Subcellular distribution in vivo of testosterone and salt extractability of nuclear androgen complexes in the prostate and prostatic adenocarcinoma: effect of estrogen treatment.

The distribution of injected [3H]testosterone into nuclear (Nt) mitochondrial-microsomal (Mp) and cytosolic fractions (Cs) obtained from hormone-dependent R-3327 Dunning tumor, dorsal prostate, ventral prostate and heart ventricle, as a non-target tissue, was studied. Since it has been suggested that salt resistant steroid receptor complexes may represent acceptor sites, extractability of nuclear androgen complexes with high KCl (0.6 M) solution was also determined. Both orchiectomized (Or) and estrogen treated (E2) rats were used. The distribution of bound radioactivity between Nt and Cs fractions was very similar in tumors, dorsal and ventral prostate, being approximately 50% and 10% in Nt and Cs, respectively. In heart the corresponding figures were 15% and 12%, respectively. The concentration of radioactivity/mg protein in nuclear KCl-extract (Ne) from tumors was approximately 10-fold higher than that in the salt-resistant (Nr) fraction and also nearly 10-fold higher than that in Cs. Similar distribution patterns were observed in dorsal and in ventral prostate, but the concentration in Ne from ventral prostate was higher than that from tumors or dorsal prostate. Both total and bound radioactivity in Cs from heart was similar to that in the tumors, whereas the concentration in Ne from heart was < 2% of that in Ne from tumors. No significant differences were found in the distribution of radioactivity in tumors or tissues obtained from Or or E2 rats.(ABSTRACT TRUNCATED AT 250 WORDS)

[Word processing of medical records and epicrises at a hospital department]. / Tekstbehandling av journaler og epikriser i en sykehusavdeling.

In January 1988 a pilot project was initiated at the Department of Neurology, University Hospital of Tromsø, to explore the possible use of word processing in routine operations of patient documentation. The project was evaluated at the end of ten months, and had resulted in a reduction of approximately 25% in the required written operations. Other notable results were reduced use of extra staff and overtime, and a general improvement in the staff's working conditions. The department's doctors reported no deterioration in the quality of the patient documentation produced using the new procedures. The successful outcome of the project was made possible by using the hospital's internal resources to develop suitable procedures for the instruction and supervision of the relevant staff, to install equipment, and to prepare a users' manual.

Estramustine-binding protein in rat and human prostate.

Estramustine phosphate, a nor-nitrogen mustard carbamate derivative of oestradiol-17 beta-phosphate, labelled with tritium in the oestradiol moiety, caused a higher concentration of radioactivity in rat prostate than did tritiated oestradiol-17 beta-phosphate or oestradiol-17 beta. Further studies in rat prostate demonstrated a protein that binds etramustine, the dephosphorylated metabolite of estramustine phosphate. The physical and chemical properties of this protein have been investigated, as has its presence in different organs of the rat and other species, including man. The finding of estramustine- binding protein in the human prostate gave rise to speculations concerning possible significance of estramustine phosphate's mechanism of action. The initial studies on estramustine-binding protein in rat and human prostate are briefly presented.

Mouse monoclonal antibodies against rat estramustine binding protein.

Hybridomas producing antibodies against rat prostatic estramustine binding protein (rEMBP) have been obtained by fusion of spleen lymphocytes from Balb/c mice, immunized with rEMBP, and SP2/0 Ag 14 mouse myeloma cells. Anti-rEMBP IgG producing cultures were identified in a solid phase ELISA using alkaline phosphatase conjugated sheep antimouse IgG. Cultures producing specific antibodies were subcloned and expanded. The produced immunoglobulins were characterized according to interaction with subunits of rEMBP. No interaction was found with [3H]estramustine-human estramustine binding protein. Following development of a radioimmunoassay, tissue distribution of rEMBP in the rat was studied. Despite high specificity, shown by selective interaction with rEMBP (F and S subunits), macromolecules interacting with anti-rEMBP were found in high concentrations in the prostrate and also in low concentrations, in other tissues of the male genital tract, and further in the adrenals, pancreas and submaxillary gland. The high specificity also makes it possible to study the F and S subunit selectively. These results show that macromolecules very similar to rEMBP are present in hormone-sensitive tissues in the rat.

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.

Influence of pituitary and adrenocortical hormones on prostatic secretion protein, a major protein in rat prostate.

Prostatic secretion protein (PSP) is an androgen-sensitive, quantitatively important protein in rat ventral prostate which has been shown to inhibit the nuclear uptake and decrease the DNA-binding capacity of the androgen-receptor complex. In the present study, the influence of the pituitary, adrenals, and gonads on the concentration of PSP in the prostate was studied. Hypophysectomy decreased the concentration of PSP to about 10% of the control level, an effect similar to that obtained by castration. No effects on prostatic wet weight or PSP concentration were observed following substitution of hypophysectomized rats with human growth hormone, rat prolactin, or rat growth hormone. On the other hand, PSP concentration as well as wet weight of the prostate were normalized in hypophysectomized rats after administration of testosterone propionate. These results are in line with a direct extrahypophyseal effect of androgens on the prostate. Adrenalectomy did not affect the concentration of PSP, nor the wet weight of the prostate. Administration of estramustine decreased the wet weight of the prostate but did not affect the prostatic concentration of PSP in normal rats. Combined treatment with testosterone propionate and estramustine seemed to increase both the wet weight and the concentration of PSP more than administration of testosterone propionate alone. These results indicate a synergistic effect between estramustine and testosterone propionate.

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.

Intracellular localization of estramustine in rat ventral prostate in vitro.

With the aim of studying the mechanism behind the effect of estramustine in the treatment of prostatic carcinoma, the intracellular fate of the drug has been investigated in rat ventral prostate in vitro. Minced tissue was incubated with [3H] estramustine under different conditions, homogenized, and submitted to isopycnic centrifugation on a sucrose gradient using the recently introduced vertical tube rotor. The subcellular localization of the drug was determined by comparison between the distribution of radioactivity in the gradient fractions and the activities of a number of marker enzymes. No metabolism of estramustine occurred as judged by thin-layer chromatography. After incubation of the minced prostate tissue for 1 hour at 30 degrees C with 0.15 microM of [3H] estramustine, most of the drug was recovered in the cytosol fractions which also contained the highest concentrations of the estramustine-binding protein. However, after incubation with 220 microM of estramustine, most drug equilibrated in heavier fractions with high concentrations of N-acetyl-beta-glucosaminidase and cathepsin B, marker enzymes for the lysosomes as well as NADPH:cytochrome c reductase, marker enzyme for the endoplasmic reticulum. Extending the incubation time and increasing the temperature reduced the amount of estramustine equilibrating in the heavy fractions and concomitantly increased the portion localized in the cytosol. During all incubation conditions, very little drug seemed to accumulate in the nuclei since the drug distribution was completely different from that of DNA. This suggests that effects other than interaction with nuclear DNA might be of importance for the cytotoxic effect of estramustine.

Influence of prostatic secretion protein on uptake of androgen-receptor complex in prostatic cell nuclei.

Prostatic secretion protein (PSP) is a major component of rat prostatic cytosol, and this protein is also found in the prostatic fluid. Purified PSP was found to inhibit the nuclear uptake of the [3H]methyltrienolone-receptor complex in vitro. Furthermore, purified PSP inhibited the binding of this androgen-receptor complex to DNA-cellulose. It is suggested that these effects of PSP may represent an intracellular control system regulating the concentration of PSP. Administration of estramustine, the dephosphorylated metabolite of the anti-cancer drug estramustine phosphate (Estracyt), to rats was found to decrease the weight of the prostate gland but to maintain the concentration of PSP, calculated as mg PSP/mg protein, at a constant level. In contrast, castration or administration of estradiol-17 beta valerate decreased the weight of the prostate gland as well as the concentration of PSP. These findings indicate that the mechanism of action of estramustine is at least partially different from that of estradiol-17 beta. Furthermore, it is suggested that estramustine may exert part of its action through its effects on the concentration of PSP.

The presence in rat and human prostate of proteins that bind steroid-cytostatic complexes.

During studies on the uptake and distribution of estramustine phosphate (Estracyt) in the rat, a major protein in the rat ventral prostate was found that binds estramustine, estromustine, and several other steroid nitrogen mustard complexes. This protein, called estramustine-binding protein, shares many physicochemical characteristics with alpha-protein, prostatic-binding protein, and prostatein, each reported to constitute a major steroid-binding protein in the rat ventral prostate. There is every indication that these four names designate one and the same protein, the binding properties of which favor the binding of lipophilic compounds, and which has shown an affinity for estramustine and closely structure-related compounds that is 100- to 1000-fold higher than for the natural steroids. Also, the human prostate has been shown to take up and bind estramustine and estromustine. However, the binding entity (or entities) is still under intense investigation.

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.

Purification and distribution of a major protein in rat prostate that binds estramustine, a nitrogen mustard derivative of estradiol-17 beta.

A protein in rat ventral prostate cytosol that binds estramustine [estradiol 3-bis(2-chloroethyl)carbamate) was purified to homogeneity by using chromatography on DEAE-cellulose, Sephadex G-100 (superfine), octyl-Sepharose (CL-4B, and polyacrylamide gel electrophoresis. The estramustine-binding protein was found to have a Mr of 46,000 as estimated by gel filtration. After analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the protein was found to consist of two subunits with Mr of about 20,000 and 18,000. After reduction of disulfide bridges, the protein was decomposed into three components with Mr of about 12,000, 11,000, and 8000. Amino acid analysis indicated that the protein is a glycoprotein. Antibodies against the protein were raised in rabbits and a radioimmunoassay was developed for it. The estramustine-binding protein constituted about 18% of the total protein in rat ventral prostate cytosol, was present in the dorsal and lateral lobes of the prostate, and was also detected in the pituitary gland, cerebral cortex, submaxillary gland, thyroid gland, adrenal gland, seminal vesicle, coagulating gland, epididymis, and preputial gland of the male rat. In female rats the protein was detected in cerebral cortex. Because the estramustine-binding protein is predominantly found in the accessory sexual glands of the male rat, it may be of importance for maintaining male fertility.

Autoradiographic studies of 3H-estramustine in the rat ventral prostate.

The alkylating agent 3H-estramustine was administered to castrated male rats and found to accumulate in the epithelium of the ventral prostate 5 and 20 min. following intravenous administration, as judged from autoradiography. Four hrs. after intravenous injection of this isotope, the radioactivity was recovered in the secretion of the prostatic lobuli. A preferential accumulation of radioactivity in prostatic secretion was also observed 2 hrs. after intramuscular administration of 3H-estramustine to intact or castrated rats. In contrast, 3H-oestradiol and 3H-testosterone, which were also taken up by the ventral prostatic epithelium, were not recovered in prostatic secretion. The present results indicate that 3H-estramustine is secreted from the prostatic cells into the lumina of the prostatic lobuli. It is speculated that a recently detected estramustine-binding protein in rat ventral prostate may be involved in this process and that the present finding may be of relevance in the understanding of the mechanism of action of the chemotherapeutic agent Estracyt (estramustine phosphate) used in the treatment of advanced prostatic carcinoma.