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Biomedical subjects

N Bruchovsky

Publications and source records attributed to N Bruchovsky.

At least 91 records · Page 5Linked to original sources
PubMed1981

Radioimmunoassay measurements of nuclear dihydrotestosterone in rat prostate. Relationship to androgen receptors and androgen-regulated responses.

The concentration of dihydrotestosterone was measured by radioimmunoassay in nuclear and cytoplasmic extracts from rat ventral prostates. In the regenerating prostates of castrated rats treated with dihydrotestosterone for 4 days, the nuclear concentration of this steroid increased from approx. 70nM to 800nM as a linear function of the injected dose, whereas the cytoplasmic concentration remained relatively constant (70-130nM). Isotope-exchange measurements of nuclear androgen receptors by using [3H]methyltrienolone indicated that, although the concentration of nuclear dihydrotestosterone was several-fold higher than the concentration of androgen receptors, they were logarithmically related. The recruitment of prostatic cells into the growth fraction and the stimulation of 5 alpha-reductase activity were more directly correlated to the nuclear concentration of androgen receptors than to the total nuclear concentration of dihydrotestosterone. Maximal restoration of a specific isoenzyme of acid phosphatase ws achieved when approx. 2000 androgen receptors were present in the prostatic nuclei; higher concentrations of nuclear androgen receptors were associated with decreased amounts of this enzyme. Hence the results imply, first, that the total amount of dihydrotestosterone accumulated by nuclei is not a direct consequence of carrier-mediated transport by androgen receptors, and, secondly, that, whereas acid phosphatase may be differentially controlled by androgens in the regenerating prostate, increases in the amount of cell proliferation and 5 alpha-reductase activity directly parallel increases in the nuclear concentration of androgen receptors.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1981

The androgenic regulation of superhelical-DNA nicking-closing enzyme in rat ventral prostate.

The activity of superhelical-DNA nicking-closing enzyme (NC enzyme) was measured in nuclei from rat ventral prostate by a fluorimetric assay based on the binding of ethidium bromide to supercoiled phage-PM2 DNA. The nuclear concentration of NC-enzyme activity declined rapidly after castration, although this response could be prevented by daily administration of dihydrotestosterone. The low NC-enzyme activity in involuted prostates (10% of normal) was restored to normal after 8-10 days of treatment with androgen. In the regenerating prostate the time course of restoration of NC-enzyme activity was not in phase with that of DNA synthesis. Examination of nucleosome repeat lengths and the arrangement of nucleosomes along the chromatin fibre revealed no differences in the structural organization of chromatin in prostates with high or low NC-enzyme activity. Together, these results suggest that the major role of NC enzyme is related to the onset and maintenance of differentiation in the prostate and that the activity of this enzyme is not expressed through gross alterations in chromatin structure.

Animals↗
PubMed1981

Partial purification of nuclear androgen receptor by micrococcal nuclease digestion of chromatin and hydrophobic interaction chromatography.

Extensive (20%) digestion of linker DNA of prostatic chromatin with micrococcal nuclease resulted in the precipitation of 95% of the nuclear androgen receptors. The receptor-enriched precipitate was dissolved in Tes buffer, pH 7.0, containing 0.6--1.2 M NaCl and analysed by hydrophobic interaction chromatography. The adsorption of receptor to omega-amino-alkyl derivatives of agarose increased with the length of the alkyl substituent indicating the presence of hydrophobic regions on the surface of the receptor molecule. Digestion of linker DNA followed by chromatography of precipitated chromatin proteins using 5-aminohexyl-agarose gave rise to a mean 93-fold purificaton of receptor with a recovery of 45%. This approach to the partial separation of nuclear androgen receptor may prove useful in conjunction with more selective purification techniques such as affinity chromatography.

Animals↗
PubMed1981

Steroid receptors in adjuvant hormonal therapy for breast cancer.

Using a computerized mathematical model the authors found that either a small [5%] or large [50%] hormone-dependent reduction in tumour cell population at the beginning of adjuvant chemotherapy for breast cancer greatly increases the probability fo achieving a cure. The fact that even a minor response to hormonal therapy early in the treatment of the tumor is effective underlines the importance of using estrogen receptors to to select potential responders in whom a cure is at least a strong theoretical possibility.

Breast Neoplasms↗
PubMed1980

Nuclear binding of androgens and acid phosphatase activity in prostatic tumors of Nb rats.

A transplantable prostatic adenocarcinoma derived from the dorsal lobe of the prostate gland of an Nb rat was analyzed for the concentration of nuclear androgen-binding sites and the presence of acid phosphatase activity. When extracts of nuclei from normal prostatic tissue were labelled with [1,2-3H]dihydrotestosterone in the absence and presence of competitor, two types of specific binding were observed: type 1 was characterized by an association constant (Ka) of 6 x 10(7) M-1 and involved a molecule that was excluded from Sephadex G-200; type 2 was characterized by a Ka of 3 x 10(8) M-1 and depended on a binding component that was retained by Sephadex G-200. Nuclei from androgen-stimulated tumors contained reduced concentrations of both androgen-binding components, whereas nuclei from autonomous tumors had only a trace amount of type 1 sites and were entirely devoid of type 2 sites. In all tumors the acid phosphatase activity per mg of protein was markedly elevated. Relative to normal, the activity of this enzyme was 140% and 350% higher in androgen-stimulated and autonomous tumors, respectively. These findings indicate that prostatic tumors are characterized by a decrease in nuclear androgen-binding, and an increase in specific activity of acid phosphatase, and also that such changes are more pronounced in autonomous than in androgen-stimulated tumors.

Acid Phosphatase↗
PubMed1980

Plasminogen activator in prostatic tumors of Nb rats.

Using a fluorometric assay, nonspecific proteolytic activity and plasminogen activator were measured in transplantable tumors of the dorsal prostate of Nb rats. Nonspecific proteolytic activity in prostatic tumors did not differ significantly from that measured in normal dorsal prostate, whereas plasminogen activator activity, undetectable in the latter tissue, was readily measurable in the tumors. Furthermore, plasminogen activator activity in prostatic tumors characterized by hormone-insensitive growth was 8-fold higher than in tumors characterized by androgen-stimulated growth. In both typess of tumors, the plasminogen activator activity per mg protein was highest in the lysosomal fractions. The results indicate that plasminogen activator may be a useful marker for discriminating between androgen-stimulated and autonomous prostatic tumors.

Adenocarcinoma↗
PubMed1980

Stromal 5 alpha-reductase activity is elevated in benign prostatic hyperplasia.

The activities of 5 alpha-reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase were assayed in homogenates of stroma and epithelium obtained from 3 normal, 9 hyperplastic and 2 carcinomatous human prostates. Irrespective of the normal or abnormal condition of the prostate, the localization of 5 alpha-reductase was predominantly in stroma whereas the reductive and oxidative activities of 3 alpha (beta)-hydroxysteroid dehydrogenase were more evenly divided between stroma and epithelium. Furthermore, the mean specific activity of 5 alpha-reductase in hyperplastic stroma at 84.6 plus or minus 13.1 (plus or minus SEM) pmol 30 min-1 mg protein-1 was almost 3 times greater (Student's t-test, P < 0.05) than the corresponding value in normal stroma, 31.6 plus or minus 7.2 pmol mg protein-1 30 min-1. We conclude that the stroma is the primary site of conversion of testosterone to dihydrotestosterone in the human prostate owing to the prevalence in stroma of 5 alpha-reductase, and that benign prostatic hyperplasia is characterized by an increased amount of stromal 5 alpha-reductase activity.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Assay of nuclear androgen receptor in human prostate.

Nuclei were recovered from normal and hyperplastic human prostates and from well differentiated carcinoma using triton N-101 and discontinuous sucrose density gradient centrifugation. The nuclei were sonicated and dissolved in 2-([2-hydroxy-1,1-bis(hydroxymethyl)ehtyl]amino)ethane sulfonic acid buffer, pH 7.0, containing 0.6 M. sodium chloride. After incubation of the nuclear extract in the presence of 2 to 20 nM. 3H-dihydrotestosterone at 4C for 18 hours, an androgen receptor was isolated by Sephadex G-25/G-200 dual-column chromatography. The receptor demonstrated greater specificity for testosterone and dihydrotestosterone than for cortisol, progesterone and 17beta-estradiol. It was characterized by a sedimentation coefficient of 3 S and a Kd of 4.5 x 10(-9) M. The mean concentration of the nuclear androgen receptor, in terms of molecules per nucleus, was normal prostate-1,000, hyperplastic prostate-1,400 and well differentiated carcinoma-1,900. With this assay the problems associated with the measurement of a cytoplasmic androgen receptor can be avoided.

Aged↗
PubMed1979

Increased ratio of 5 alpha-reductase: 3 alpha (beta)-hydroxysteroid dehydrogenase activities in the hyperplastic human prostate.

The activities of 5 alpha-reductase and 3 alpha (beta)-hydroxysteroid dehydrogenase were assayed in homogenates of eight normal, 21 hyperplastic and four carcinomatous human prostates. Samples consisting of 300--500 microgram tissue protein in Tris buffer, pH 7.0, were incubated at 37 degrees C for 30 min in the presence of 50 nM-[3H]androgen and an NADPH-generating system started with 5 X 10(-4)M-NADP. The yield of 5 alpha- and 3 alpha-reduced metabolites, as established by using t.l.c. and g.l.c., gave an estimate of enzyme activity. The formation of metabolites denoting 5 alpha-reductase activity in normal, hyperplastic and carcinomatous tissue respectively was 28.8 +/- 47 (S.E.M.), 76.8 +/- 8.9 and 3.5 +/- 0.7 pmol 30 min-1 mg protein-1; similarly, that denoting 3 alpha (beta)-hydroxysteroid dehydrogenase activity was 69.3 +/- 6.7, 46.6 +/- 5.7 and 38.8 +/- 22.1 pmol 30 min-1 mg protein-1. In all normal prostates 5 alpha-reductase activity was lower than 3 alpha (beta)-hydroxysteroid dehydrogenase activity. Conversely, in 18 out of 21 hyperplastic prostates, 5 alpha-reductase activity was higher than 3 alpha (beta)-hydroxysteroid dehydrogenase activity. The effect of the increase in 5 alpha-reductase activity without a compensatory change in 3 alpha (beta)-hydroxysteroid dehydrogenase activity was to alter the mean ratio between 5 alpha-reductase and 3 alpha (beta)-hydroxysteriod dehydrogenase activities from 0.47 +/- 0.11 in the normal prostate to 1.84 +/- 0,19 in hyperplastic tissue. It is inferred that this change may predispose the hyperplastic prostate to asymmetrical rates of androgen metabolism and thereby contribute to the abnormal accumulation of dihydrotestosterone.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Androgen receptors in benign and malignant prostatic tissue.

A new procedure has been developed to purify prostatic cell nuclei and to achieve a high yield of nuclear material for study. This technique allowed the investigation of the enzyme 5-alpha-reductase and its relationship to the translocation of dihydrotestosterone and androgen receptors into the nucleus of the prostatic cell. The studies revealed that 5-alpha-reductase was localized chiefly to the stromal components of all types of prostatic tissue. Although there is a higher concentration of dihydrotestosterone in carcinoma tissue the 5-alpha-reductase activity is low. Studies on the effect of androgens and antiandrogens on nuclear uptake of androgen revealed that androgens are potent stimulants of receptor translocation, while antiandrogens stimulate little or no translocation.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1978

Mechanisms of replenishment of nuclear androgen receptor in rat ventral prostate.

1. The concentration of androgen receptor in the nucleus of the prostatic cell is rapidly elevated by the administration in vivo of 2mug of [(3)H]testosterone to 1-day-castrated rats. From a concentration of 2300 receptors/nucleus at 5min after intravenous injection of hormone, there is an increase to 21000 receptors/nucleus at 60min. At the same time, the amount of binding of androgen in the cytoplasm remains constant at a relatively low value. 2. An identical dose of [(3)H]testosterone administered to 7-day-castrated rats produces a much smaller change in the concentration of nuclear receptor, from 700 receptors/nucleus at 5min to only 4300 receptors/nucleus at 60min. Thus the reservoir from which nuclear receptor is replenished is considerably smaller in regressed prostatic cells. Again, the amount of binding of androgen in the cytoplasm remains unchanged at a low value over the experimental time course of 60min. 3. In contrast with the scant labelling of cytoplasmic receptor achieved by injecting animals with [(3)H]testosterone, labelling in vitro, by incubation of tissue slices with radioisotope, indicates that prostate of 1-day-castrated animals actually contains 21400 receptors/cell in the cytoplasmic compartment, and prostate of 7-day-castrated animals 3000 receptors/cell. 4. Owing to the similarity between the concentration of nuclear receptor measured in vivo and the concentration of cytoplasmic receptor measured in vitro, the labelling techniques in vivo and in vitro were used in sequence to demonstrate the movement of most of the cytoplasmic receptor into the nucleus. In the 5-60min interval after the administration of [(3)H]testosterone to 1-day-castrated rats, a decrease of 17400 receptor molecules in the cytoplasm is exactly mirrored by an increase of 17200 receptor molecules in the nucleus. 5. These results imply that, in prostate of 1-day-castrated rats, nuclear receptor is replenished exclusively by translocation of cytoplasmic receptor. However, in the regressed prostate of 7-day-castrated rats, only about 25% of the nuclear receptor is replenished through translocation of existing cytoplasmic receptor. The remainder is ultimately synthesized during new rounds of cell division induced by hormone.

Animals↗
PubMed1976

The relationship between adrogen receptors and the hormonally controlled responses of rat ventral prostate.

1. The administration of dihydrotestosterone to rats orchidectomized 7 days previously stimulated the synthesis of nuclear receptor in prostatic cells several hours in advance of DNA synthesis and mitosis. 2. The synthesis of nuclear receptor is tightly coupled to cell proliferation; consequently, in resting cells, there is no further net synthesis of nuclear receptor above the maximum of approx. 8000 molecules/cell. 3. After orchidectomy a rapid decline in the concentration of free androgen in the nuceus and a slower decline in the concentration of nuclear receptor are observed. 4. Owing to the apparent scarcity of receptor-inactivating factors in the nucleus, and the inverse relationship between amounts of nuclear and cytoplasmic receptors, it is concluded that the nuclear receptor is discharged into the cytoplasm after orchidectomy. 5. The formation of the cytoplasmic receptor is an early event preceding the onset of cellular autolysis. 6. Regressing prostate develops the capacity to eliminate cytoplasmic receptor, and this capacity is retained by the regenerating prostate for at least 14 days. 7. The synthesis of nuclear receptor in early G1 phase may control the entry of cells into the cell cycle and the prolonged retention of receptor in the nucleus may prevent the activation of autophagic processes.

Animals↗