Estrogen and progesterone receptors in human gallbladder

Gallbladder disease is more prevalent in women than men. Estrogen therapy has been associated with an increased incidence of gallbladder disease in both sexes. Further, increased progesterone levels have been implicated in impairment of gallbladder motility in pregnancy. Because sex hormones often exert their action through specific receptors, we investigated whether human gallbladder contains receptors for estrogen and progesterone. Binding of radiolabeled hormones in cytosol and nuclei prepared from human gallbladder of both sexes is indicative of the presence of receptors for both estrogen and progesterone. The binding is saturable, of high affinity and highly specific for its particular type of hormone but not other steroids. Fractionation of sodium molybdate-stabilized gallbladder cytosol on Sephadex G-100 demonstrates that the labeled hormones are not bound to defined proteins such as sex steroid binding globulin or albumin. These studies indicate that human gallbladder contains both estrogen and progesterone receptors, that the presence of these receptors may explain the sensitivity of gallbladder tissue to these hormones and that certain aspects of gallbladder function may be mediated by the interaction of steroid hormones with these receptors.     

Estradiol and progesterone receptors in human breast cancer

The concentration of estradiol and progesterone receptors in tumor cytosol from human breast cancer patients represents an important factor for the selection of patients for endocrine therapy and for the prognosis of the disease. This report presents results obtained by measurement of estradiol receptor in 157 breast tumors by the dextran coated charcoal method in media containing sodium molybdate (102 cases) or glycerol (55 cases). Results obtained when these two reagents were present separately in the assay media were similar, at least when the frequencies of estradiol receptor positive cases and the mean values for estradiol receptor concentrations were compared. Concomitant analyses for estradiol and progesterone receptors were done in 25 primary mammary carcinomas by the method of dextran coated charcoal using single saturating doses of hormones and cytosols obtained in a glycerol containing buffer. The results are discussed in relation with the steroid hormone mechanism of action.     

Estrogen and progesterone receptors in the human vagina

Estrogen (E) and progesterone (Pg) receptor (R) levels were determined in the human vagina in relation to menopausal status, day of ovarian cycle and pregnancy. The results obtained confirmed that the human vagina contains ER and, in addition, demonstrated for the first time the presence of PgR in this organ in humans. In cycling women, ER and PgR did not vary significantly during the ovarian cycle; however low (less than or equal to 10 fmoles/mg cytosol protein) concentrations of PgR were more frequently (6 out of 8 cases) detected during the secretory phase. No substantial difference was seen in ER and PgR values between anterior and posterior wall of the vagina. In postmenopausal patients the levels of ER (range: 10-83 fmoles/mg) were similar to those found in premenopause (range: 12-78 fmoles/mg). As regards PgR, the majority (14 out of 20) of vaginae were devoid of PgR, 4 had a very low (less than or equal to 6 fmoles/mg) PgR content and only 2 cases had a PgR level higher than 10 fmol/mg cytosol protein. In pregnant patients (6th to 8th week) ER were found in all vaginae, while PgR were present only in some cases (3 out of 8). It was concluded that the behavior of ER in the human vagina seems different from that in the human endometrium, since ER levels do not vary in relation to changes in the concentrations of sexual hormones in the circulation. On the contrary, PgR levels appear to depend on blood estradiol and progesterone concentration, as in other target tissues.     

Estradiol and progesterone receptors in human cutaneous melanoma

The presence of estradiol and progesterone receptors (ER and PR, respectively) was assessed in 24 removed cutaneous melanomas, adapting the routine procedure used for the detection of the presence of these steroid hormone receptors in breast cancers. The study included only those cases which were not subjected to any anticancer therapy before surgery. The ER and PR values were comparable to those found in breast cancer and the tumors thus investigated could be classified in the same four distinct groups, namely ER+PR+, ER+PR-, ER-PR+, and ER-PR-. Each group is expected to exhibit a specific rate of response to endocrine therapy. No relation was found between the presence of steroid receptors and the type of tumor tissues (benign and primary tumors, recurrences or metastases), or the sex of the patients. Because of the small number of cases in each age group we could not correlate the levels of ER+ and PR+ with the age of the patients. Saturation analysis, competition studies and Scatchard analysis were performed in order to determine the characteristics of ER. Our data suggest that cutaneous melanoma cytosols contain a saturable, high affinity and low capacity, specific binding component for estradiol. Further investigations are required to show that estrogen responsive tissues are functional in either melanocytes or melanomas from any species.     

High expression of NPY receptors in the human testis

NPY receptors represent novel molecular therapeutic targets in cancer and obesity. However, the extent of NPY receptor expression in normal human tissues is poorly investigated. Based on the role of NPY in reproductive functions, the NPY receptor expression was studied in 25 normal human testes and, additionally, 24 testicular tumors using NPY receptor autoradiography. In the normal testis, Leydig cells strongly expressed NPY receptor subtype Y2, and small arterial blood vessels Y1. Y2 receptors were found to be functional with agonist-stimulated [(35)S]GTPγS binding autoradiography. Full functional integrity of the NPY system was further suggested by the immunohistochemical detection of NPY peptide in nerve fibers directly adjacent to Leydig cells and arteries. Germ cell tumors expressed Y1 and Y2 on tumor cells in 33% and Y1 on intratumoral blood vessels in 50%. Based on its strong NPY receptor expression in Leydig cells and blood vessels, the normal human testis represents a potentially important physiological and pharmalogical NPY target.     

Androgen and progesterone receptors in shark (Squalus) testis: characteristics and stage-related distribution.

Although testosterone (T) is essential for the normal completion of spermatogenesis, the exact T-sensitive control points are still unknown. Using staged tissues (premeiotic, PrM; meiotic, M; and postmeiotic, PoM) from zonal testes of the spiny dogfish Squalus acanthias, and standard [3H] steroid binding analysis, we characterized a T-binding component with physiochemical characteristics resembling classical androgen receptors (AR). [3H]T binding was of high affinity (dissociation constant = 4.4 x 10(-9) M), limited capacity (maximum binding, 94 fmol/g tissue) and relatively stable (t1/2 = 4 h at 4 C). The T-binding component was present in both cytosolic and nuclear extracts, adhered to DNA-cellulose, and displayed predicted sedimentation properties of an activated receptor in vivo or in vitro (5.06S). T, 5 alpha-dihydrotestosterone (DHT), and mibolerone (Mib), but not methyltrienelone (R1881), competed well for [3H]T binding; however, progesterone (P) was equivalent to T in its ability to displace tracer. Subsequent analysis of [3H] P binding revealed a P-binding component that was present in nuclear and cytosolic extracts, adhered to DNA, but differed from AR in its inability to bind Mib. Competition studies in which excess radioinert Mib was used to block AR revealed ligand specificity characteristics of progesterone receptors (PR): promegestone (R5020) greater than P greater than deoxycorticosterone, but T, DHT, dexamethasone, and corticosterone were ineffective competitors. Also, a nonlinear Scatchard plot was obtained, suggesting two P-binding activities, which differed in their binding affinities (dissociation constant = 0.88 vs. 5.9 x 10(-9) M) and capacities (66 vs. 210 fmol/g tissue). Conversely, using [3H]Mib to avoid interference from PR, we confirmed that T, DHT, and P were equivalent in their ability to displace ligand from AR. Comparison of tissues by stage of spermatogenesis revealed different distribution patterns for AR (PrM greater than M much greater than PoM) vs. PR (PoM much greater than M = PrM). These data provide definitive evidence for separate testicular T- and P-binding mechanisms and indicate the presence of temporally distinct sets of steroid-regulated genes.     

Evidence for the role of phospholipids in follicle-stimulating hormone binding to membrane-bound and soluble receptors from calf testis

The role of phospholipids in the interaction of FSH with receptors in the calf testis was explored by studying the effects of phospholipase-C (PL-C) on the binding of radioiodinated human FSH ([125I]iodo-hFSH) to three previously characterized but different preparations of FSH receptor: a membrane fraction derived from calf testis homogenate, a buffer-soluble receptor present in the cytosol of the calf testis homogenate, and a detergent-soluble receptor prepared from the membrane fraction by extraction with Triton X-100. Prior incubation with PL-C markedly reduced specific [125I]iodo-hFSH binding to the membrane-bound and buffer-soluble receptors. This loss in binding was associated with hydrolysis of phospholipids, was prevented by the addition of o-phenanthroline, an inhibitor of PL-C, but not by the addition of a protease inhibitor, and could not be reproduced by the addition of the products of PL-C hydrolysis. Enzyme treatment did not dissociate [125I]iodo-hFSH already bound to the buffer-soluble receptor and dissociated only 20% of [125I]iodo-hFSH bound to membrane receptor. PL-C treatment did not reduce [125I]iodo-hFSH binding to detergent-solubilized receptor, nor did it hydrolyze constituent phospholipids. The apparent resistance of the detergent-solubilized receptor to PL-C treatment was studied by incubating membranes with or without PL-C before detergent solubilization. Triton X-100 itself (2%) inhibited phospholipid hydrolysis by PL-C, but this could be overcome by reducing the detergent concentration 10-fold by dilution. Despite a marked decrease in specific [125I]iodo-hFSH binding to PL-C-treated membranes compared to that of untreated controls, the total amount of [125I]iodo-hFSH binding to Triton X-100 extracts of each membrane preparations was not significantly different. Addition of Triton X-100 to the buffer-soluble receptor restored 40% of the hormone binding that had initially been lost concomitant with enzymic hydrolysis of membrane phospholipids. It appears that constituent phospholipids play an important role in the interaction of FSH with membrane-bound or solubilized receptor and that Triton X-100 is able to substitute, although imperfectly, for phospholipids in that regard, probably because of its related amphipathic properties.     

Expression and functional consequences of oestrogen and progesterone receptors in human insulinomas

The expression of steroid receptors by tumours offers a therapeutic advantage if functionally responsive to exogenous hormones. Insulinomas represent a highly symptomatic group of pancreatic tumours and the steroid receptor status of these tumours is poorly understood. The object of the study was to characterise the sex steroid receptor status of human insulinomas and to investigate whether sex steroids alter insulin expression therein. At our tertiary referral University Hospital, archival and prospective tissues from 25 insulinoma patients collected over 14 years were analysed for oestrogen receptor-alpha (ERalpha), oestrogen receptor beta (ERbeta) and progesterone receptor (PR) expression. Tissue explants of insulinoma and control pancreatic tissue from two new insulinoma patients were cultured and treated with oestrogen and progesterone and insulin expression measured by RT-PCR and ELISA. The main outcome measures were established before data collection and included sex steroid receptor status of tumours and insulin expression in fresh tissue in response to exogenous sex steroids. PR was expressed in 24 out of 25, ERalpha in 10 out of 25 and ERbeta in 21 out of 25 insulinomas. In fresh insulinoma cultures, insulin expression was increased by oestrogen or progesterone, whereas no significant effect was observed in adjacent pancreatic tissue. This study demonstrates widespread expression of sex steroid receptors on human insulinoma tissue and provides in vitro evidence of functionality with increased expression of insulin by insulinoma explants in response to exogenous oestrogen or progesterone. Confirmation of these results may provide a therapeutic mechanism for reducing symptomatic insulin secretion by receptor blockade.     

Spontaneous solubilization of membrane-bound human testis angiotensin-converting enzyme expressed in Chinese hamster ovary cells.

The testis isozyme of angiotensin-converting enzyme (ACE; EC 3.4.15.1) is a membrane-bound protein that, apart from the first 35 N-terminal residues, is identical to the C-terminal half of somatic ACE and contains the same putative C-terminal membrane anchor. Stable transfection of Chinese hamster ovary (CHO) cells with an expression vector containing the full-length human testis ACE cDNA results in the expression of two forms of recombinant human testis ACE (hTACE): membrane-bound ACE and, surprisingly, large quantities (up to 3 mg/liter) of soluble hTACE in the conditioned medium. Both forms are fully active and are physicochemically similar. However, by phase separation in Triton X-114, the soluble enzyme is hydrophilic, as is an anchor-minus mutant hTACE recovered from the medium of CHO cells transfected with a vector that contains a 3'-truncated testis ACE cDNA lacking the sequence encoding the membrane anchor. In contrast, the membrane-bound hTACE is amphipathic but is converted to a hydrophilic form on treatment with trypsin. The data establish that in ACE the hydrophobic sequence near the C terminus is necessary for membrane anchoring. Moreover, in CHO cells, membrane-bound hTACE is apparently solubilized by proteolytic cleavage of this anchor. A similar mechanism may account for the release of endothelial ACE in vivo to generate serum ACE and more generally for the constitutive processing and solubilization of analogously anchored proteins such as the amyloid precursor protein, among others. The release of membrane-bound ACE in CHO cells may, therefore, provide a useful system for the study of membrane-protein-solubilizing proteases.     

Simultaneous measurement of progesterone and androgen receptors in human prostate: a microassay

The characteristics of binding of radiolabeled progesterone, promegestone [17 alpha,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione (R5020)], medroxyprogesterone acetate (4-pregnen-6 alpha-methyl-17 alpha-ol-3,20-dione acetate), and methyltrienolone [17 beta-hydroxy-17 alpha-methyl-4,9,11-estratriene-3-one (MT)] to the progesterone receptor in human prostatic cytosol have been compared. MT binds to both androgen and progesterone receptors with high affinity (Kd = 0.9 and 0.6 nM, respectively). The binding of MT to the progesterone receptor can be blocked by adding an excess of unlabeled triamcinolone acetonide [9 alpha-fluoro-11 beta, 16 alpha, 17 alpha,21-tetrahydroxy-1,4-pregnadiene-3,20-dione-16,17-acetonide (TAC)]. The difference between the binding of [3H]MT in the absence and presence of TAC (i.e. [MT - (MT + TAC)] represents specific binding of MT to the progesterone receptor. Ligand specificity studies demonstrated that this binding was typical of a progesterone receptor. Furthermore, progesterone receptor levels measured in this way were comparable to those obtained using progesterone, R5020, or medroxyprogesterone acetate as labeled ligands. Progesterone receptor quantitation from the difference MT - (MT + TAC) is of particular advantage when simultaneous quantitation of progesterone and androgen receptors is desired in small tissue specimens since only three sets of incubations are required: [3H]MT, [3H]MT plus unlabeled TAC, and [3H]MT plus unlabeled MT (to measure nonspecific binding). Conditions are described for the application of this methodology to a microassay. A marked underestimate of progesterone receptor content was observed when incubation was terminated with hydroxylapatite compared to that measured when dextran-coated charcoal was used. The presence of comparable amounts of progesterone and androgen receptors in human prostatic cytosol deserves further investigation.     

Studies of the human testis. VII. Conversion of pregnenolone to progesterone.

Delta5-3beta-Hydroxysteroid dehydrogenase (EC.1.1.1.145) and steroid delta-isomerase (EC.5.3.3.1) were extracted from frozen human testicular tissue and co-precipitated by addition of ammonium sulfate. The activities of both enzymes were localized in the 0-40% (NH4)2SO4 fraction. The enzyme preparation catalyzed conversion of pregnenolone, 17alpha-hydroxypregnenolone, dehydroepiandrosterone, and androstenediol to the corresponding delta4-3-oxosteroid. Since isomerization appeared not to be the rate-limiting step of the overall reaction, measurement of activity of delta5-3beta-hydroxysteroid dehydrogenase was related to the amount of delta4-3-oxosteroid produced from the corresponding delta5-3beta-hydroxysteroid. Delta5-3beta-Hydroxysteroid dehydrogenase required NAD for maximal activity. The Michaelis constants (Km) for NAD were 50 muM, 33 muM and 14 muM, respectively for the dehydrogenation of pregnenolone, 17alpha-hydroxypregnenolone, androstenediol and dehydroepiandrosterone. Km values for each substrate were: pregnenolone 10 muM, 17alpha-hydroxypregnenolone and dehydroepiandrosterone 2.5 muM and androstenediol 3.0 muM. Human testicular delta5-3beta-hydroxysteroid dehydrogenase was inhibited by most of the steroids procued by the testis. The following steroids acted as competitive inhibitors with pregnenolone: 17alpha-hydroxypregenolone (Ki = 1.3 muM), androstenediol (Ki = 2.4 muM), dehydroepiandrosterone (Ki = 0.74 muM), 20alpha-dihydroprogesterone (Ki = 1.1 muM) estrone (Ki = 0.33 muM) and estradiol-17beta (Ki = 0.87 muM). 17alpha-Hydroxyprogesterone, testosterone and androstenedione showed mixed-type inhibition of the enzyme for pregnenolone. Progesterone and NADH were noncompetitive inhibitors of the enzyme for pregnenolone. Ki values, with respect to prenenolone, were 7.4 muM for progesterone and 150 muM for NADH. NADH, however, acted competitively with NAD and Ki value was 30 muM.     

Estrogen and progesterone receptors in esophageal carcinoma

SUMMARY.  Information is sparse and contradictory in the literature regarding the role of estrogen receptor (ER) and progesterone receptor (PR) in esophageal carcinoma. This study was conducted over a period of 18 months from September 2004 with the primary aim of determining the PR, ER alpha (ERα) and ER beta (ERβ) status of esophageal carcinoma and normal esophageal mucosa (NEM). The receptor status was correlated with tumor type, tumor differentiation and tumor stage. A total of 45 patients with histologically proven squamous cell carcinoma (SCC) ( n  = 30) and adenocarcinoma (AC) ( n  = 15) were studied. Receptor status was detected by immunohistochemistry (IHC) and semiquantitative assessment was done by quick score method of endoscopic biopsy specimens. The mean age for SCC and AC were not significantly different. The gender ratio in favor of males was 3 : 2 for SCC and 4 : 1 for AC. None of the specimens from SCC or AC showed positivity for PR both in NEM and tumor tissue. Likewise none of the specimens were positive for ERα by IHC. The mean ERβ score for AC was significantly higher than SCC. For SCC it was seen that ERβ positivity in tumor cells increases with dedifferentiation and increasing tumor stage. This trend was seen for AC as well. ERβ is over-expressed in poorly differentiated SCC and AC compared to NEM. Thus ERβ may be a marker for poor biological behavior, that is dedifferentiation or higher stage of disease. In view of these findings we propose a large-scale prospective, longitudinal interventional study using selective estrogen modulators.     

Immunohistochemical analysis of human uterine estrogen and progesterone receptors throughout the menstrual cycle

Estrogen receptors (ER) and progesterone receptors (PgR) were studied immunohistochemically using specific antireceptor monoclonal antibodies in uterine tissue samples from 33 women in various stages of the menstrual cycle. Immunohistochemical localization was quantified as to intensity of staining and tissue distribution in glandular epithelium, stroma, and myometrium, and the results were compared with those of standard ligand binding assays. In all samples ER and PgR localized within the nuclei of target cells. The maximal concentrations of ER and PgR occurred in the mid- to late proliferative phase of the menstrual cycle. ER content declined throughout the secretory phase. In contrast, PgR content underwent unexpectedly complex and dyssynchronous fluctuations during the secretory phase of the menstrual cycle. Specifically, the glandular epithelium had diminished PgR content, while the stroma and myometrium maintained a significant PgR content. PgR and perhaps ER are not concordant in different cell types within the uterus. Segregation of function through alteration of receptor content may be an important mechanism in steroid-dependent growth and differentiation of target tissues.     

Estrogen and progesterone receptors in ovarian epithelial tumors

Epidemiological studies have indicated a relationship between ovarian cancer and gonadal steroid hormones. In the present study immunohistochemical localization in combination with morphometry were used to characterize changes in the pattern of expression for estrogen receptor alpha (ERalpha), estrogen receptor beta (ERbeta), and progesterone receptor (PR), in epithelial cells of normal ovaries, and in benign, borderline and malignant ovarian tumors of different types (n=53). Positive correlations with immunoreactivity of the cell proliferation-marker, Ki67, and the apoptosis-related marker of genetic instability, p53, between the different tumor types were also found. A simultaneous expression of ERalpha, ERbeta and PR in epithelial cells of all histopathological tumor types was noted, with the notable exception of all mucinous tumors who remained ERbeta-positive, but ERalpha- and PR-negative. Epithelial cells in ovarian cancer tissue showed significantly lower mean immunoreactivity of ERbeta and PR, but not ERalpha, than in normal ovarian tissue. These novel findings may provide a rationale for the development of new diagnostic and possibly therapeutic strategies.     

Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes

Summary Human blood monocytes when cultured on hydrophobic Teflon membranes differentiate into mature macrophages. The expression of transferrin receptors was monitored by monoclonal antibody (OKT9) binding as detected by immunperoxidase staining. Whereas monocytes were negative, an increasing percentage of macrophages, starting from day 2 in culture, labelled with the anti-transferrin receptor antibody as these cells undergo differentiation. After completion of maturation more than 90% of macrophages expressed transferrin receptors. While 90–95% of macrophages from broncho-alveolar lavage fluids labelled with the OKT9 antibody, only a minor portion of macrophages obtained from peritoneal and pleural cavities did so. In parallel, intracellular ferritin in cells of the monocyte-macrophage lineage increased from 10ng/10⁶ cells to 350–1,500ng/10⁶ cells during maturation in vitro. Alveolar macrophages proved to have the highest ferritin content which ranged from 355–8,400ng/10⁶.The results may indicate that iron uptake and storage is a function of cells at late stages of macrophage maturation and that the occurrence of surface receptors for transferrin can be regarded as differentiation dependent marker.Supported by Deutsche Forschungsgemeinschaft