SUMMARY

A method for the determination of oestrone sulphate, oestradiol-17β and oestrone in human peripheral plasma is described, and the accuracy, specificity, precision and sensitivity of each analysis is defined.

Free oestrogens were extracted from plasma with ether. Oestradiol-17β was isolated by chromatography on silica gel. Oestrone, similarly purified, was reduced to oestradiol-17β and rechromatographed. Oestrone sulphate in the residual plasma was solvolysed and the oestrone liberated was isolated and reduced to oestradiol-17β. All three fractions were estimated separately by competitive protein binding with rabbit uterine cytosol. Manipulative losses were assessed from recovery of [³H]oestradiol-17β and [³H]oestrone added to the plasma and of [³H]oestrone sulphate added to the ether extracted plasma.

The concentration of oestrone sulphate in peripheral plasma of six men greatly exceeded that of oestradiol-17β or oestrone. The mean concentrations were respectively, 72, 2·3 and 3·6 ng/100 ml. Analysis of samples collected daily during a menstrual cycle from each of two women also showed that the concentration of oestrone sulphate was much greater than that of oestradiol-17β or oestrone. Changes in the concentrations of all three oestrogens occurred at similar times in the cycle. At mid-cycle, the mean concentrations were: oestrone sulphate, 308 ng/100 ml; oestradiol-17β, 57 ng/100 ml; oestrone, 15 ng/100 ml.

In rat mammary tumours and uteri it was found that reaction time, ionic strength, reproductive state and storage in liquid nitrogen affected the form of the oestrogen receptor detected by sucrose density-gradient analysis using a vertical-tube rotor. A standard method of gradient analysis was defined. The sedimentation profile of the oestrogen receptor was compared in three types of experimental rat mammary tumour of known hormonal sensitivity. These were two lines of ovary-independent transplantable tumours and those tumours induced by dimethylbenz(a)anthracene (DMBA) which are mainly ovary-dependent. In all three types of tumour the 8S receptor was the predominant molecular form (32 out of 36 tumours induced by DMBA and 11 out of 13 transplantable tumours). Sedimentation profiles of the oestrogen receptor were also examined in 46 human breast carcinomata found to be receptor-positive by the dextran-coated charcoal technique. Of these, 17 were found to be receptor-negative by gradient analysis and 28 out of 29 receptor-positive tumours contained 8S receptor. It was concluded that (1) 8S receptor is the predominant molecular form in both human and rat mammary tumours, (2) molecular form of receptor is not related to ovarian dependence in these rat mammary tumours and (3) gradient analysis seems unlikely to provide additional discrimination in the prediction of response to endocrine therapy over that provided by simpler methods.

Dimethylbenz(a)anthracene (DMBA)-induced and transplanted rat mammary tumours (2 lines) were examined for oestrogen receptor activity, and for sensitivity to hormones in vivo (by ovariectomy) and in vitro (by tissue culture). In vivo, the growth of all tumours induced by the administration of DMBA in random-bred Sprague–Dawley rats was found to be dependent on the ovary, whilst in all transplanted tumours (12 TG-3 and six TG-5 lines), maintained in an inbred strain of Sprague–Dawley rats, growth was found to be independent of the ovary. In vitro, the capacity for DNA synthesis in DMBA-induced tumours was better maintained after 24 h when insulin (10 μg/ml) and corticosterone (5 μg/ml) or insulin, corticosterone and prolactin (each 5 μg/ml) were present in the medium (five out of 12 and eight out of 11 tumours respectively); no effect of hormones in the media was detected after 48 h. In the transplanted tumours, no effect of hormones on DNA synthesis was detected after either 24 or 48 h of culture. Synthesis of lecithin was not detectably influenced by the presence of hormones in either DMBA-induced or transplanted tumours. Oestrogen receptor concentrations were, on average, significantly higher in the DMBA-induced tumours than in either line of transplanted tumour. For 22 DMBA-induced tumours and 15 transplanted tumours, the effect of hormones in vitro (`response') was directly correlated with receptor concentration at time 0 (Spearman's ρ = + 0·59) and inversely correlated with the rate of DNA synthesis (`basal') at time 0 (Spearman's ρ = −0·62). No single parameter or pair of parameters permitted accurate distinction between the tumour types.

Forty-eight rat mammary tumours and 25 human breast carcinomata were examined for (a) oestrogen receptor activity and (b) capacity for intranuclear translocation of oestrogen. Receptor activity was determined by saturation analysis using charcoal adsorption to separate free and bound hormone. The capacity for intranuclear translocation was determined by incubation of tumour slices in Krebs–Ringer bicarbonate solution containing [³H]oestradiol-17β at a physiological concentration, in the presence and absence of a large excess of non-radioactive oestradiol, at 37 °C. Saturable nuclear uptake of [³H]oestradiol (= translocation) in boiled or receptor-negative tissues was minimal, i.e. <12 fmol/mg DNA per 2 h and in receptor-positive tissue was reduced by 85% when the temperature was lowered to 0°C. In 27 ovary-independent transplantable tumours and 21 dimethylbenz(a)anthracene-induced tumours (predominantly ovary dependent) saturable nuclear uptake was strongly correlated with level of oestrogen receptor activity (Spearman's rank correlation coefficient r= +0·73, P<0·001). Nineteen of these 48 tumours were examined further: 60–80% of the saturable uptake was precipitable with protamine sulphate and this fraction of the total saturable uptake was also highly correlated with receptor level (Spearman's r = +0·87, P<0·001). In the 25 human tumours studied, saturable nuclear uptake was again correlated With receptor level (Spearman's r = +0·75, P<0·001). These studies suggest that saturable transfer of oestradiol from the extracellular medium through the cytoplasm to the cell nucleus is mediated by the oestrogen receptor in the rat and human tumours examined. They provide no evidence for any defect of the receptor mechanism before nuclear binding in tumours which are receptor positive but hormone insensitive.

ABSTRACT

The receptor for epidermal growth factor (EGF) was characterized in the particulate fraction from human benign prostatic hyperplasia (BPH) and was present in 85% of tissues analysed. The uptake of ¹²⁵I-labelled EGF by BPH was dependent on both time and temperature, with maximum specific uptake achieved after incubation for 90 min at 37 °C. Binding characteristics revealed two classes of binding sites of higher (mean dissociation constant (Kd)±s.d.= 0·8 ± 0·2 nmol/l) and lower (Kd = 7·6 ±2·8 nmol/l) affinities. Competition studies demonstrated the specificity of the receptor assay since the binding of labelled EGF was abolished with excess unlabelled EGF but not with excess unlabelled human GH, human insulin, venom nerve growth factor, human FSH, human LH and human prolactin. There was a complex biphasic relationship between specific binding and protein concentration in the range 0·1–8 mg/ml. Subcellular fractionation of BPH homogenates demonstrated that the bulk of the specific binding was confined to the 800 g (crude heavy pellet) and 15 000 g (mitochondrial pellet) fractions. The 105 000 g (microsomal pellet) and the 105 000 g (cytosol fraction) exhibited low and variable binding capacities for the growth factor. The presence of EGF receptor was also confirmed by immunocytochemical staining of frozen sections from BPH using monoclonal antibody specific for EGF receptors. A positive correlation between ¹²⁵I-labelled EGF binding and the intensity of staining was found. The presence of a specific EGF-binding receptor protein in human BPH tissues suggests that EGF may play a role in the pathogenesis of human BPH.

J. Endocr. (1987) 113, 147–153

ABSTRACT

Since uterine leiomyomata (fibroids) are not found in conditions where oestradiol is either absent or present only in low concentrations, oestradiol is considered to be an important factor in the control of fibroid growth. To investigate whether this is due to a direct effect on the tissue, oestradiol and progestogen receptors were measured in tissue removed at hysterectomy from 12 normally cycling women and 13 women who had received the gonadotrophin-releasing hormone (GnRH) agonist Zoladex (ICI 118630) as a subcutaneous depot given at monthly intervals for 3 months preoperatively and a third group of three women who had received the antioestrogen tamoxifen (20 mg daily) for 3 months before surgery. Both unoccupied oestradiol receptors (measured by separating bound from free hormone with dextran-coated charcoal; DCC) and 'total' receptor populations (as measured by an enzyme immunoassay) were measured in each fibroid and adjoining myometrium. There was significantly more binding of both oestradiol and progestogen to fibroid than to myometrium in both the control (P <0·01) and agonist-treated groups (P <0·05). Oestradiol binding to fibroids in women treated with Zoladex exceeded that in the normally cycling women (P <0·05) which in turn exceeded that in the tamoxifen-treated group (P <0·05). However, the binding of progestogen, measured by DCC showed the reverse trend. These results may be explained by the low circulating oestradiol concentration in the GnRH agonist-treated women leading to low receptor occupancy.

Journal of Endocrinology (1989) 121, 389–396