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B S Katzenellenbogen

Publications and source records attributed to B S Katzenellenbogen.

At least 163 records · Page 9Linked to original sources
PubMed1984

Biological activity and receptor binding of a strongly interacting estrogen in human breast cancer cells.

A substantial proportion of human breast cancers contain estrogen receptors, and it is believed that the growth of some of these tumors and their synthesis of specific proteins are stimulated by estrogens. Since natural estrogens, such as estradiol, react reversibly with estrogen receptors, it was of interest to determine the biological consequences that would result from very strong, possibly irreversible interaction of an estradiol-based ligand with the estrogen receptor of breast cancer cells. For these studies, we have examined the receptor interactions and biological character of 11 beta- chloromethylestradiol (CME) and 11 beta- bromomethylestradiol (BME) as potential estradiol-based affinity labeling ligands in MCF-7 human breast cancer cells which contain high levels of estrogen receptors. The apparent relative binding affinities of CME and BME for MCF-7 estrogen receptor measured by competitive binding assay are 230 and 15%, respectively, whereas the affinity of estradiol is considered 100%. Incubation of receptor preparations from MCF-7 cells or rat uteri with CME at 21 degrees results in a concentration- and time-dependent decrease in receptor content measured by exchange assays with [3H]estradiol. This may be due to covalent attachment of CME to receptor and is termed "inactivation." Inactivation of 80 to 85% of the receptors occurs within 30 min at 21 degrees by exposure to 5 or 20 nM CME, with 2 nM giving 20 to 40% inactivation. This receptor inactivation is prevented by preincubation with 2000 nM estradiol, indicating that the interaction of CME is occurring at the estradiol binding site on the receptor. MCF-7 cells incubated with 20 nM CME show a rapid loss of cytosol receptor sites and no accumulation of receptors detectable by exchange assay in the nucleus, while 20 nM estradiol shows nuclear localization of receptor. BME, in contrast, inactivates only a portion (approximately 40%) of estrogen receptors. CME and BME both behave as estrogen agonists. They stimulate the proliferation of MCF-7 cells and increase cellular progesterone receptor content and plasminogen activator activity. CME is at least as potent as estradiol on a molar basis in increasing all of these activities, while BME shows a biopotency of only 1% of that of estradiol or CME. Hence, although CME reacts very strongly and apparently irreversibly with estrogen receptors in MCF-7 cells, it still behaves as a potent estrogen agonist.

Animals↗
PubMed1983

Plasminogen activators in tissues of the immature and estrogen-stimulated rat uterus and in uterine luminal fluid: characterization and properties.

We have characterized the molecular properties of the plasminogen activators in different cell types comprising the immature and the estrogen-stimulated rat uterus and in rat uterine luminal fluid. There were two plasminogen activators in the immature (day 20) rat uterus with apparent molecular weights, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, of 70,000 and 46,000. Both plasminogen activators were present in epithelial and in stromal plus myometrial cell fractions of the immature uterus, and after stimulation by 17 beta-estradiol, no new plasminogen activators were detected in either cell fraction. The Michaelis constants (Km) for the activation of dog plasminogen by extracts from epithelial cells and from stromal plus myometrial cells obtained from either immature or 17 beta-estradiol-stimulated uteri were similar (approximately 11 microM). The maximal velocity (Vmax), normalized to protein concentration, increased 2.5-fold in the stromal plus myometrial cell fraction and 6.5-fold in the epithelial cell fraction, upon hormone stimulation (2 micrograms 17 beta-estradiol/day X rat for 3 days). The greatest concentration of plasminogen activator activity was found in the luminal fluid from estrogen-stimulated uteri, where the Vmax per mg protein was more than 10-fold greater than that in the cell fractions from estrogen-stimulated uteri. The plasminogen activator activity of luminal fluid was inhibited by diisopropyl fluorophosphate and rho-nitrophenyl rho-guanidinobenzoate, was not inhibited by human alpha-1-proteinase inhibitor and human antithrombin III, and was inhibited by high, but not low, concentrations of soybean trypsin inhibitor and bovine pancreatic trypsin inhibitor. These studies indicate that the plasminogen activators in different cell types comprising the uterus are similar and show that the estrogen enhancement of uterine plasminogen activator activity is the result of an increase in Vmax. The presence, upon hormone stimulation, of an apparent concentration gradient of increasing plasminogen activator activity through the uterus from myometrium to epithelium to luminal fluid may be a reflection of the dynamic role of this protease in the physiology of the uterus.

Animals↗
PubMed1983

Modulation of progestin binding activity in cultured human breast carcinoma cells: the effect of serum type and concentration.

Progesterone receptor levels in MCF-7 human breast cancer cells increase as a specific response to estrogen and to some nonsteroidal antiestrogens. In the present study we demonstrate that the type and quantity of serum present during culture of these cells modifies the level of progestin binding activity, but not the level of estradiol binding activity. MCF-7 cells maintained in media supplemented with 5% charcoal-dextran treated calf serum (CDCS) contain 0.3 - 0.4 pmol of cytosol progesterone receptor (PRc) per mg DNA. When cells previously maintained in 5% CDCS-media are shifted to media containing 5% charcoal-dextran treated fetal calf serum (CDFCS), the level of progestin binding increases after day 16, and stabilizes at 2 - 3 pmol/mg DNA at days 30 to 40. Shifting these cells back to 5% CDCS-media, reduces PRc to 0.2 - 0.4 pmol/mg DNA within 3 days. This reduction is dose dependent with a half-optimal decrease at 1% CDCS, and a full decrease at 2% CDCS (4d incubation). Nuclear progestin binding was uniformly low (0.2 - 0.4 pmol/mg DNA) and unaffected by type or concentration of serum, and no consistent change in cytosol or nuclear estrogen receptor levels was observed. These cytoplasmic progestin binding sites are translocated to the nucleus by progesterone, and are similar to estradiol (E2) induced sites by Scatchard binding and sucrose gradient analysis. Similar serum-dependent changes are also observed in the T47D human breast cancer cell line where growth in CDFCS-media results in 4-fold higher progestin binding levels than observed in CDCS-media. Our findings suggest the presence of non-dialyzable stimulatory factor(s) in CDFCS that influence the progestin receptor level and highlight the fact that serum components can alter dramatically the cellular progestin binding activity.

Binding Sites↗
PubMed1983

Antiestrogen pharmacology and mechanism of action.

Antiestrogens are nonsteroidal compounds that are capable of antagonizing many of the actions of estrogens. They have long half-lives in serum, and during their action in vivo, the antiestrogens C1628, U23,469 and tamoxifen undergo bioactivation to more polar forms that have a higher affinity for the estrogen receptor. Utilizing the high affinity antiestrogens C1628M and trans-hydroxytamoxifen in radiolabeled form, we find differences in the interaction of the estradiol receptor complex and antiestrogen receptor complex with DNA cellulose and differences in their association in the nucleus. In MCF-7 human breast cancer cells, there are marked differences in the sedimentation rate and chromatographic properties of the nuclear estrogen receptor when complexed with antiestrogen (5.5 +/- 0.06 s, 5.93 +/- 0.20 nm Stokes radius, 137,000 mol. wt) as opposed to estrogen (4.1 +/- 0.03 s, 4.84 +/- 0.20 nm Stokes radius, 83,000 mol. wt). Our data are consistent with the association of the antiestrogen receptor with an additional nuclear protein of approx. 55,000 mol. wt. This antiestrogen promoted change in receptor association with another cellular component, which may function as a possible nuclear "blocking protein" and the alterations in antiestrogen-receptor association with DNA and nuclei, may be important aspects of the estrogen antagonist and growth inhibiting properties of these compounds. In addition to binding directly to the estrogen receptor, antiestrogens can be found associated with binding sites that are distinct from the estrogen receptor. These sites are present in a wide variety of estrogen target and non-target tissues in the rat, and in estrogen receptor positive and negative human breast cancer cell lines (MCF-7, T47D, MDA-MD-231) that differ markedly in their sensitivity to antiestrogens. These sites have a subcellular distribution and ligand binding specificity very different from those of the estrogen receptor. While their wide distribution and order of binding affinities for different antiestrogens raise questions about their role in mediating directly the classically recognized estrogen antagonism of antiestrogens, these sites might alter the apparent distribution volume of antiestrogens and hence their biological potency and pharmacokinetics.

Animals↗
PubMed1982

The intranuclear distribution of rat uterine estrogen receptors determined after nuclease treatment and chromatin fractionation.

The intranuclear locations at which rat uterine estrogen receptors interact with chromatin have been probed using digestions performed with DNAase I and micrococcal nuclease. Exposure to nuclease has been controlled to effect limited to extensive digestion of nuclear DNA under conditions which maintain the integrity of the [3H] estradiol-receptor complex. The effect of divalent cation concentration on the release of estrogen receptors fron nuclease-treated chromatin was examined and found to be of consequence above 2 mM. Exposure to nuclease released nuclear estrogen receptors from chromatin, with DNAase I being more efficient than micrococcal nuclease in mediating this release. The release of the bulk of nuclear estrogen receptors closely paralleled the nuclease-mediated digestion of chromatin DNA. At 1 h after exposure to estrogen, substantial quantities of uterine estrogen receptors (80-90%) were distributed in chromatin fractions which, on the basis of fractionation terminology, have been termed 'transcriptionally inactive' by convention. Enrichment of estrogen receptors in chromatin which has been termed ' transcriptionally active' only occurred with 10-20% of the estrogen receptors. Hence, our findings support a model where, at early times after estrogen exposure, receptors from the rat uterus are enriched to only a minor extent in chromatin to which 'transcriptional activity' is generally assigned while the bulk of receptors are localized in chromatin which is generally considered 'transcriptionally inactive'.

Animals↗
PubMed1982

Antiestrogen basicity--activity relationships: a comparison of the estrogen receptor binding and antiuterotrophic potencies of several analogues of (Z)-1,2-diphenyl-1-[4-[2-(dimethylamino)ethoxy]phenyl]-1-butene (tamoxifen, Nolvadex) having altered basicity.

A series of N-substituted (Z)-1,2-diphenyl-1-[4-(2-aminoethoxy)phenyl]-1-butenes, analogues of the antiestrogen tamoxifen (Nolvadex), in which the side-chain basicity is varied over a wide range, has been prepared to probe the importance of basicity in evoking estrogen antagonism. All of the compounds, except the pyrrole analogue 14, were found to possess significant antiestrogenic activity in the rat, as measured by their ability to inhibit estrogen-induced uterine growth. This implies that in the tamoxifen series the level of side-chain basicity, at least in the Lowry-Brønsted sense, is not a determining factor in the estrogen antagonist potencies of these compounds.

Animals↗
PubMed1982

Uterine plasminogen activator activity: modulation by steroid hormones.

We have used a sensitive and quantitative assay to investigate the hormonal regulation of plasminogen activator (PA) activity in the rat uterus. PA activity is increased 5-fold (per U protein or DNA) by low physiological (0.1 micrograms) doses of estradiol, with increases in activity first observed at approximately 12 h. The stimulation of PA activity shows strict specificity among the steroid hormones, being stimulated by estrogens only or by high doses of dihydrotestosterone, which are known to affect the estrogen receptor system, and this stimulation is suppressed markedly by triphenylethylene antiestrogens. Comparative dose-response studies with a variety of estrogens of different uterotropic potencies indicate a good correlation between the potencies of different estrogens in stimulating PA activity and uterine growth (diethylstilbestrol = 17 beta-estradiol greater than estrone = 17 alpha-estradiol greater than estriol), with the exception of the zearalanol estrogen P-1496, which was consistently a potent stimulator of PA activity while being a very weak uterotropic agent. These studies suggest that increases in uterine PA levels may serve as a good marker of estrogen action in the uterus. Although the role of PA in uterine function remains unknown at present, its relatively large increase (up to 25-fold increase in content per uterus) may play a role in tissue remodeling during uterine growth.

Animals↗
PubMed1981

Interaction of a high affinity anti-estrogen (alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbene, CI628M) with uterine estrogen receptors.

The polar metabolite of the anti-estrogen alpha-[4-pyrrolidinoethoxy]phenyl-4-methoxy-alpha'-nitrostilbene (CI628), that is selectively accumulated by the nuclear estrogen receptor in vivo, is identified as the corresponding free phenol (alpha-[4-pyrrolidinoethoxyl]phenyl-4-hydroxy-alpha'-nitrostilbene (CI628M) and has been prepared in high specific activity tritium-labeled form to study its interaction with the rat uterine estrogen receptor. CI628M has an affinity for the cytosol receptor that is comparable to that of estradiol (Kd (CI628M) = 0.16 nM; Kd (estradiol) = 0.28 nM), and the binding of these two compounds is mutually competitive. Dissociation of the CI628M complex with the estrogen receptor is somewhat slower than that of the estradiol complex, and prewarming the complexes at 28 degrees C causes a characteristic decrease in the rate of dissociation. On low salt sucrose gradients, the CI628M and estradiol cytoplasmic receptor complexes both sediment as 8S species; their sedimentation profiles on high salt gradients are somewhat different, however. While the estradiol receptor complex shows mainly a 4.6S species that shifts to a 5.8S species upon warming, the unwarmed CI628M complex is predominantly 5.8S and becomes somewhat more disperse upon warming. Under high salt conditions, there is a progressive shift of the 4.6S to the 5.8S species as cytosol concentration increases; the CI628M complex shows a greater tendency to form the more rapidly sedimenting form at all cytosol concentrations. After in vivo injection of [3H]CI628M, the uterine nuclear receptor sediments as a characteristic 5S species. Cytosol receptor complexes with estradiol and CI628M, maintained at 0 degrees C, show very little binding to ATP-Sepharose, while after warming, 50-60% of both complexes are bound by the resin. Both complexes also show similar elution profiles from DNA-cellulose columns; however, the extent of binding of the [3H]CI628M receptor complex to the column is consistently only 40-50% that of the estradiol receptor complex. These results indicate that the properties of the uterine estrogen receptor complexed with estradiol and with the high affinity anti-estrogen CI628M are very similar. The differences observed in the behavior of the CI628M cytosol receptor complex on high salt sucrose gradients and on DNA-cellulose columns, though quantitative rather than qualitative, may be indicative of differences in receptor properties that are important to the antagonist activity of this compound.

Animals↗