Other steroid receptor assays

When only limited amounts of cells are available (e.g., of human leukocytes for estimation of corticoid receptors or of human skin fibroblasts for estimation of androgen receptors) an estimation of receptors in separated cell fractions might not be feasible. Total numbers of binding sites per cell may be obtainend if different concentrations of suitable highly specific ligands are used and incubation conditions of intact cells are carefully calibrated. In addition a good separation between medium with free steroid or steroids loosely adsorbed to the cell surface and steroid bound to the receptor inside the cell is essential. 

In one procedure [24] cells are rapidly centrifuged through an oil layer with a density in between the density of cells and medium. In this way cells and medium are not only separated within seconds, preventing redistribution of bound steroid, but also steroid adhering to the cell surface is removed by the lipophilic separation layer. 

Information on the rate of synthesis or turnover of receptors can be obtained from studies with dense amino acids, e.g., in studies with cultured cells. The isolated steroid receptor complexes are separated by density gradient centrifugation and the presence of a faster sedimenting form of the receptor, containing the dense ami-noacids is monitored [25], 

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Steroid hormones achieve their effects on target tissues through intracellular receptor proteins. According to recent views, oestrogen and progestin receptors are localized in the nuclear compartment of the cells, whereas glucocorticoid receptors may reside in both the cytoplasm and the nucleus. Determination of the intracellular localization of androgen receptors awaits the development of (monoclonal) antibodies which will enable immunohistochemical studies. The molecular aspects of the mechanism of action of steroid hormones will be covered in other chapters [1-3] in this volume. The present chapter deals with the characterization, assay and purification of steroid receptors. 

HPLC is a method for the analysis and characterization of steroid receptors based on characteristics such as molecular weight. Qualitative relationships and multiple forms of the receptor can be maintained by the rapid gel-exclusion system, and contaminants can be readily identified (P2). Advantages of using HPLC include rapid analysis time, minimal receptor modification, improved resolution, and high reproducibility. Unfortunately the HPLC assay is tedious, requiring saturation analysis for each sample and quantitative validity has not been established for this procedure. Other disadvantages associated with HPLC methods include the requirement for expensive equipment and sophisticated technical skills. Therefore, HPLC analysis of receptor proteins is currently used only in research studies. 

False-positive results of ER assays (ER-positive tumor but no response to endocrine therapy) are more common than are false-negative results. The most frequent explanation is heterogeneity of tumor with biopsy of a site that is not representative of the other tumor deposits. In addition to this problem, evidence exists that some tumor cells have receptor defects distal to the initial binding step (e.g., variant cells are able to bind steroid in the cytoplasm but not transport the receptor to the nucleus). 

Organometallic derivatives of steroid hormones have been used to study ligand-receptor interactions for quite some time, SERMs with anti-tumor activity have already been discussed in Section 1.31.3.1. Other applications were developed for metallo-immuno assays and are discussed in Section 1.31.5.3. For such applications, binding of the organometallic compound to a receptor is necessarily implied but not always proved or quantified. 

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