-
Endocrinology 06/1981; 108(5):1703-9. · 4.46 Impact Factor
-
Journal of Biological Chemistry 11/1980; 255(20):9699-705. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In MCF-7 human breast cancer cells about 75% of the cell's total estrogen receptor sites were unexpectedly found in nuclei not bound with estrogens [13]. These findings were based on studies with crude nuclei and it was originally proposed that these unfilled nuclear estrogen receptors might be biologically active in the absence of estrogen [23]. In order to examine the subcellular distribution of receptor more critically, we have prepared highly purified MCF-7 nuclei, assessed their purity by electron microscopy and ratios, and compared their estrogen receptor content with less pure subcellular fractions. In estrogen-withdrawn cells the percentage of the total unfilled receptors found in nuclear fractions (Rn) decreased dramatically from 30% (1.81 pmol/mg DNA) in the crudest preparation to only 3% (0.19 pmol/mg DNA) in highly purified nuclei. (This decrease is not due to an irreversible loss of receptor sites, as receptors lost from nuclei were recovered in the cytosol.) By contrast, after 1 h incubation in vivo with estradiol, the number of estrogen-filled receptor sites (RnE) found in the crudest nuclear preparation (3.28 pmol/mg DNA) and in the most highly purified nuclei (2.66 pmol/mg DNA) were nearly the same. It is very likely that the high level of unfilled receptors found in crude nuclei is a contamination of the preparation with cytoplasmic receptor. 1.1. Electron microscopy shows gross contamination of crude nuclei with whole cells and cytoplasmic attachments. Purification of nuclei removes attaching cytoplasm which is parallelled by a dramatic decrease in unfilled receptor sites contained in the nuclear preparations.2.2. Multiple extractions with Tris buffer (without KCl) releases as much as 72% of the receptor contained in crude nuclei and treatment with 0.2% Triton X-100 extracts more than half.3.3. Tris-extractable receptor from crude nuclei sediments identically with cytosol receptor as a single 8S peak on low salt sucrose gradients.4.4. Unfilled receptor does not bind in vitro to purified nuclei.Although we cannot entirely exclude the possibility that unfilled receptors are nuclear in origin but may leak from nuclei during cell fractionation, these results are consistent with the localization of unfilled receptor largely in the cytoplasm of MCF-7 cells, as found in normal target tissues such as the rat uterus.
Experimental Cell Research 06/1980; 127(1):197-213. · 3.58 Impact Factor
-
Endocrinology 05/1979; 104(4):1007-12. · 4.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Estrogen antagonists are widely used in the treatment of breast cancer, and studies of their mechanism of action may provide clues to an understanding of tumor growth regulation and mechanisms of normal estrogen action. We have used human breast cancer cells in long term culture as an in vitro model to study the roles of estradiol and the antiestrogens, tamoxifen and nafoxidine, on cell growth and progesterone receptor (PgR) induction. Tamoxifen is found to have dual dose-dependent estrogenic/antiestrogenic properties. With 1 micrometer tamoxifen, cell growth and PgR induction are suppressed. These effects are reversed by estradiol. At lower doses (less than 0.1 micrometer), however, tamoxifen is a potent estrogen and rapidly induces (24--48 h) PgR, which increases 4- to 10-fold after 4--6 days and falls if tamoxifen is removed. Induction of PgR by estradiol is weaker but follows a similar time course. Tamoxifen-induced PgR is similar to that induced by estradiol; it sediments at 8S on sucrose density gradients, is a tight binder (R5020 Kd, 1.7 micrometer at 4 C and 0.87 nM at 15 C), and can be translocated to the nucleus by R5020. The dual properties of tamoxifen are not due to metabolic formation of an active antiestrogen from a prohormone precursor. In contrast, the action of the antiestrogen nafoxidine is not biphasic in MCF-7 cells; it does not induce PgR over a wide dose range and at high doses, the compound inhibits cell growth.
Endocrinology 12/1978; 103(5):1742-51. · 4.46 Impact Factor
-
Journal of Biological Chemistry 12/1978; 253(22):8185-91. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The interactions of phytoestrogens with estrogen receptors were studied in the human breast cancer cell line, MCF-7. The compounds tested were coumestrol, genistein, and formononetin and the mycotoxins, zearalenone and its reduced derivative, zearalenol. All but formononetin compete for binding of [3H]-estradiol to unfilled cytoplasmic estrogen receptor or unfilled nuclear estrogen receptor sites. Relative binding affinities are zearalenol HMP (high melting point isomer) greater than zearalenol LMP (low melting point isomer) greater than zearalenone = coumestrol greater than genistein greater than formononetin. Dissociation constants estimated from competition curves show that binding affinities are high. In contrast to estradiol, phytoestrogens bind only weakly to sex steroid-binding globulin; they also do not bind to corticosteroid-binding globulin. These compounds translocate the cytoplasmic estrogen receptor and bind to unfilled nuclear estrogen receptors in whole cells. Bound nuclear receptors are then processed in a manner similar to estradiol in a step which rapidly decreases total cellular estrogen receptors. The phytoestrogens are also biologically active; they can markedly enhance tumor cell proliferation. In sum, phytoestrogens interact with the estrogen receptors of human breast cancer cells in culture and, therefore, may affect estrogen-mediated events in these cells.
Endocrinology 12/1978; 103(5):1860-7. · 4.46 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: This communication describes a novel effect of actinomycin D (AcD) in inhibiting the nuclear processing or turnover of estrogen receptors in the human breast cancer cell line. MCF-7. In the absence of AcD, estradiol treatment results in rapid (5 min) hormone binding and translocation of unfilled cytoplasmic receptors (Rc) and binding of unfilled nuclear receptors (Rn). Thereafter, filled nuclear receptors (RnE) progressively deplete and, by 3 to 5 h, 70% are lost or processed. We now show that 1 to 2 micrometer AcD or chromomycin A3, both of which intercalate at G-C base-pairs on DNA, selectively and completely block RnE processing. In contrast, estrogen binding, translocation of receptor complex, and RnE accumulation in the nucleus are completely insensitive to AcD inhibition. At 1 to 2 micrometer, all other intercalators and inhibitors tested, including other inhibitors of transcription and replication, or inhibitors of translocation or of other functions, fail to prevent binding, translocation, or the nuclear processing step. AcD intranslocation, or the nuclear processing step. AcD inhibition of RnE processing is dependent on dose; at lower doses (100 nM decreasing to 1 nm), progressively greater RnE depletion occurs. AcD completely prevents RnE depletion if given together with or within 30 min after estradiol; at any time between 30 min and 5 h after estradiol, the processing of RnE is stopped instantly by addition of AcD. Because of the complexity of actinomycin action, several mechanisms can be proposed to explain its effect on nuclear ER levels.
Journal of Biological Chemistry 10/1978; 253(18):6319-22. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Nine human breast cancer cell lines in permanent tissue culture and currently available to researchers have been assayed for their content of cytoplasmic estrogen receptors, progesterone receptors, androgen receptors, and glucocorticoid receptors, as well as for the presence of unfilled or hormone-filled nuclear estrogen receptors. Receptor distribution varied considerably among the nine lines and differed from the expected distribution predicted from solid tumors. We find that estrogen receptor, when present, is usually localized in the nucleus as unfilled nuclear estrogen receptor. Progesterone receptor is correlated with presence of unfilled nuclear estrogen receptor. Glucocorticoid receptors are ubiquitous; they were found in all cell lines tested. The distribution of androgen receptor and progesterone receptor differed, suggesting that these proteins are dissimilar.
Cancer Research 09/1978; 38(8):2434-7. · 7.86 Impact Factor
-
Journal of Steroid Biochemistry 06/1978; 9(5):461-6.
-
Journal of Biological Chemistry 05/1978; 253(7):2223-8. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The role of cytoplasmic estrogen receptor (ER) assays in determining therapeutic strategies for advanced breast cancer is certainly well established. The use of ER assays in the primary breast tumor specimen to predict for early recurrence and ultimate survival is a new finding, however, and will probably be employed in future trials of adjuvant therapy. The prevalence and significance of nuclear-bound ER still requires additional clarification. Our previous suggestion that progesterone receptor measurements might be a useful marker for hormone dependence in advanced breast cancer is gaining support and may soon have a place in routine therapeutic decision-making. The emphasis on early adjuvant therapy has hastened the search for a safe endocrine therapy that would have good patient compliance and achieve remission rates comparable to previous agents and procedures. Antiestrogens show promise of meeting these requirements. We are now beginning an era in which primary and secondary systemic therapies for breast cancer can be based on sound biologic principles. The empirical approach is outdated.
Metabolism 05/1978; 27(4):487-501. · 2.66 Impact Factor
-
Current topics in experimental endocrinology 02/1978; 3:93-129.
-
[show abstract]
[hide abstract]
ABSTRACT: The human breast cancer cell line MCF-7 does not require estrogen for growth, but paradoxically its growth is inhibited by antiestrogens. Our results show that, unlike normal target cells, MCF-7 cells carry most of their estrogen receptors in their nuclei even when these receptors are not charged with estrogens. The receptors for androgen and for progesterone, on the other hand, are localized in the cytoplasm as usual. Therefore, it is possible that the growth of these abnormal cells is stimulated by estrogen receptor in spite of the absence of the hormone and that the binding of antiestrogen molecules antagonize this stimulation.
Science 06/1977; 196(4290):663-4. · 31.20 Impact Factor
-
Pathobiology annual 02/1977; 7:191-211.
-
[show abstract]
[hide abstract]
ABSTRACT: We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor (measured with R5020-3H), about 40 fm/mg protein 5alpha-dihydrotestosterone (5alpha-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 x 10(-10)M (E2), 1 x 10(-9)M (R5020), 2.8 x 10(-10)M (5alpha-DHT) and 8 x 10(-9)M (dexamethasone). No cross competition was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent in vitro model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.
Steroids 01/1976; 26(6):785-95. · 2.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We have identified a specific progesterone receptor in 11 of 33 human breast cancer cytosols. Since progesterone itself binds to glucocorticoid receptor, to corticosteroid binding globulin (CBG), and to nonspecific components as well as to its own receptor, we have used a synthetic progestin, R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), whose binding specificity is restricted to progesterone receptor. Bound R5020 sediments at 8 S in sucrose gradients; binding is competed by excess unlabeled R5020 or progesterone. The receptor is distinct from glucocorticoid receptor and CBG as determined by competition studies using dexamethasone and hydrocortisone. The dissociation constant for R5020 obtained by Scatchard analysis of dextran-coated charcoal assays is approximately 2 times 10- minus 9 M.
Steroids 05/1975; 25(4):497-505. · 2.83 Impact Factor
-
Journal of Steroid Biochemistry 7(11-12):875-82.
-
[show abstract]
[hide abstract]
ABSTRACT: We have identified receptors for glucocorticoids, progestins, and androgens in a human breast tumor cell line (MCF-7) known to have estrogen receptor. Sucrose density gradients show that MCF-7 cytosol contains approximately 100 fm/mg protein estradiol (E2-3H) receptor, more than 300 fm/mg protein progesterone receptor [measured with R5020-3H), about 40 fm/mg protein 5α-dihydrotestosterone (5α-DHT-3H) receptor, and 800 fm/mg glucocorticoid receptor (measured with dexamethasone-3H). Dissociation constants obtained by Scatchard analyses were approximately 0.6 × 10−10M (E2), 1 × 10−9M (R5020), 2.8 ×10−10M (5α-DHT) and 8 × 10−9M (dexamethasone). No cross competi- tion was found for estrogen receptor, but progestins competed for androgen and glucocorticoid binding. The androgen, but not the glucocorticoid, partially competed for R5020 binding to progesterone receptor. This first demonstration of 4 classes of steroid receptors in human breast cancer means that MCF-7 may be an excellent model for studying the mechanism of tumor response to endocrine therapy as well as the complex relationships between binding and biological actions of these hormones.
Steroids.
-
[show abstract]
[hide abstract]
ABSTRACT: Breast cancer is often hormone responsive, since growth or regression of tumors can often be modulated by appropriate endocrine manipulations. Estrogen and progesterone are major hormones involved in regulation of breast cancer tumor growth. Considerable insight into the mechanism of action of these hormones on tumor growth stimulation has been provided by demonstration of specific receptors for each. The inference that each hormone acts independently through its receptor to control tumor growth is belied by current studies which show that certain hormones are capable of regulating the receptor sites, metabolism, or nuclear translocation of others. This may begin to explain the complex hormonal interactions and requirements of normal and neoplastic breast tissues. Considerable progress has thus been made in understanding the basis for success of various ablative therapies.The pharmacologie actions of estrogens and progestins in causing breast tumor regression is much less well understood. The role of hormone receptor sites has not been established in the mechanism of tumor regression caused by these pharmacological therapies. Nevertheless, when estrogen receptors are absent in a tumor, we can with accuracy pre.dict that endocrine therapies will fail, whereas when ER is present the likelihood of a successful response to pharmacological or ablative therapy is high.Receptor sites seem to be a common denominator and useful marker for hormone dependence or hormone responsiveness, irrespective of their actual role in the tumor regression process. Further investigations into the receptor functions should lead to new approaches in the endocrine management of patients with breast cancer.
Journal of Steroid Biochemistry.
