W. de Boer

Mechanism of androgen action: Recent observations on the domain structure of androgen receptors and the induction of EGF-receptors by androgens in prostate tumor cells☆

In this paper two different aspects of androgen action are reviewed. Polyacrylamide gel electrophoresis of androgen receptors, photoaffinity labeled with R1881 showed that receptors isolated from both human prostate cells and calf uterine cytosol cells are proteins with a molecular mass of approx 110 kD. Purification to homogeneity of this form of the receptor from calf uterus also yielded a 110 kD protein. A molecular model for the DNA-binding form of the receptor is presented in which one polypeptide comprises three active domains: one for ligand binding, one for interaction with nuclear acceptor sites, and a third domain which modulates nuclear interaction. Mild digestion with chymotrypsin or a protease from rat prostates removes the modulating domain and leaves the ligand binding and nuclear interaction domain intact. Trypsin treatment yields a fragment of lower molecular mass containing the ligand binding domain with some affinity for RNA, but not DNA. In vitro studies with a human prostate tumor cell line (LNCaP), suggest that androgens not only directly effect cell growth, but also act indirectly. Both epidermal growth factor (EGF) and androgens stimulate cell growth. In addition androgens stimulate synthesis of receptors for EGF. Thus androgens effect tumor cell growth by autocrine or paracrine mechanisms by making the cells more sensitive for growth factor mediated stimuli.

Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways

AbstractObjective:The aim of this study was to analyze a possible contribution of human neutrophil defensins and secretory leukocyte proteinase inhibitor (SLPI) to the induction of airway epithelial changes such as squamous cell metaplasia. Materials and methods:The presence of these molecules and the number of proliferating (Ki-67-positive) epithelial cells was analyzed by immunohistochemistry in bronchial epithelium from subjects with (n = 15) or without (n = 14) chronic obstructive pulmonary disease (COPD). Results:Our data demonstrate higher numbers of defensin-positive (p = 0.0001), elastase-positive (p = 0.0001) and Ki-67-positive (p = 0.0001) cells in areas with squamous cell metaplasia as compared to areas with intact or damaged epithelium, while the reverse was observed for SLPI expression (p = 0.002). No differences were observed between subjects with or without COPD, nor between current smokers and those that had stopped smoking. Conclusions:These data are in line with a role of defensins in the hyperproliferative phenotype of squamous metaplastic lesions in the airways. This role does not seem to be restricted to patients with COPD.

Photoaffinity labelling of androgen receptors with 17β-hydroxy-17α-[3H]methyl-4,9,11-estratrien-3-one

Abstract The synthetic androgen 17β-hydroxy-17α-[ 3 H]methyl-4,9,11-estratrien-3-one (R1881) has been used as photoaffinity label to characterize andorgen receptors in calf uterus and rat prostate. Polyacrylamide gel electrophoresis under denaturing conditions showed that the DNA-binding form of the androgen receptor in calf uterus cytosol is a protein with a molecular mass of 98 kD. In rat prostate cytosol an androgen receptor with a molecular mass of 46 kD could be photoaffinity labelled with R1881. The photoaffinity labelling procedure described here provides a method for studying the hormone binding domain of androgen receptors in partial purified preprations.

Image analysis and quantification in lung tissue

On 9–10 September 1999, an international workshop on image analysis and quantification in lung tissue was held at the Leiden University Medical Center, Leiden, The Netherlands. Participants with expertise in pulmonary and/or pathology research discussed the validity and applicability of techniques used for quantitative examination of inflammatory cell patterns and gene expression in bronchial or parenchymal tissue in studies focusing on asthma and chronic obstructive pulmonary disease (COPD). Differences in techniques for tissue sampling and processing, immunohistochemistry, cell counting and densitometry are hampering the comparison of data between various laboratories. The main goals of the workshop were to make an inventory of the techniques that are currently available for each of these aspects, and in particular to address the validity and unresolved problems of using digital image analysis (DIA) as opposed to manual scoring methods for cell counting and assessment of gene and protein expression. Obviously, tissue sampling and handling, fixation and (immunohistochemical) staining, and microscope settings, are having a large impact on any quantitative analysis. In addition, careful choices will have to be made of the commercially available optical and recording systems as well as the application software in order to optimize quantitative DIA. Finally, it appears to be of equal importance to reach consensus on which histological areas are to be analysed. The current proceedings highlight recent advances and state of the art knowledge on digital image analysis for lung tissue, and summarize the established issues and remaining questions raised during the course of the workshop.

Testicular Estradiol Receptors in the Rat

Specific high affinity saturable binding proteins for oestradiol-17beta have been demonstrated in the cytoplasm of liver, adrenal, pituitary, prostate, epididymis and testis interstitial tissue of the rat. Specific testicular oestradiol receptors were present in cytoplasmic and nuclear fractions of interstitial tissue, but not in seminiferous tubules of rat testis. After in vivo or in vitro administration of estradiol the steroid was bound by the nuclear fraction of interstitial tissue only in the presence of the cytoplasmic receptor. Using agar gel electrophoresis the cytoplasmic oestradiol receptor from rat testis could be separated from the testicular androgen binding protein (ABP) and from a specific androgen receptor.

The testis: Biochemical actions of trophic hormones and steroids on steroid production and spermatogenesis

Abstract The present paper summarizes recent new information on steroid production and spermatogenesis in the rat testis, with respect to: 1. The cellular and subcellular localization of testicular steroid production. 2. Biochemical factors which appear to be related to the effect of trophic homones on testicular steroid production. 3. Some biochemical observations on Sertoli cells and germinal cells in the seminiferous tubules and relationships with steroid hormones.

Multiparameter analysis of primary epithelial cultures grown on cyclopore membranes.

The use of porous membranes as culture support for epithelial cells has previously been shown to cause functional differentiation of these cells mimicking an in vivo condition, in contrast to culture on plastic. The different materials of which the membranes are made also have different properties, such as transparency, rigidity, and retention of molecules. Cyclopore membranes (polyethylene terephtalate) are permeable, transparent, rigid, and have low protein retention. In this study we examined the applicability of assessing multiple parameters on a single culture of primary epithelial cells on a Cyclopore membrane. Cultures of transitional epithelial cells on these membranes differentiate into an organoid-like epithelium. We were able to perform morphometric analysis during and after cell culture and to quantitate proliferation and differentiation by double immunoenzymatic staining. On these cultures, quantitative radiochemical analysis could also be achieved, retaining the morphology and the immunohistochemical staining. Cross-sections of paraffin-embedded and plastic-embedded cultures were analyzed qualitatively by light and transmission electron microscopy, respectively. Finally, cytokeratins in these cultures could also be visualized by immunofluorescence analysis. This suitability for simultaneous assessment of both qualitative and quantitative parameters on a single cell culture grown on a Cyclopore membrane reduces the need of biological materials and may lead to better insight into physiological processes.

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.

Hyperplasia of epithelium adjacent to transitional cell carcinoma can be induced by growth factors through paracrine pathways

Hyperplasia of transitional cell epithelium adjacent to human transitional cell carcinomas (TCC) is a common finding in pathology. This hyperplasia may be a precancerous aberration. Alternatively, it has been suggested that the hyperplasia is due to paracrine action of tumour-derived growth factors. In this study we tested the latter hypothesis using the mouse tumorigenic TCC cell line NUC-1. Transplantation of NUC-1 tumour cells into the urinary bladder submucosa of syngeneic mice in vivo induced hyperplasia of normal adjacent urothelium in all tested mice. Implantation of normal mouse bladder mucosa did not induce urothelial hyperplasia. In vitro, conditioned medium of NUC-1 cells induced the proliferation of the mouse urothelial cell line g/G, which closely resembles normal urothelial cells. This induction was inhibited by transforming growth factor β1 (TGFβ1). Similarly, TGFβ1 inhibited the fibroblast growth factor-1 (FGF-1) and FGF-2 induced proliferation of g/G cells. Chemico-physical examination, bioassays with conditioned media, and RNA analysis of NUC-1 cells revealed that these cells secreted a growth factor with FGF-like properties. These results indicate that epithelial hyperplasia surrounding carcinomas is not necessarily a precancerous aberration, but may result from direct paracrine action of tumour-derived growth factors.

Characterization of Steroid Receptors Using Ion Exchange Fast Protein Liquid Chromatography (FPLC)

Abstract Fast Protein Liquid Chromatography (FPLC) on a Mono Q anion exchange column (Pharmacia) was used to characterize receptors for androgens, estrogens and progesterone. In the presence of 20 mM molybdate androgen, estrogen and progesterone receptors eluted as a single peak at 0.32 M NaCl. The recoveries of receptors after FPLC ranged from 75% – 95%, while a 30 – 70 fold purification was obtained. In calf uterine cytosol kept at 0°C two differently charged forms of the 4S estrogen receptor were identified. After heat induced transformation of the same cytosol one major peak was eluted at a higher ionic strength as was found for both 4S isoforms and which sedimented at 5S in high salt sucrose gradients.