Horm Res 35: 259–300, 1979.PubMedGoogle Scholar
  10. 10.
    Katzenellenbogen BS, Pavlik EJ, Robertson DW, Katzenellenbogen JA: Interaction of a high affinity antiestrogen (α-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-α′-nitro-stilbene, CI628M) with uterine estrogen receptors. J Biol Chem 256: 2980–2915, 1981Google Scholar
  11. 11.
    Lippman M, Bolan G, Huff K: The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture. Cancer Res 36: 4595–4601, 1976PubMedGoogle Scholar
  12. 12.
    Horwitz KB, McGuire WL: Estrogen control of progesterone receptor in human breast cancer. J Biol Chem 253: 2223–2228, 1978PubMedGoogle Scholar
  13. 13.
    Katzenellenbogen BS, Norman MJ, Eckert RE, Peltz SW, Mangel WF: Bioactivities, estrogen receptor interactions, and plasminogen activator-inducing activities of tamoxifen and hydroxy-tamoxifen isomers in MCF-7 human breast cancer cells. Cancer Res 44: 112–119, 1984PubMedGoogle Scholar
  14. 14.
    Faye JE, Lassere B, Bayard F: Antiestrogen specific high affinity saturable binding sites in rat uterine cytosol. Biochem Biophys Res Commun 93: 1225–1231, 1980PubMedGoogle Scholar
  15. 15.
    Gulino A, Pasqualini JR: Heterogeneity of binding sites for tamoxifen and tamoxifen derivatives in estrogen target and nontarget fetal organs of guinea pig. Cancer Res 42: 1913–1921, 1982PubMedGoogle Scholar
  16. 16.
    Murphy LC, Sutherland RL: Modifications in the aminoether side chain of clomiphene influence affinity for a specific antiestrogen binding site in MCF-7 cell cytosol. Biochem Biophys Res Commun 100: 1353–1369, 1981PubMedGoogle Scholar
  17. 17.
    Sudo K, Monsma FJ Jr, Katzenellenbogen BS: Antiestrogen binding sites distinct from the estrogen receptor: subcellular localization, ligand specificity, and distribution in tissues of the rat. Endocrinology 112: 425–434, 1983PubMedGoogle Scholar
  18. 18.
    Sutherland RL, Foo MS: Differential binding of antiestrogens by rat uterine and chick oviduct cytosol. Biochem Biophys Res Commun 92: 183–191, 1979Google Scholar
  19. 19.
    Sutherland RL, Foo MS, Green MD, Waybourne AM, Krozowski ZS: High affinity antiestrogen binding site distinct from the estrogen receptor. Nature (Lond) 288: 273–275, 1980Google Scholar
  20. 20.
    Sutherland RL, Murphy LC: The binding of tamoxifen to human mammary carcinoma cytosol. Eur J Cancer 16: 1141–1148, 1980PubMedGoogle Scholar
  21. 21.
    Miller MA, Katzenellenbogen BS: Characterization and quantitation of antiestrogen binding sites in estrogen recep-tor-positive and -negative human breast cancer cell lines. Cancer Res 43: 3094–3100, 1983PubMedGoogle Scholar
  22. 22.
    Katzenellenbogen JA, Tatee T, Robertson DW: Preparation of tritium-labeled 4-hydroxy-α-[p-(2-(N-pyrro-lidinyl-ethoxy-phenyl]-α′-nitrostilbene (CN-928), a biolog-ically-important metabolite of the antiestrogen CI628. J Labelled Comp Radiopharm 18: 865–879, 1981Google Scholar
  23. 23.
    Robertson DW, Katzenellenbogen JA: Synthesis of the E and Z isomers of the antiestrogen tamoxifen and its metabolite, hydroxytamoxifen, in tritium-labeled form. J Org Chem 47: 2387–2393, 1982Google Scholar
  24. 24.
    Keene J, Sweet F, Ruh MF, Ruh TS: Interaction of the radiolabeled high affinity antiestrogen [3H]H1285 with the cytoplasmic oestrogen receptor. Biochem J 217: 819–826, 1984PubMedGoogle Scholar
  25. 25.
    Sutherland RL, Green MD, Hall RE, Reddel RR, Taylor IW: Tamoxifen induces accumulation of MCF-7 human mammary carcinoma cells in the G0-G1 phase of the cell cycle. Europ J Cancer Clin Oncol 19: 615–621, 1983PubMedGoogle Scholar
  26. 26.
    Osborne CK, Boldt DH, Estrada P: Human breast cancer cell cycle synchronization by estrogens and antiestrogens in culture. Cancer Res 44: 1433–1439, 1984PubMedGoogle Scholar
  27. 27.
    Butler WB, Kirkland WL, Jorgensen TL: Induction of plasminogen activator by estrogen in human breast cancer cell line (MCF-7). Biochem Biophys Res Commun 90: 1328–1334, 1979PubMedGoogle Scholar
  28. 28.
    Huff KK, Lippman ME: Hormonal control of plasminogen activator secretion in ZR-75-1 human breast cancer cells in culture. Endocrinology 114: 1702–1710, 1984PubMedGoogle Scholar
  29. 29.
    Ryan TJ, Seeger JI, Kumar SA, Dickerman HW: Estradiol preferentially enhances tissue plasminogen activators of MCF-7 breast cancer cells. J. Biol Chem 259: 14324–14327, 1984PubMedGoogle Scholar
  30. 30.
    Horwitz KB, McGuire WL: Estrogen control of progesterone receptor in human breast cancer. J Biol Chem 253: 2223–2228, 1978PubMedGoogle Scholar
  31. 31.
    Westley B, Rochefort H: A secreted glycoprotein induced by estrogen in human breast cancer cell lines. Cell 20: 353–362, 1980PubMedGoogle Scholar
  32. 32.
    Edwards DP, Adams DJ, McGuire WL: Estradiol stimulates synthesis of a major intracellular protein in a human breast cancer cell line (MCF-7). Breast Cancer Res Treat 1: 209–223, 1981PubMedGoogle Scholar
  33. 33.
    Katzenellenbogen BS, Miller MA, Eckert RL, Sudo K: Antiestrogen pharmacology and mechanism of action. J Steroid Biochem 19: 59–68, 1983PubMedGoogle Scholar
  34. 34.
    Adam HK: Review of pharmacokinetics and metabolism of ‘Nolvadex’ (tamoxifen). In Sutherland RL, Jordan VC (eds): Non-Steroidal Antiestrogens. Academic Press, Sydney, Australia, p 59–74, 1981Google Scholar
  35. 35.
    Robertson DW, Katzenellenbogen JA, Long DJ, Rorke EA, Katzenellenbogen BS: Tamoxifen antiestrogens. A comparison of the activity, pharmacokinetics, and metabolic activation of the cis and trans isomers of tamoxifen. J Steroid Biochem 16: 1–13, 1982PubMedGoogle Scholar
  36. 36.
    Miller MA, Greene GL, Katzenellenbogen BS: Estrogen receptor transformation in MCF-7 breast cancer cells: characterization by immunochemical and sedimentation analyses. Endocrinology 114: 296–298, 1984PubMedGoogle Scholar
  37. 37.
    Miller MA, Mullick A, Katzenellenbogen BS: Crosslinking and density shift experiments to study the subunit nature of the 5S nuclear estrogen receptor complex in MCF-7 breast cancer cells. Proceedings, VII International Congress of Endocrinology, Quebec, Abstract 1599, p. 1060, 1984Google Scholar
  38. 38.
    Evans E, Baskevitch PP, Rochefort H: Estrogen receptor-DNA interaction: difference between activation by estrogen and antiestrogen. Europ J Biochem 128: 185–191, 1982PubMedGoogle Scholar
  39. 39.
    Horwitz KB, McGuire WL: Nuclear mechanisms of estrogen action. Effects of estradiol and antiestrogens on estrogen receptors and nuclear receptor processing. J Biol Chem 253: 8185–8191, 1978PubMedGoogle Scholar
  40. 40.
    Ruh TS, Baudendistel LJ: Antiestrogen modulation of the salt-resistant form of the nuclear estrogen receptor. Endocrinology 102: 1838–1846, 1978PubMedGoogle Scholar
  41. 41.
    Rochefort H, Borgna JL: Differences between the activation of the oestrogen receptor by oestrogen and by antioestrogen. Nature 292: 257–259, 1981PubMedGoogle Scholar
  42. 42.
    Sasson S, Notides AC: Inhibition of the estrogen receptor's positive cooperative [3H]estradiol binding by the antagonist, clomiphene. J Biol Chem 257: 11540–11545, 1982PubMedGoogle Scholar
  43. 43.
    Tate AC, Greene GL, DeSombre ER, Jensen EV, Jordan VC: Differences between estrogen- and antiestrogen-estrogen receptor complexes from human breast tumors identified with antibody raised against the estrogen receptor. Cancer Res 44: 1012–1018, 1984PubMedGoogle Scholar
  44. 44.
    Katzenellenbogen JA, Carlson KE, Heinman DF, Robertson DW, Wei LL, Katzenellenbogen BS: Efficient and highly selective covalent labeling of the estrogen receptor with [3H]tamoxifen aziridine. J Biol Chem 258: 3487–3495, 1983PubMedGoogle Scholar
  45. 45.
    Monsma FJ Jr, Katzenellenbogen BS, Miller MA, Ziegler YS, Katzenellenbogen JA: Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently-attaching antiestrogen. Endocrinology 115: 143–153, 1984PubMedGoogle Scholar
  46. 46.
    Eckert RL, Mullick A, Rorke EA, Katzenellenbogen BS: Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique. Endocrinology 114: 629–637, 1984PubMedGoogle Scholar
  47. 47.
    Scholl S, Lippman ME: The estrogen receptor in MCF-7 cells: evidence from dense amino acid labeling for rapid turnover and a model of activated nuclear receptor. Endocrinology 115: 1295–1302, 1984PubMedGoogle Scholar
  48. 48.
    Kon OL: An antiestrogen-binding protein in human tissues. J Biol Chem 258: 3173–3177, 1983PubMedGoogle Scholar
  49. 49.
    Adelman MR, Blobel G, Sabatini DD: Nondestructive separation of rat liver rough microsomes into ribosomal and membranous components. Methods Enzymol 31: 201–215, 1974PubMedGoogle Scholar
  50. 50.
    Watts CKW, Sutherland RL: High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver. Biochem Biophys Res Commun 120: 109–115, 1984PubMedGoogle Scholar
  51. 51.
    Watts CKW, Murphy LC, Sutherland RL: Microsomal binding sites for nonsteroidal antiestrogens in MCF-7 human mammary carcinoma cells. Demonstration of high affinity and narrow specificity for basic ether derivatives of triphenylethylene. J Biol Chem 259: 4233–4229, 1984Google Scholar
  52. 52.
    Miller MA, Lippman ME, Katzenellenbogen BS: Antiestrogen binding in antiestrogen growth-resistant estrogenresponsive clonal variants of MCF-7 human breast cancer cells. Cancer Res 44: 5038–5045, 1984PubMedGoogle Scholar
  53. 53.
    Faye JC, Jozan S, Redeuilh G, Baulieu EE, Bayard F: Physicochemical and genetic evidence for specific antiestrogen binding sites. Proc Natl Acad Sci USA 80: 3158–3162, 1983PubMedGoogle Scholar
  54. 54.
    Tatee T, Carlson KE, Katzenellenbogen JA, Robertson DW, Katzenellenbogen BS: Antiestrogens and antiestrogen metabolites: preparation of tritium-labeled U23,469 and characterization and synthesis of a biolog-ically-important metabolite. J Med Chem 22: 1509–1517, 1979PubMedGoogle Scholar
  55. 55.
    Ruenitz PC, Bagley JR, Pope CW: Some chemical and biochemical aspects of liver microsomal metabolism of tamoxifen. Drug Metab and Disposition 12: 478–483, 1984Google Scholar
  56. 56.
    Clark JH, Winneker RC, Guthrie SC, Markaverich BM: Endogenous ligand for the triphenylethylene antiestrogen binding site. Endocrinology 113: 1167–1169, 1983PubMedGoogle Scholar
  57. 57.
    Murphy PR, Butts C, Lazier CB: Triphenylethylene anti-estrogen-binding sites in cockerel liver nuclei: evidence for an endogenous ligand. Endocrinology 115: 420–426, 1984PubMedGoogle Scholar

Copyright information

© Martinus Nijhoff Publishers Publishers 1985

Authors and Affiliations

  • Benita S. Katzenellenbogen
    • 1
  • Margaret Ann Miller
    • 1
  • Alaka Mullick
    • 1
  • Yhun Yhong Sheen
    • 1
  1. 1.Department of Physiology and Biophysics, 524 Burill HallUniversity of Illinois College of Medicine at Urbana-ChampaignUrbanaUSA

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