Eugene R. DeSombre

Estrogen-binding substances of target tissues.

Abstract A variety of evidence supports the concept that the interaction of estradiol with uterine tissue takes place by a two stage mechanism. The hormone associates spontaneously with an extranuclear protein to form an 8S complex, made up of 4S subunits, which possesses a strong affinity for nuclei. In the nucleus a new estradiol-protein complex, extractable as a 5S unit, is formed from the 8S complex by a temperature-dependent process which consumes 8S receptor. The concept of “induced derepression” is proposed to explain how the nuclear transformation of 4S to a 5S estradiol-protein complex might initiate uterine growth.

Receptors Reconsidered: A 20-Year Perspective

Publisher Summary The original discovery of steroid hormone receptors and essentially all information concerning their interaction and function in target cells have depended on experiments in which a radioactive steroid serves as a marker for the receptor protein to which it binds. The female reproductive tissues, such as uterus, vagina, and anterior pituitary, contain a characteristic estrogen-binding component which was first indicated by their striking ability to take up and retain tritiated hexestrol and estradiol after the administration of physiological doses of these substances to immature animals. This chapter discusses the biochemical mechanism by which the reaction of estradiol and other estrogenic hormones with receptor substances elicits hormonal response has been the subject of extensive investigation. The activated steroid-receptor complex is translocated to the nucleus where it binds to chromatin and in some way modulates RNA synthesis which appears to be characteristically restricted in hormone-dependent tissues.

Tissue-specific stimulation of RNA synthesis by transformed estradiol-receptor complex.

Summary Exposure to the estradiol-receptor complex of uterine cytosol increases the RNA synthetic capacity of purified uterine nuclei but not of liver or kidney nuclei. This stimulation is effected only by complex in which the binding unit of the receptor has undergone estrogen-induced conversion from the native (4S) to the transformed (5S) form. It is suggested that an important function of estrogenic hormone is to promote transformation of the receptor protein to an active form which can enter the nucleus and stimulate RNA synthesis.

Estrophile to nucleophile in two easy steps

Abstract A variety of self-consistent experimental evidence supports the concept that estrogcnic hormones interact with their target tissues by a multi-stage mechanism in which the hormone first associates with the 4S binding unit of an 8S extranuclear receptor protein, activating it to undergo conversion to a 5S form. This temperature-dependent transformation takes place when the receptor is bound to estradiol, but not when it is uncomplexed or bound to estrone. The transformed hormone-receptor complex migrates to the nucleus where it associates with an acceptor site in the chromatin. Analytical amounts of the 5S nuclear complex and a calciumstabilized 4S cytosol complex have been isolated from calf uterus in apparently pure state, opening the way for the large scale purification of these different forms of the receptor protein. The estrogen-receptor interaction sequence appears to be involved in the acceleration of biosynthetic reactions in hormone-dependent tissues. Exposure of isolated uterine nuclei either to transformed complex or to native complex under conditions where transformation can take place causes a significant increase in their ability to synthesize RNA. Similar treatment does not enhance the already high RNA synthetic capacity of liver or kidney nuclei. It is suggested that an important function of the hormone is to promote the conversion of the receptor protein to an active form which can enter the nucleus and alleviate a defficiency in RNA synthesis, characteristic of estrogen-responsive tissues.

Structural plasticity in the oestrogen receptor ligand-binding domain

The steroid hormone receptors are characterized by binding to relatively rigid, inflexible endogenous steroid ligands. Other members of the nuclear receptor superfamily bind to conformationally flexible lipids and show a corresponding degree of elasticity in the ligand‐binding pocket. Here, we report the X‐ray crystal structure of the oestrogen receptor α (ERα) bound to an oestradiol derivative with a prosthetic group, ortho‐ trifluoromethlyphenylvinyl, which binds in a novel extended pocket in the ligand‐binding domain. Unlike ER antagonists with bulky side groups, this derivative is enclosed in the ligand‐binding pocket, and acts as a potent agonist. This work shows that steroid hormone receptors can interact with a wider array of pharmacophores than previously thought through structural plasticity in the ligand‐binding pocket.

Hormone dependency in breast cancer.

Hormone deprivation by surgical ablation of endocrine glands affords the most effective treatment presently available for advanced breast cancer in those patients whose tumors are of the hormone-dependent type. Less than half of premenopausal patients and a smaller fraction of postmenopausal patients respond to endocrine ablation. The estrogen receptor (estrophilin) content of an excised specimen of the tumor can provide information useful for selecting optimal therapy. Of 123 women with advanced breast cancer whose without ovarian function who responded to hormone therapy had 750 or more fmol/gm of tumor tissue. Of premenopausal patients only those with 300 fmol or more per gm of tumor tissue responded. Therefore 750 fmol/gm of tumor tissue for postmenopausal and 250 for premenopausal patients have been resported as positive or receptor-rich. Those with less have been clarified as receptor-poor. Nearly 66% of patients whose cancers are reported as receptor-rich for estrophilin can expect to benefit from endocrine therapy. Those with lesser amounts of estrophilin have very little chance of benefiting from hormone therapy and are better treated by chemotherapy. Of more than 1200 women whose breast cancers have been assayed about 30% were classified as receptor-rich and 70% as intermediate or receptor-poor. The simultaneous presence of a progesterone receptor has also been shown to be of importance in some cases. Routinely characterizing primary breast cancers at the time of mastectomy is recommended. Subsequent recurrences or metastases have been shown to have estrophilin content similar to the primary tumor. In a discussion the receptor-rich and receptor-poor classification was thought by some to be misleading. Wider use of antiestrogens was recommended instead of ablative surgery.

Estrophilin assays in breast cancer: Quantitative features and application to the mastectomy specimen

Knowledge of the tumor content of estrogen receptor, called estrophilin, has proved to be of significant clinical value in human breast cancer. Although most breast cancer tissues contain cytosol estrophilin, essentially only patients whose cancers have moderate to high levels of estrophilin, designated estrophilinrich, are found to respond to endocrine therapy. About two‐thirds of patients with estrophilin‐rich cancers obtain objective benefit from endocrine therapy. Present results indicate that predictions of response to endocrine therapy at time of recurrence can be based on the estrophilin assay of the primary lesion. Nonetheless, changes in estrophilin content during the course of disease are not uncommon. Despite an occasional patient in whom multiple samples separated by a long time have unchanged estrophilin content, there is a general tendency toward decreased estrophilin content with time.

Estrogen-independent activation of the receptor protein of calf uterine cytosol

Abstract Precipitation of the estrogen receptor protein of calf uterine cytosol by ammonium sulfate at 2°C, either in the presence or absence of estradiol, is accompanied by its conversion from native (3. 8 S) to a transformed (5. 5 S) state. This product, complexed with estradiol, resembles that previously obtained, either by warming uterine cytosol with estradiol or by extracting nuclei from hormone-treated uteri, in its ability to bind to uterine nuclei and to increase their RNA polymerase activity. The transformed protein, uncomplexed with steroid, also stimulates RNA synthesis, supporting the hypothesis that receptor transformation, effected physiologically by the hormone, plays a key role in estrogen action.

Hormone receptor assays: clinical usefulness in the management of carcinoma of the breast.

The revision of the subcellular model of hormone action is described, with an incorporation of potential autocrine mechanisms. A general overview of available assay methodologies considers the major disadvantages of earlier methods and describes in detail the current methodologies (sucrose gradient analysis, dextran-coated charcoal assays, ER-EIA, ERICA). A major concern with clinical correlations of response to hormone receptor levels is the quality assurance of the multicentric programs. Results from national and international programs are considered. The clinical correlations are divided into four major categories: (1) the response to hormone deprivation (oophorectomy or adrenalectomy), (2) the development of specific agents which exploit receptor mechanisms (antiestrogens) or inhibit steroid biosynthesis (aminoglutehimide), (3) the rates of recurrence of tumors following mastectomy, and (4) the correlation of hormone receptors with current adjuvant therapies.

Identification of Ligands with Bicyclic Scaffolds Provides Insights into Mechanisms of Estrogen Receptor Subtype Selectivity

Estrogen receptors α (ERα) and β (ERβ) have distinct functions and differential expression in certain tissues. These differences have stimulated the search for subtype-selective ligands. Therapeutically, such ligands offer the potential to target specific tissues or pathways regulated by one receptor subtype without affecting the other. As reagents, they can be utilized to probe the physiological functions of the ER subtypes to provide information complementary to that obtained from knock-out animals. A fluorescence resonance energy transfer-based assay was used to screen a 10,000-compound chemical library for ER agonists. From the screen, we identified a family of ERβ-selective agonists whose members contain bulky oxabicyclic scaffolds in place of the planar scaffolds common to most ER ligands. These agonists are 10–50-fold selective for ERβ in competitive binding assays and up to 60-fold selective in transactivation assays. The weak uterotrophic activity of these ligands in immature rats and their ability to stimulate expression of an ERβ regulated gene in human U2OS osteosarcoma cells provides more physiological evidence of their ERβ-selective nature. To provide insight into the molecular mechanisms of their activity and selectivity, we determined the crystal structures of the ERα ligand-binding domain (LBD) and a peptide from the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator complexed with the ligands OBCP-3M, OBCP-2M, and OBCP-1M. These structures illustrate how the bicyclic scaffolds of these ligands are accommodated in the flexible ligand-binding pocket of ER. A comparison of these structures with existing ER structures suggests that the ERβ selectivity of OBCP ligands can be attributed to a combination of their interactions with Met-336 in ERβ and Met-421 in ERα. These bicyclic ligands show promise as lead compounds that can target ERβ. In addition, our understanding of the molecular determinants of their subtype selectivity provides a useful starting point for developing other ER modulators belonging to this relatively new structural class.