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Biomedical subjects

N Bruchovsky

Publications and source records attributed to N Bruchovsky.

At least 73 records · Page 4Linked to original sources
PubMed1987

DNA and protein components of nuclear acceptor sites for androgen receptors in the rat prostate.

Androgen receptor-acceptor complexes in nuclei from rat ventral prostates were cross-linked in situ with formaldehyde and partially purified using affinity chromatography. To isolate acceptor DNA, the cross-linked receptor-acceptor complexes in formaldehyde-treated chromatin samples were adsorbed to dihydrotestosterone-17 beta-succinyl agarose, eluted with 75 microM dihydrotestosterone-1% SDS, digested with proteinase K and extracted with phenol-chloroform. After 32P end-labelling and PAGE, this DNA contained two distinct bands of DNA (about 300 and 400 base pairs respectively) which were unique relative to the total prostatic DNA. As an alternative approach for characterizing acceptor DNA, the DNA in prostatic nuclei and cross-linked chromatin was labelled with 32P by nick translation and analysed in glycerol density gradients for associations with cross-linked androgen receptors. A symmetrical 7s peak of 32P-DNA with a small amount of coincident receptor was observed in the gradients after mild trypsin treatment. In the absence of trypsin treatment, both the cross-linked receptors and the labelled DNA sedimented to the bottom of the gradients. Isolation of acceptor proteins involved iodination of cross-linked chromatin with 125I and androgen affinity chromatography. A comparison of the relative efficiency of retention and elution of 125I-proteins from different affinity columns revealed that testosterone-17 beta-succinyl agarose was potentially most suitable for purification of acceptor proteins. After electrophoresis on SDS-polyacrylamide gels, the eluates from this type of affinity matrix were found to contain two major peaks of 125I-labelled proteins--one corresponding to a protein with a similar molecular weight as the nuclear androgen receptor (33,000 Da); the other having a molecular weight of 20,000 Da. While the precise identity of this latter entity is unknown, its enrichment and retention by the affinity gel implies that it is closely associated with the androgen receptor and may be a component of the acceptor sites.

Animals↗
PubMed1986

Changes in endonuclease activity during growth and regression of the Shionogi mammary carcinoma.

Autodigestion of nuclei isolated from the androgen-dependent Shionogi mouse mammary carcinoma revealed the presence of endogenous endonuclease activity which was stimulated by Ca2+ and Mg2+ and generated a repeating pattern of DNA fragments with an average monomeric size of 179 base pairs. The nuclear concentration of endonuclease activity declined rapidly after castration to a level 45% of that measured in tumours from non-castrated hosts. Most of the endonuclease activity (80%) could be extracted in a soluble form after sonication of the nuclei and subsequent centrifugation. The reduced concentration of endonuclease activity in regressing tumours following castration was not due to changes in chromatin template conformation and could be restored to normal levels within 1 day of androgen treatment. Examination of DNA synthetic activity and the concentration of nuclear androgen receptors during tumour regression indicated that the decrease in endonuclease activity paralleled the declining rate of DNA synthesis but was preceded by a loss of nuclear androgen receptors. Together these results suggest that the endonuclease activity of the Shionogi carcinoma is more likely involved with androgen-stimulated cell proliferation rather than with the autophagic mechanism responsible for tumour regression and cell death.

Androgens↗
PubMed1985

In situ cross-linking of androgen receptors to nuclear acceptor sites of rat prostate with formaldehyde.

Androgen receptors were attached covalently in situ to their nuclear acceptor sites with the contact site cross-linker, formaldehyde. Chromatin, prepared from sonicated nuclei of rat prostate, was labeled by isotope exchange with [3H]dihydrotestosterone and found to contain 19,000 +/- 900 (mean +/- S.E.) salt-extractable androgen receptors/nucleus which sedimented in the 3-4 S region of 7.6-76% (v/v) glycerol gradients and at a density of approximately 1.28-1.35 g/ml in CsCl gradients. After incubation of the chromatin with 0.5% (w/v) formaldehyde for 1 h at 4 degrees C, there was a 90% reduction in the concentration of free androgen receptors and an increase in the density of the androgen binding sites recovered from CsCl gradients. Extensive digestion of the cross-linked chromatin with micrococcal nuclease liberated 18% of the androgen receptors as 3-4 S entities and caused an overall decrease in the density of the receptor-acceptor complexes. Ribonuclease digestions had no effect on the androgen receptors cross-linked to chromatin. Mild digestion of the cross-linked preparations with trypsin, alone or in combination with micrococcal nuclease, resulted in the release of 74% and 97% of the androgen receptors, respectively. Together, these findings imply that two classes of receptor-acceptor complexes are present in prostatic chromatin--one, containing about 20% of the androgen receptors in which the receptors are in direct contact with DNA but not with proteins and the other, containing most of the androgen receptors in which the receptors are adjacent to acceptor proteins but not to DNA.

Animals↗
PubMed1985

Elevated growth hormone levels in sera from breast cancer patients.

The concentrations of growth hormone (GH) and prolactin (PRL) in the serum of 42 breast cancer patients were determined by radioimmunoassay (RIA). Forty percent of the patients had elevated GH levels while only 17% had elevated PRL levels. These findings suggest a relationship between GH and breast cancer; a weaker correlation exists between PRL and this malignancy. In addition, total lactogens in the serum were measured by a bioassay (BA). The BA/RIA (GH + PRL) ratio was greater in the breast cancer patients than the controls, indicating that variant forms of the hormones with higher than normal biological activity might be present.

Adult↗
PubMed1985

Bioactive forms of serum lactogens: effects of treatment of prolactinoma patients with bromocriptine.

An in vitro bioassay for lactogenic hormones, based on the specific mitogenic effects of lactogens on cultured Nb2 node rat lymphoma cells, was used to measure the levels of lactogens in whole serum and in fractionated serum samples from three patients with prolactin (PRL) secreting pituitary adenomas. Under basal conditions, after pituitary stimulation, and following treatment with bromocriptine which greatly decreased the serum PRL levels, the bioassay (BA) results closely paralleled fluctuations in the sum of radioimmunoassay (RIA) estimates of serum PRL and growth hormone concentrations. The extreme sensitivity (10 pg/ml) of the BA facilitated measurement of PRL in fractions obtained after Sephadex G-100 chromatography of only 0.1 to 0.3 ml of sera from both untreated and bromocriptine-treated prolactinoma patients. In both types of samples, similar heterogenous patterns of bioactive PRL were observed, although most of the recovered activity appeared to be due to monomeric PRL. BA and RIA of fractions obtained after DEAE cellulose chromatography of sera from untreated prolactinoma patients revealed two PRL peaks, the first one containing 75% of the activity. Therapy with bromocriptine led to a reduction in the total serum PRL activity and, in particular, in the activity of the second peak.

Adenoma↗
PubMed1985

Variability of androgen-related phenotypes in the Shionogi mammary carcinoma during growth, involution, recurrence, and progression to hormonal independence.

Several parameters of androgen action were measured in hormone-dependent Shionogi carcinoma cells during phases of growth, regression, and recurrence. In the parental C1 line under steady state conditions, dihydrotestosterone is localized exclusively in the nucleus while testosterone is confined almost entirely to the cytoplasm. After castration, the concentration of testosterone declines more rapidly than that of dihydrotestosterone. Spontaneous recurrent growth is not accompanied by significant elevation of the whole-tissue concentration of either androgen. Neither are changes observed in the concentration of cytoplasmic receptor or in the rate of uptake of androgens into the nucleus. However, relapse is associated with the appearance of a glucose-6-phosphate dehydrogenase double-enzyme phenotype and a loss of responsiveness to androgen withdrawal. The autonomous C3 variant line which is devoid of androgen-related markers is characterized by a deficiency of androgen retention by whole tissue and possibly a permeability defect of the plasma membrane. This variant tends to express a glucose-6-phosphate dehydrogenase double-enzyme phenotype. In contrast, the autonomous C4 variant line retains the ability to concentrate modest levels of testosterone in whole tissue and high levels of dihydrotestosterone in the nucleus. Although the number of nuclear binding sites is the same as that observed in the parental C1 line, the concentration of cytoplasmic receptor and the rate of nuclear uptake of androgens are relatively decreased. Expression of a glucose-6-phosphate dehydrogenase double-enzyme phenotype is less frequent than in the autonomous C3 variant line. The above results suggest that a recurrent tumor may contain hormone-sensitive cells which resume growth in an androgen-depleted environment. They also imply that progression from the androgen-dependent to the autonomous condition involves the selection and outgrowth of hormone-insensitive cells of variable phenotype.

Androgens↗
PubMed1984

Vinblastine and vincristine--growth-inhibitory effects correlate with their retention by cultured Nb 2 node lymphoma cells.

Vinblastine (VLB) and vincristine (VCR) have been compared with respect to their inhibitory effects on the growth of cultured Nb2 node rat lymphoma cells and their binding and release by these cells. When examined over a wide concentration range, the two alkaloids were found to be almost equally effective in inhibiting culture growth, providing they were continuously present in the incubation medium. A striking difference was found, however, when the cells were incubated for short periods with the alkaloids and then restored to alkaloid-free medium for further incubation. Cells treated with VCR (5 X 10(-9) g/ml) for 3 h and washed free of extracellular alkaloid failed to resume normal proliferation during a subsequent 48 h incubation period in alkaloid-free medium. In contrast, cells treated with VLB at a higher concentration (5 X 10(-8) g/ml) and for a longer time (7 h), rapidly resumed growth in alkaloid-free medium. Binding studies, using (3H)VLB and (3H)VCR, showed that when cells were incubated continuously with the alkaloids at a given concentration, they bound equal amounts of the drugs. When labelled cells were resuspended in alkaloid-free medium, VLB was released very readily by the cells, whereas VCR was tenaciously retained. The findings indicate that the growth-inhibitory effects of VLB and VCR are related to the amount of alkaloid associated with the cells and that the long-lasting effects of VCR, relative to VLB, are very likely due to the retention of VCR by the cells when the extracellular alkaloid levels decline. The study also suggests that, in the treatment of malignancies, VLB could be more effective if its extracellular levels were maintained, e.g. by continuous administration.

Animals↗
PubMed1984

Increased activity of plasminogen activators during involution of the rat ventral prostate.

Plasminogen activator was measured in the ventral prostates of non-castrated, castrated, and androgen-treated rats to determine whether changes in this activity correlated with the process of glandular involution. While the activity was very low in cytosolic extracts from the prostates of non-castrated rats, 2 days following castration the plasminogen activator activity increased in a near-linear fashion such that by day 7 it was 10-fold higher in terms of specific activity (per mg of protein) and cellular concentration (per mg of DNA). During this interval there was a rapid decrease in the cell population of the prostates. Treatment of the 7-day castrated rats with the potent androgen, dihydrotestosterone, both reduced the plasminogen activator activity and restored the cell number in a dose-related manner. Gel electrophoretic analysis revealed two major bands of plasminogen activator activity in the cytosolic extracts from 4- and 7-day castrated rats, plus additional minor bands in samples from 10- and 14-day castrated rats. Approx. 10% of the cellular concentration of plasminogen activator activity was recovered in association with an 18000g pellet fraction from the prostates; this fraction showed less heterogeneity of the plasminogen activator forms as observed by gel electrophoresis. Inhibitor studies indicated that the 18000g pellet fraction from the prostates of non-castrated rats possessed some plasminogen activator inhibitor activity, but the relative concentration of the inhibitor activity was small. We conclude that the involution of the prostate is probably associated with increased synthesis of plasminogen activators through a de-repression process which may involve loss of androgen receptors.

Animals↗
PubMed1984

Bioassay and radioimmunoassay of lactogens in sera from children.

Lactogen levels in sera from children have been determined using the Nb 2 lymphoma cell bioassay (BA) and conventional radioimmunoassay (RIA). Assays were done on samples obtained under basal conditions and after pituitary stimulation induced by insulin or arginine administration. There was a close correspondence between BA and RIA results (r = 0.94; n = 43). The average ratio of the BA and RIA estimates of the lactogen levels (BA/RIA) was 0.86 +/- 0.13 (mean +/- SD) and 0.82 +/- 0.17 for basal and stimulated conditions, respectively. The increased secretion of lactogens after pituitary stimulation was not found to be associated with a change in the BA/RIA ratio.

Adolescent↗
PubMed1984

Synthesis and evaluation of immobilized androgens for affinity chromatography in the purification of nuclear androgen receptor.

Seven biospecific adsorbents containing immobilized androgens were synthesized: dihydrotestosterone-17 beta-succinyl agarose, and both the unsubstituted and the 17 beta-acetyl derivatives of dihydrotestosterone-7 alpha-undecanoyl agarose, testosterone-7 alpha-undecanoyl agarose, and 19-nortestosterone-7 alpha-undecanoyl agarose. The retention capacities for nuclear androgen receptor were generally between 40-80% with little variation in reproducibility; the amount of binding was greatest with dihydrotestosterone-17 beta-succinyl agarose and dihydrotestosterone-17 beta-acetoxy-7 alpha-undecanoyl agarose. Rapid flow rates were obtained with all gels, and no tendency for decomposition was observed over a period of 1 year. Factors that affected retention included the concentration of immobilized androgen, length of the linker arm, occupation of receptor sites, interval of contact with the gel, and temperature of incubation. Chemical dissociation of androgens from androgen receptor complexes with 0.2 mM mersalyl increased the retention of receptor by dihydrotestosterone-17 beta-succinyl agarose. Two elutants showed promise for the dissociation of gel-bound receptor: 1) 0.2 mM mersalyl in the presence of 1.5 mg/ml of ovalbumin; 2) 10% (v/v) dimethylformamide:water containing 30 microM [1,2-3H] dihydrotestosterone and 0.5 M sodium thiocyanate.

Androgens↗
PubMed1983

Isolation of 3 S androgen receptors from salt-resistant fractions and nuclear matrices of prostatic nuclei after mild trypsin digestion.

The physical properties of two types of androgen-binding sites in prostatic nuclei were compared and found to be identical. The first type was released from chromatin by micrococcal nuclease digestion and solution in 0.6 M NaCl; the second resisted such treatment and remained associated with nuclear structures. After in vivo administration of [1,2-3H]testosterone to 24-h castrated rats and sonication of purified nuclei, 90% of the nuclear radioactivity was extracted with nuclease/salt treatment and was found by sucrose density gradient analysis to be associated with a 3 S androgen receptor. If sonication was omitted, 50 to 60% of the nuclear radioactivity was recovered in the nuclease/salt-resistant pellets or bound to nuclear matrices. Mild digestion of either of these particulate fractions with trypsin resulted in the release of a 3 S androgen receptor. After in vitro isotope-exchange labeling with [1,2-3H]dihydrotestosterone, the sedimentation coefficient, steroid specificity, and dissociation constant of the androgen receptors released by trypsin digestion of nuclease/salt-resistant pellets or nuclear matrices were similar to those of the receptors extracted by nuclease/salt treatment. These results indicate first, that all androgen-binding sites in prostatic nuclei can be released, either with nuclease/salt or trypsin digestion procedures to yield a 3 S androgen receptor with uniform binding characteristics, and second, that the androgen receptors are distributed between two intra-nuclear pools--one containing about 10,000 molecules/nucleus sensitive to micrococcal nuclease digestion and salt and the other containing about 8,000 to 13,000 androgen receptors tightly bound to the nuclear matrix.

Animals↗
PubMed1983

5 alpha-reductase activity in stroma and epithelium of rat prostate and epididymis. A contribution to elucidation of the mechanism for development of hyperplastic growth of prostatic tissue.

The 5 alpha-reductase activity was assayed in homogenates of stroma and epithelium in the rat ventral prostate and epididymis. Samples consisting of a 0.3 mg/ml tissue protein in TES buffer, pH 7.0 were incubated at 37 degrees C for 30 min in the presence of 50 nM [1,2-3H]testosterone and a NADPH-generating system started with 5 x 10(-4) M NADP. The yield of 5 alpha-reduced metabolites, as established by using thin-layer chromatography, gave an estimate of enzyme activity. Whereas the specific activity of 5 alpha-reductase was highest in prostatic stroma and epididymal epithelium, most of the total enzyme activity was associated with the epithelium in both the prostate and epididymis. The effect of dihydrotestosterone on specific activity of 5 alpha-reductase was studied by administering the hormone to 7-day castrated rats. In prostate, the specific activity of both stromal and epithelium forms of the enzyme reached a maximum after 4 days of treatment. In epididymis only the epithelial form of 5 alpha-reductase underwent a major change in specific activity, the latter peaking after 8-12 days of treatment. Furthermore, while the total activity of 5 alpha-reductase in the prostatic tissue fractions could be induced by as much as 4-fold the normal control values, the epididymal enzyme could not be induced above the normal level either in the stroma or the epithelium. This may explain the relative resistance of epididymis to abnormal growth stimulation under the influence of hormones.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1983

Kinetic analysis of 5 alpha-reductase isoenzymes in benign prostatic hyperplasia (BPH).

Kinetic parameters (KM and Vmax) of the 5 alpha-reductase activities in homogenates of stroma and epithelium, isolated from BPH tissue, were determined using both testosterone and progesterone as substrate. The mean KM values for stromal 5 alpha-reductase, at 67.9 nM and 27.7 nM for testosterone and progesterone respectively, were 3-4-fold higher than the comparable KM estimates for the epithelial enzyme. The KM estimates for the epithelial 5 alpha-reductase showed little variation whereas those measured in the stromal homogenates could be subgrouped at less than 50 nM and greater than 100 nM. The mean Vmax for the stromal 5 alpha-reductase was approximately 235 pmol/30 min/mg protein irrespective of the substrate used; a value 10-fold higher than the Vmax of the epithelial 5 alpha-reductase. Preliminary experiments with inhibitors of 5 alpha-reductase demonstrated that the stromal and epithelial enzymes differed in their relative sensitivity to these compounds. Three major conclusions can be drawn from these results: first, that progesterone is a better substrate for prostatic 5 alpha-reductase than testosterone; second, that BPH tissue has at least two isoenzymes of 5 alpha-reductase--one in the epithelium and one (or more) in the stroma; and third, in hyperplastic prostates, most of the 5 alpha-reductase molecules are located in the stroma.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1982

Plasminogen activator activity in human prostate and breast tumors: relationship to steroid receptors.

Using a fluorometric assay based on the activation of human plasminogen, plasminogen activator (PA) activity was measured in cytosolic and lysosomal extracts prepared from normal, benign hyperplastic (BPH), and carcinomatous human prostates. In all three types of prostatic tissue, the lysosomal extracts had much higher concentrations of PA activity than did the cytosolic extracts. The mean PA activity in lysosomal extracts of the carcinomatous prostates was 170% and 85% higher than that measured in normal and BPH prostates, respectively (Student's t-test, p less than .05). With prostatic carcinoma and BPH specimens there was an inverse (negative) relationship between lysosomal PA activity and the nuclear concentration of androgen-receptors (correlation coefficient, -0.84). By comparison in specimens of human breast tumors, there were weakly positive correlations between PA activity and either estrogen (ER) or progestin (PR) receptors (correlation coefficients of + 0.23 and + 0.54, respectively). While as a group ER+, PR+ breast tumors had higher PA activity that ER+, PR- or ER-, PR- tumors, the differences were not statistically significant (Student's t-test, p greater than .05). Thus in breast tumors, it is uncertain whether high levels of PA activity are indicative of hormonal dependence. However, our findings with prostatic tumors infer that in contrast, high concentrations of this enzyme may reflect a malignant phenotype characterized by a decrease in both androgen responsiveness and differentiation.

Aged↗
PubMed1982

Mechanisms of action of androgens and antiandrogens: effects of antiandrogens on translocation of cytoplasmic androgen receptor and nuclear abundance of dihydrotestosterone.

The effects of antiandrogens on the translocation of androgen receptor and the accumulation of nuclear dihydrotestosterone in rat ventral prostate were investigated. Using an isotope-exchange assay and radioimmunoassay it was found that 1 hr after the injection of 70 nmol of dihydrotestosterone into 1-day castrated rats, 19,000 +/- 2.350 (mean +/- SE) receptor molecules and 99,300 +/- 8,200 molecules of dihydrotestosterone were recovered per nucleus. When equivalent doses of antiandrogens were substituted for dihydrotestosterone only 0-1,500 receptor molecules were translocated. After injection of antiandrogens followed 1 hr later by injection of dihydrotestosterone, the inhibition of translocation given by the reduction in the mean numbers of molecules of receptor and dihydrotestosterone, respectively, was as follows: 17 beta-estradiol (0, 0); megestrol acetate (2000, 0); diethylstilbestrol (6000, 4000); cyproterone acetate (5500, 27000); flutamide (10000, 29000); R2956 (10000, 28000). With the exception of the effect of cyproterone acetate on receptor translocation, the reductions produced by the latter three antiandrogens were all statistically significant (P less than 0.5). Since less than 20% of the dihydrotestosterone in the nucleus is bound to receptor in dihydrotestosterone-treated rats, and since the concentrations of receptor and dihydrotestosterone are unequally affected by some antiandrogens, we infer that the nuclear abundance of dihydrotestosterone may be regulated in part through nonreceptor-mediated processes.

Androgen Antagonists↗
PubMed1982

Concentration and preservation of nuclear androgen receptor by lyophilisation.

Nuclei from rat ventral prostate were disrupted by sonication and treated with micrococcal nuclease to precipitate nuclear proteins including the androgen receptor. The precipitate was dissolved in 0.6--1.2 M ammonium bicarbonate buffer, pH 7.6, with no loss of receptor when compared to the conventional Tes buffer, pH 7.0 containing 0.6--1.2 M NaCl. Lyophilisation of the solubilised protein did not produce any qualitative or quantitative differences in the recovery of receptor relative to a non-lyophilised control preparation, both of which were analysed for binding properties by Sephadex G-25/G-100 dual-column chromatography. Over longer periods of storage at -80 degrees C, the rate of inactivation of receptor was found to be 6% per week. The stability of the lyophilised receptor was improved by the inclusion of MgCl2 and SH-reducing agents in the ammonium bicarbonate buffer. Recovery improved also with increasing ionic strength of the buffer used to dissolve the lyophilised receptor.

Animals↗