Binding characteristics of a major protein in rat ventral prostate cytosol that interacts with estramustine, a nitrogen mustard derivative of 17 beta-estradiol.

The tissue distribution of [3H]estramustine, the dephosphorylated metabolite of estramustine phosphate (Estracyt), in the male rat was compared to that of [3H]estradiol 30 min and 2 hr following i.p. administration. In contrast to estradiol, estramustine was found to be efficiently concentrated in the ventral prostate gland by a soluble protein. The binding characteristics of this protein were studied in vitro using cytosol preparations of the gland. With a dextran-coated charcoal technique, the protein was found to bind estramustine with a broad pH optimum between pH 7 and pH 8.5, with an apparent Kd of 10 to 30 nM, and with a binding capacity of about 5 nmol/mg cytosol protein. The estramustine/protein complex was not retained by DNA-cellulose. None of the natural steroids tested inhibited the binding of 10 nM [3H]estramustine by more than 35% (progesterone), even when added in 4500-fold excess. The presence of a nitrogen mustard moiety at position 3 of the steroid was necessary for high-affinity binding to the protein. The protein was calculated to constitute about 20% of the total cytosol protein content.

Ontogeny of the rat hepatic receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin and its endocrine indepence.

The ontogeny of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor was studied in Sprague-Dawley rats by quantitation of the receptor in liver cytosol using isoelectric focusing in polyacrylamide gel. No differences by sex in the receptor concentration were seen at any of the ages studied. Newborn, 21-day-old, and 42-day-old rats contained significantly more receptor in the liver cytosol than did 56-day-old rats. There was no significant difference in the receptor concentration in liver cytosol from 7-day-old rats compared to that from 56-day-old rats. The maximum receptor concentration was found in cytosol from 21-day-old rats [36.1 +/- 24.0 (S.D.) fmol/mg protein]. Adult rats (56 days old) contained the lowest concentration of receptor (13.3 +/- 6.3 fmol/mg protein). The level of TCDD receptor in liver cytosol from adult rats was not significantly changed by orchiectomy, ovariectomy, adrenalectomy, or hypophysectomy. The maximum for TCDD receptor concentration at puberty corresponds to the reported maximum for the induction of aryl hydrocarbon hydroxylase activity. However, no further conclusion can as yet be drawn concerning the regulation of the TCDD receptor.

Partial characterization and "quantitation" of a human prostatic estramustine-binding protein.

The [3H]estramustine-binding macromolecule in human prostate was partially characterized using a number of chromatographic procedures. Although human estramustine-binding protein (HEMBP) had a marked tendency to aggregate in several systems, a molecular weight of about 54,000 was determined by gel filtration on Sephacryl S-200 Superfine and high-performance liquid chromatography. A sedimentation-coefficient of about 3.6S was obtained for HEMBP when analyzed by sucrose density gradient centrifugation. Isoelectric focusing in polyacrylamide gels indicated an isoelectric point of 4.7 to 4.8, which was in agreement with the elution position of HEMBP following chromatofocusing on Polybuffer Exchanger 94. Furthermore, HEMBP was eluted from diethylaminoethyl-Sepharose with 0.23 M KCl, was retained by concanavalin A-Sepharose (indicating that HEMBP is a glycoprotein), but did not interact with Affi-Gel Blue. Special efforts were concentrated on establishing that HEMBP was a species distinct from human serum albumin. Separation between the [3H]estramustine-labeled HEMBP and the [3H]estramustine-human serum albumin complex was obtained both on sucrose density gradients by chromatography on Affi-Gel Blue, by chromatofocusing, by gel filtration, by isoelectric focusing, and on concanavalin A-Sepharose by affinity chromatography. Twenty-two of 27 human benign hyperplastic prostate cytosol samples were found to contain protein immunochemically similar to estramustine-binding protein (EMBP) purified from rat ventral prostate as determined by the EMBP radioimmunoassay method. Concentrations from 0.2 to 139.6 ng EMBP per mg of total cytosolic protein (mean, 19.3) were determined. Furthermore, four of seven prostatic cancer specimens as well as two of two normal prostatic specimens were also found to contain rat EMBP-immunoreactive material. The unequivocal demonstration of the presence of a HEMBP is of great potential interest in consideration of estramustine phosphate (Estracyt) therapy against prostatic carcinoma. It is not inconceivable that the concentration of HEMBP in the carcinomatous tissue will be of significance in determining the drug uptake in the malignant tissue.

Correlation between clinical response to hormone therapy and steroid receptor content in prostatic cancer.

Hormonal therapy is the dominating form of treatment for prostatic carcinoma. The majority of cases (80%) are well controlled for varying times with this regimen. However, thus far there have been no adequate methods to predict in which cases hormonal therapy is of less benefit. Measurement of cancer tissue content of intracellular hormone receptors constitutes progress toward a more individualized therapy in prostatic carcinoma. In this study biopsies from 16 cancer patients were taken before therapy was given, and the specimens were analyzed with regard to content of specific methyltrienolone-binding sites. A correlation has been made between receptor content and clinical response to hormonal therapy in each case. Twelve specimens contained measurable amounts of steroid receptors. Of these, one patient died during irradiation therapy before onset of hormonal treatment. However, of the remaining 11 patients, 9 responded well to hormones (9/11 approximately 82%). The two receptor-positive nonresponders had the lowest measurable receptor levels in the series. Four specimens contained no detectable amounts of receptors. Three of these patients showed no response to therapy (3/4 = 75%) but one was "false negative." Our data indicate that steroid receptor analysis may become a valuable diagnostic tool in individualizing the therapy for prostatic cancer.

Influence of sex hormones on prostatic secretion protein, a major protein in rat prostate.

Prostatic secretion protein (PSP) or estramustine-binding protein is a major protein in rat ventral prostate. The amount of PSP was measured per mg of cytosolic protein at different ages and after castration or administration of sex hormones. The amount of PSP is relatively low before puberty (25 microgram/mg of protein) but increases at about 28 days of age to about 670 microgram/mg of protein and then decreases to a constant level of about 300 to 400 microgram/mg of protein, which is stable until at least 9 months of age. Following castration, the amount of PSP decreased relatively slowly, but 6 days after castration less than 20% of the original amount of PSP was detected. Treatment with testosterone propionate (1 mg/day) for 2 weeks (starting 2 weeks after castration) restored precastration levels of PSP. It is concluded that PSP is an androgen-sensitive protein, and it is suggested that PSP should be considered as a probe for estimation of androgenic action on the prostate. PSP is similar to the so-called prostatic binding protein as well as to prostatein, and it is quite possible that the three proteins represent one and the same entity.

Fractionation of the rat ventral prostate with respect to isolation and exocytosis of the prostatic secretion protein.

The ventral prostate was fractionated into one mitochondrial and three microsomal fractions. The different fractions were characterized morphologically and chemically. An interesting finding was that upon homogenization the endoplasmic reticulum membranes often turned 'inside-out' giving rise to microsomes with ribosomes attached to the inside of the vesicles. The secretion of the prostatic secretion protein was studied by means of isotopic pulse labeling using radioactive leucine. Peak radioactivity in the microsomal fraction was obtained at 2 h after injection with a relatively rapid fall. The radioactivity in the secretory fluid displayed a continuous increase up to 8 h followed by a plateau. When prostatic secretion protein was purified from secretory fluid and microsomes using a Con A-Sepharose column it showed a typical precursor-product relationship with an early peak at 60 min in microsomal prostatic secretion protein followed by a peak in secretory fluid at 4 h. Vinblastine blocked the release of labeled secretion protein into the secretory fluid, a phenomenon characteristic for secretory proteins which are exocytosed by means of fusion between secretory granules and the plasma membrane. Following intravenous injection of [3H]estramustine, accumulation was seen in the secretory fluid. Some estramustine probably binds to newly synthesized prostatic secretion protein and follows the same route of intracellular transport and extracellular discharge as does prostatic secretion protein.

Partial characterization of [3H]methyltrienolone binding in rat prostate cytosol.

3H-labelled R 1881 (methyltrienolone) was bound with high affinity (Kd, 2 . 10(-9) M) and low capacity (11--15 fmol/mg protein)in prostate cytosol from rats castrated 18 h earlier. The binding was inhibited by 5alpha-dihydrotestosterone, testosterone, cyproterone acetate and LEO 1727, but not by estradiol-17beta, R 5020 or corticosterone. The 3H-labelled R 1881-receptor complex was excluded from a Sephadex G=200 column, had a sedimentation coefficient of 3.5 S, was eluted from a DEAE-cellulose column at 0.2 M KCl concentration and precipitated between 20--40% of saturation of ammonium sulfate. 20% of specifically bound 3H-labelled R 1881 was retained by DNA-cellulose at low ionic strength. The fraction which was not bound to DNA-cellulose did not bind either, after rechromatography, not even following heating at 20 degrees C for 20 min or following fractionation with ammonium sulphate. In summary, the results indicate that the 3H-labelled R 1881-receptor complex in rat prostate cytosol has characteristics similar to those of the 5alpha-[3H]dihydrotestosterone-receptor complex.

Interaction of the hepatic receptor protein for 2,3,7,8-tetrachlorodibenzo-rho-dioxin with DNA.

2,3,7,8-Tetrachlorodibenzo-rho-dioxin (TCDD) binds to a specific, high-affinity, low-capacity protein in rat liver cytosol. The TCDD-receptor complex is a large molecule with a Stokes radius of 6.6 nm as determined by gel filtration on calibrated columns. The receptor complex sediments at 5.0 S on glycerol gradients. The calculated molecular weight from the physical parameters was 136 000 and the frictional ratio 1.79. The TCDD-receptor complex binds to DNA-cellulose without preceding heat activation or incubation at high ionic strength. The receptor must first bind TCDD before it can interact with DNA. The DNA-binding ability can be removed from the TCDD receptor by limited proteolysis with trypsin. This treatment does not affect the TCDD-binding site of the receptor. The proteolytic fragment of the TCDD-receptor complex containing the TCDD-binding site but not the ability to bind to DNA appears to be approximately the same size as the native receptor, as judged from chromatography of Sepharose CL-6B and glycerol gradient centrifugation.

High-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cell nuclei from rat liver.

The intranuclear binding of radioactive 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver has been studied both in vivo and in vitro. Following the intravenous administration of [1,6-3H]TCDD, a maximum uptake by cell nuclei could be observed at 2 h after injection with a concurrent decrease in the cytosolic uptake. Using linear sucrose density gradient centrifugation, dextran-coated charcoal adsorption assay, DEAE-Sepharose ion-exchange chromatography, competition, enzymatic and saturation studies, a high-affinity binding protein for TCDD in liver cell nuclei could be demonstrated both in vivo and after an exchange in vitro of intravenously administered unlabelled 2,3,7,8- tetrachlorodibenzofuran (TCDBF) for [3H]TCDD. Sucrose density gradient analysis showed a size of 4-5 S for both the cytosolic and nuclear TCDD binding entity. The specific binding of [3H]TCDD to nuclear components was heat labile and saturable and had an equilibrium dissociation constant of 1.05 nM. Based on a differential susceptibility to specific hydrolases, i.e. DNAase, RNAase, trypsin and pronase, the binding entity appears to be a 4-5 S salt-extractable protein.

Steroid receptor content in cytosol from normal and hyperplastic human prostates.

Analyses of steroid hormone receptors were performed using a dextran-coated charcoal technique in cytosolic preparations from 40 cases of benign prostatic hyperplasia (BPH) and 10 normal human prostates. Binding data were calculated according to Scathchard. In all BPH specimens, receptors for the synthetic androgen methyltrienolone (MT) were found (mean maximum number of binding sites, 566 fmol/mg DNA; mean Kd, 0.61 nM), and 25 of 28 samples contained progestin [17 alpha, 21-dimethyl-19-nor-4.8-pregnadiene-3,20-dione (R5020)] receptors (mean maximum number of binding sites, 420 fmol/mg DNA; mean Kd, 0.39 nM). No specimen contained glucocorticoid [dexamethasone (9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha, 21-trihydroxy-1,4-pregnadiene-3,20-dione); n = 16] or estrogen [17 beta-estradiol or 11 beta-methoxy-17 alpha-ethynyl-17 beta-estradiol (R2858); n = 26] receptors. No correlations were found between receptor content and age of the patients, weight of adenomas, or percentage of different cell types within the specimens. MT receptors were found in all normal prostates, while 5 of the specimens lacked progestin receptors. Estrogen receptors were found in 3 of the normal prostates, whereas none contained glucocorticoid receptors. The ligand specificity of the MT receptor in a normal prostate with minor amounts of progestin receptors was typical of an androgen receptor, and the ligand specificity of the R5020 receptor in a BPH specimen was typical of a progestin receptor. MT and R5020 had approximately the same affinity for the progestin receptor, whereas the relative binding affinity of R5020 for the androgen receptor was below 0.02 compared to that of MT. The androgen receptor was found to be more stable during repeated freezing and thawing than the progestin receptor.

Prediction of tumor response to endocrine therapy in prostatic carcinoma based on steroid receptor assay.

Measurement of estrogen receptor content in human breast cancer has become a valuable tool to predict the response of a tumor to endocrine manipulations. The aim of this study was to see whether steroid receptor assay could also be used to predict the value of endocrine therapy in human prostatic carcinoma. Biopsies from 25 primary tumors were analyzed with regard to quantity of methyltrienolone (a synthetic androgen) binding to the receptor 20 specimens were receptor-positive and 5 were receptor-negative. The correlation between receptor content and response to endocrine treatment was approximately approximately equal to 80%. The steroid receptor profiles of 5 lymph node metastases were also analyzed. 4/5 contained methyltrienolone receptors and 2/5 contained progestin receptors. 3/5 were glucocorticoid receptor-positive while all specimens were estrogen receptor-negative.

Pilot study on the growth inhibition by estramustine phosphate (Estracyt) of rat mammary tumours sensitive and insensitive of oestrogen.

Rat mammary tumours induced by 7,12-dimethylbenz(a)anthracene had a higher concentration of 3H and 14C than muscle after injection of estramustine phosphate labelled with 3H in the oestrogen moiety and 14C in the alkylating moiety. Thin-layer chromatography showed that dephosphorylated estramustine phosphate was present in the tumours but no free oestradiol-17beta. The uptake of the drug in the tumours was parallelled by a dose dependant retardation of tumour growth and a prevention of tumour number increase. Estramustine phosphate also retarded growth of mammary tumours resistant to treatment with oestradiol-17 beta. It is concluded that estramustine phosphate has a greater effect on tumour growth than oestrogen.

Treatment of advanced prostatic carcinoma with estramustine phosphate (Estracyt).

Estramustine phosphate (Estracyt) was used in the treatment of 154 patients with carcinoma of the prostate in stage IV. Sixty-three patients were given Estracyt from the outset (primary treatment group) and 91 had previously received some other endocrine therapy (secondary treatment group). All of the patients were observed for more than one year. The drug was given intravenously and/or orally. Objective remissions occurred in 46 (73.0%) of the 63 patients in the primary treatment group and subjective remissions in all the objective responders and in 12 additional patients (92.0%). The corresponding figures for the secondary treatment group were 28 (30.7%) and 52 (57.1%) of 91. The side-effects were negligible, and the drug was well tolerated. No cumulative toxic effect was observed in patients who had been receiving the treatment for more than five years. In our opinion the compound is valuable in the treatment of advanced prostatic carcinoma (stage IV).