Wade V. Welshons

Remodeling the estrogen receptor model

The estrogen receptor model has revised to make it compatible with new data on subcellular localization of the receptor and physical characteristics of solubilized versus immobilized receptors. Our current model suggests that receptors, with or without bound estrogen, are present in the nuclear fraction. Furthermore, the receptor behaves as if it were immobilized or bound to some nuclear constituent at all times. Thus, the association of the estrogen-free receptor (unbound receptor) to a target site in the nucleus is considered to be the critical event in defining the nature of the response to estrogenic hormones.

Adaptation of estrogen-dependent MCF-7 cells to low estrogen (phenol red-free) culture

When human breast cancer-derived MCF-7 cells were maintained in low estrogen medium (phenol red-free), the cells adapted to grow without added estrogen, but growth could be inhibited by antiestrogen in the medium. Estrogen-stimulated progesterone receptor levels remained basal but could be stimulated by estradiol. Estrogen receptor content increased steadily during adaptation, which may model the increasing levels of estrogen receptor observed in breast cancer with increasing patient age. The mechanism of the adaptation to low estrogen medium is unclear; however, cell lines such as MCF-7 may need to be cultured in the presence of an estrogen such as phenol red in order to maintain a stable estrogen-sensitive phenotype. On the other hand, maintenance of estrogen-dependent cells in low estrogen media may convert them to dependence on factors which are not currently understood. This may ultimately increase their value as models of hormone action.

Estrogenic activity of phenol red

It has recently been reported that phenol red, a pH indicator present in most tissue culture media, is a weak estrogen that can stimulate some estrogen-sensitive cells. However, the relative impact of phenol red on various cell lines is controversial. We examined the effect of phenol red on several estrogen-responsive cell systems that we use to study estrogen action. These included estrogenic stimulation of progesterone receptor and growth in human breast cancer-derived MCF-7 cells, stimulation of growth in human breast cancer-derived T47D cells, stimulation of prolactin synthesis in primary cultures of immature rat pituitary cells, and stimulation of progesterone receptor in primary cultures of immature rat uterine cells. Estrogenic responses in MCF-7 cells were the most sensitive to the presence of phenol red, while the other three cell cultures showed lesser effects of the indicator. In addition to intrinsic differences in cell responses, there were several other factors involved. These included differences in the estrogenic activity of phenol red-containing media and phenol red itself from different commercial suppliers, and differences in the concentration of free phenol red in final media due to binding of the indicator by serum. Higher concentrations of serum reduced the impact of phenol red on estrogenic responses in primary pituitary cells. Phenol red added to rat uterine cytosol competed with estradiol for binding to the estrogen receptor (relative binding affinity (RBA) approx. 0.001), and the acidic and basic forms of the indicator showed similar activity. Some commercial phenol red samples inhibited cell growth at levels of 100 mg/l; these effects were toxic rather than antiestrogenic, because growth inhibition could not be competitively reversed by an excess of estradiol. The amount of the indicator bound to serum in the final media, the source of the phenol red and the sensitivity of different cell types to the indicator ultimately determine its influence to the response of cells in tissue culture.

Evolution of a Model of Estrogen Action

Publisher Summary This chapter illustrates the evolution of a model of estrogen action. The unoccupied estrogen receptor (no estrogen ligand) is thought to be a nuclear protein bound to nuclear components by low affinity interactions. Cytoplasmic exclusion may also influence the nuclear localization. Estrogens are lipophilic and therefore can diffuse through cell membranes, cytoplasm, and nuclear envelope to interact with the nuclear receptor. As a result of this interaction rapid changes occur in the conformation of the receptor protein. These conformational changes result in the new physical properties, including a higher affinity for nuclear components which prevents low salt extraction of the transformed estrogen–receptor complex. The nature of the interaction between estrogen–receptor and nucleus is still unknown but nuclear components involved could include chromatin proteins, the nuclear matrix, DNA, or the various combinations of these. However, that estrogen binding to the receptor causes increased rates of transcription of a variety of genes, depending upon the respective target cell.

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.

Nuclear Location of Estrogen Receptors

Publisher Summary This chapter discusses that cell enucleation and immunocytochemistry have provided direct evidence that the estrogen receptor is located exclusively in the nucleus of the cell and that little unoccupied receptor is found in the cytoplasm. It presents experiments in which cell enucleation techniques are used to separate cytoplasm from the nucleus of the cell without breaking open the cell and diluting its contents. Both the cytoplast and the remainder of the cell that contains the nucleus (nucleoplast) are alive and are surrounded by an intact plasma membrane. If the unoccupied receptor is a cytoplasmic protein, one would expect to find it in the cytoplasts and to find less receptor in the nucleoplasts, from which cytoplasm has been removed. However, little receptor was found in cytoplasts; instead, the unoccupied receptor was found in nucleus-containing fractions.

Estrogen Receptors as Nuclear Proteins

The estrogen receptor appears to be a nuclear protein regardless of whether or not it is occupied by an estrogenic ligand (1). As illustrated in Figure 1, the estrogen receptor is thought to be bound with low affinity to some nuclear component. Thus the unoccupied receptor is not in a soluble form in the intact nucleus but is readily extracted into dilute aqueous buffers upon homogenization. When receptor-containing cells or tissues are exposed to estrogens, the estrogen-receptor complex undergoes a conformational change resulting in an increased affinity for nuclear components. Therefore the estrogen-receptor complex can be extracted only with buffers containing high salt concentrations (0.4 M NaC1). We believe that the estrogen-induced conformational change is the critical event in estrogen action.