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B S Katzenellenbogen

Publications and source records attributed to B S Katzenellenbogen.

At least 145 records · Page 8Linked to original sources
PubMed1986

Dynamics of estrogen receptor turnover in uterine cells in vitro and in uteri in vivo.

The rate at which the estrogen receptor (ER) is synthesized and degraded (turns over) in target cells is a major factor regulating the levels of ER and cell sensitivity to estrogen. We have used the ability of tamoxifen aziridine [(TAZ)(Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl]1,2-diphenyl-1-buten e] to affinity label the ER in intact cells to study the dynamics of ER turnover in uterine cells in vitro and in intact rat uteri in vivo. In primary cultures of rat uterine cells (from 21-day-old rats), ER is degraded with a half-life of 3-4 h, as determined by pulse-chase experiments in which ER in cells are covalently labeled with [3H]TAZ (20 nM) and then exposed to a chase of 10(-6) M estradiol. Density shift experiments, in which uterine cells are exposed for different time periods to medium containing dense (15N,13C,2H) amino acids and the shift of receptor from a normal density to a more dense species is analyzed on sucrose gradients, also confirm this rapid turnover for the uterine ER. A similar half-life is obtained for ER in rat uteri in vivo, after intralumenal installation of [3H]TAZ to label effectively ER covalently. Analysis of nuclear ER on sodium dodecyl sulfate-polyacrylamide gels during the chase period reveals the loss of the 65,000 mol wt [3H]TAZ-labeled receptor species; no lower mol wt [3H]TAZ-labeled fragments are observed. Likewise, immunoblot analyses of receptor with the ER monoclonal antibody H222Sp gamma reveal only the 65,000 mol wt receptor species, with no evidence of any smaller receptor forms in cells. Interestingly, incubation of uterine cells with 10(-5) M cycloheximide (which inhibits 98% of protein synthesis) completely arrests ER turnover, suggesting that ER turnover, which is rapid, is probably dependent upon the synthesis of other rapidly turning over proteins. Hence, we conclude that ER turnover is rapid in uterine cells, that turnover of uterine ER occurs at a rate similar to that previously reported for ER turnover in MCF-7 human breast cancer cells, and that ER turnover is similar in uterine cells under in vitro and in vivo conditions. These findings indicate that the receptor is a dynamic and rapidly turning over protein whose level could be adjusted quickly by alterations in the rate of synthesis and/or degradation.

Affinity Labels↗
PubMed1986

Antiestrogen action in the medial basal hypothalamus and pituitary of immature female rats: insights concerning relationships among estrogen, dopamine, and prolactin.

We have examined the effects of nonsteroidal antiestrogens (AEs) and estradiol (E) on dopamine (DA) levels and turnover rates in the medial basal hypothalamus (MBH) and on serum and pituitary PRL to gain insight into DA-PRL-E/AE interrelationships. In 21-day-old female rats, E was found to increase MBH DA levels and turnover and serum PRL concentrations in a time- and concentration-dependent manner. Changes were observed by 1 day, and after 3 days of E treatment (1 microgram/day), MBH DA levels increased 2-fold (to 1300 pg/mg tissue), and DA turnover rates increased 5-fold (to 1170 pg/mg tissue . h). The AEs tamoxifen, monohydroxytamoxifen, CI628, and LY117018 (50 micrograms/day for 3 days) weakly stimulated uterine weight gain and significantly suppressed the uterotropic action of E. The AEs LY117018, monohydroxytamoxifen, CI628, and tamoxifen competed with E for binding to the MBH estrogen receptor and displayed relative binding affinities of 190%, 185%, 6.7%, and 1.4%, with E set at 100%; these affinities are similar to those found for uterine estrogen receptors. The AEs increased DA turnover rates only 2-fold, and they antagonized the E-induced 5-fold increase in DA turnover rates very successfully. In animals treated with bromocriptine, E and AE failed to increase the low serum PRL levels, yet they evoked significant (approximately 2-fold) increases in DA turnover rates and nearly 2-fold increases in MBH DA content. Hence, a part of the actions of E and AE on MBH DA appears to be exerted independently of changes in circulating PRL and may occur by direct action of these compounds on the estrogen receptor system present in the MBH. In addition, these studies reveal that AEs behave as partial estrogen agonists/antagonists in terms of their effects on MBH DA turnover.

Age Factors↗
PubMed1986

Characterization of estrogen and progesterone receptors and the dissociated regulation of growth and progesterone receptor stimulation by estrogen in MDA-MB-134 human breast cancer cells.

We have examined the properties of the estrogen receptor and progesterone receptor in MDA-MB-134 human breast cells and have evaluated the effects of estrogen on cell proliferation and progesterone receptor levels in these cells as indices of hormonal sensitivity. These cells contain high levels of estrogen receptor (approximately 1.5 pmol/mg DNA) and low levels of progesterone receptor (0.15 pmol/mg DNA). More than 80% of the estrogen receptor is found in the nuclear fraction in the absence of estrogen, and the Kd of the receptor for estradiol is approximately 1.5 X 10(-10) M. Upon exposure to estradiol, the receptors become occupied, but there is no processing or apparent decrease in either nuclear or total cellular estrogen receptor content, as can be seen in MCF-7 human breast cancer cells. The nuclear estrogen receptor sediments as a 4.6 S species on high salt sucrose gradients, and it can be detected on sodium dodecyl sulfate-polyacrylamide gel immunoblot analysis as a species of molecular weight 65,000, identical to that of the MCF-7 estrogen receptor, using the monoclonal antibodies D75P3 gamma and H222Sp gamma prepared against the MCF-7 estrogen receptor. The estrogen receptor shows binding selectivity for estrogens and antiestrogens, and its affinity for ligands follows the order diethylstilbestrol (190%) greater than estradiol (100%) greater than estriol (13%) greater than tamoxifen (3%), as expected for estrogen receptor. Hence the receptor appears normal in many of its physicochemical properties and in terms of its binding affinity and specificity for estrogens and antiestrogens. Control cells contain low levels of progesterone receptor that display high affinity (Kd = 6 X 10(-9) M) for the synthetic progestin R5020, but exposure to estradiol (10(-11)-10(-7)M) fails to increase cellular progesterone receptor levels. In contrast, estradiol markedly stimulates the rate of cell proliferation, while tamoxifen suppresses the growth of control and of estradiol treated cells. Hence, our data show that these cells, which contain substantial levels of estrogen receptor, respond to estrogen with enhanced cell proliferation but fail to have their progesterone receptor level modulated by estradiol. These cells represent an interesting and unusual situation in which estrogenic regulation of proliferation and the stimulation of progesterone receptor are dissociated. These cells should prove useful in further evaluation of estrogenic regulation of cell proliferation and specific protein synthesis in human breast cancer.

Antibodies, Monoclonal↗
PubMed1985

Antiestrogen action in breast cancer cells: modulation of proliferation and protein synthesis, and interaction with estrogen receptors and additional antiestrogen binding sites.

Antiestrogens have proven to be effective in controlling the growth of hormone-responsive breast cancers. At the concentrations of antiestrogens achieved in the blood of breast cancer patients taking antiestrogens (up to 2 X 10(-6) M), antiestrogens selectively inhibit the proliferation of estrogen receptor-containing breast cancer cells, and this inhibition is reversible by estradiol. Antiestrogens also inhibit estrogen-stimulation of several specific protein synthetic activities in breast cancer cells, including increases in plasminogen activator activity, progesterone receptor levels and production of several secreted glycoproteins and intracellular proteins. Antiestrogens bind with high affinity to the estrogen receptor and to additional microsomal binding sites to which estrogens do not bind. These latter sites, called antiestrogen binding sites (AEBS), are present in equal concentrations in estrogen receptor-positive and -negative breast cancer cells and are present in a wide variety of tissues, with highest concentrations being found in the liver. The antiestrogenic and growth suppressive potencies of a variety of antiestrogens correlate best with their affinity for estrogen receptor and not with affinity for AEBS. Antiestrogens undergo bioactivation and metabolism in vivo and hydroxylated forms of the antiestrogen have markedly enhanced affinities for the estrogen receptor. Detailed studies with high affinity radiolabelled antiestrogens indicate that antiestrogens induce important conformational changes in receptor that are reflected in the enhanced maintenance of a 5 S form of the estrogen receptor complex; reduced interaction with DNA; and altered activation and dissociation kinetics of the antiestrogen-estrogen receptor complex. These conformational changes effected by antiestrogens likely result in different interactions with chromatin, causing altered cell proliferation and protein synthesis. Analyses of the rates of synthesis and turnover of the estrogen receptor through pulse-chase experiments utilizing the covalently attaching antiestrogen, tamoxifen aziridine, and studies employing dense amino acid labeling of estrogen receptor reveal that the antiestrogen-occupied receptor is degraded at a rate (t 1/2 = 4 h) similar to that of the control unoccupied receptor. Hence, antiestrogens do not prevent estrogen receptor synthesis and they do not either accelerate or block estrogen receptor degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1985

Biological activities of tamoxifen aziridine, an antiestrogen-based affinity label for the estrogen receptor, in vivo and in vitro.

Tamoxifen aziridine (TA), an antiestrogen-based affinity label for the estrogen receptor, is highly selective and efficient in its covalent binding to the estrogen receptor (Katzenellenbogen et al., J. biol. Chem. 258 (1983) 3487-3495). Thus, it was of interest to investigate the biological character and potency of this compound and, in particular, to determine if the irreversible attachment of this tamoxifen-derived compound to the estrogen receptor would result in enhanced antiestrogenic properties or in unusual biological activity. The effect of tamoxifen aziridine and tamoxifen (Tam), the parent compound which is an antiestrogen that binds reversibly to the estrogen receptor, were compared with respect to their effects on uterine growth, growth of dimethylbenzanthracene (DMBA)-induced mammary tumors in rats, and proliferation and plasminogen activator activity of MCF-7 human breast cancer cells. In immature (day 20) rats, Tam and TA behaved as weak estrogen agonists and estrogen antagonists in that Tam or TA alone increased uterine weight to levels lower than that evoked by estradiol (E2), and both were able to suppress the stimulation of uterine weight evoked by E2. Administration of Tam and TA via Alzet minipumps (25 or 200 micrograms/rat/day) to mature rats bearing DMBA-induced mammary tumors resulted in marked regression and/or disappearance of most tumors. Uterine weights were also suppressed in these mature rats by Tam and TA. Tam was slightly more potent than TA in evoking tumor regression and in suppressing uterine weights in these in vivo studies. In MCF-7 human breast cancer cells in culture, Tam and TA suppressed cell proliferation and evoked no increase in plasminogen activator activity by themselves, while being very effective in preventing plasminogen activator activity stimulation by E2. Thus, TA displayed a bioactivity profile similar to that of Tam, the reversibly binding ligand, in vitro and in vivo. The covalent attachment of TA to the receptor does not, therefore, markedly alter the biological character or potency of the antiestrogen receptor complex.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1985

Facile geometric isomerization of phenolic non-steroidal estrogens and antiestrogens: limitations to the interpretation of experiments characterizing the activity of individual isomers.

In many estrogen responsive systems the isomers of tamoxifen are known to have different biological character-the trans isomer is generally an antagonist and the cis isomer an agonist. Attempts to similarly characterize the isomers of hydroxytamoxifen (which differ greatly in their affinity for the estrogen receptor) are shown to be complicated by their facile isomerization. This isomerization was studied in cultures of estrogen receptor positive MCF-7 human breast cancer cells and monitored by HPLC under reversed phase conditions. Hydroxytamoxifen isomers that are initially 99% pure, undergo a time and temperature dependent isomerization, so that after 2 days in tissue culture medium at 37 degrees C they have isomerized to the extent of 20%. This isomerization occurs in the cell-free medium alone and cannot be attributed to a metabolic conversion by the cells. The isomerization occurs much more slowly at 4 than at 37 degrees C and can be reduced considerably by various antioxidants (butylated hydroxytoluene, ascorbate, alpha-tocopherol, retinoic acid and retinal); however, at concentrations that block isomerization, these antioxidants are toxic to the cells. Although the medium contains both the cis and trans isomers of hydroxytamoxifen, the MCF-7 cells preferentially accumulate the trans isomer and the material associated with the nuclear estrogen receptor is, in all cases, mainly the higher affinity trans isomer. A similar preference of the estrogen receptor for the trans isomer is seen with diethylstilbestrol, resulting again in almost exclusive accumulation of the trans isomer in the receptor binding site. These experiments indicate the importance of verifying the isomer compositions of easily isomerizable non-steroidal estrogens and antiestrogens, such as diethylstilbestrol and hydroxytamoxifen, both in stock solutions and in experimental samples (especially those derived from receptor-associated material), so as to ascertain that the activity of the individual isomers is being correctly assigned.

Animals↗
PubMed1985

Characterization of the subunit nature of nuclear estrogen receptors by chemical cross-linking and dense amino acid labeling.

We have used chemical cross-linking and dense amino acid labeling of estrogen receptors to characterize the subunit nature and rate of turnover of nuclear 5S estrogen-receptor complexes. When MCF-7 human breast cancer cells are incubated with [3H]estradiol or [3H]antiestrogen [alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilbe ne (CI628M) or (Z)-1-[4-(2-[N-aziridinyl]ethoxy)phenyl] 1,2-diphenyl-1-butene (tamoxifen aziridine)] and nuclear estrogen-receptor complexes are extracted with 0.6 M KCl and then chemically cross-linked with the cross-linker 2-iminothiolane, the cross-linked receptor complexes sediment as a 5.4S species on 3 M urea-containing sucrose gradients, while the noncross-linked species are 4S. Sodium dodecyl sulfate-polyacrylamide gel analyses of these cross-linked nuclear receptor complexes labeled with the covalently attaching ligand [3H]tamoxifen aziridine reveal a species of about 130,000 mol wt, while the noncross-linked or the cross-linked but mercaptan-cleaved receptor is 65,000 mol wt. Both receptor species are also detectable by interaction with an immunoadsorbent column containing antireceptor monoclonal antibody. For analyses of receptor turnover rates, cells exposed for different time periods to medium containing dense (15N, 13C, and 2H) amino acids were labeled with [3H]antiestrogen [1-[4-(2-dimethylaminoethoxy)phenyl]1-[4-hydroxyphenyl] 2-phenylbut-1-(2)ene (trans-hydroxytamoxifen) or CI628M] or [3H]estradiol, and salt-extracted nuclear estrogen receptors were analyzed on sucrose gradients. The normal density 5S form shifted to a broader, more dense peak at 2 and 4 h and finally, by 8-10 h, to a more dense, sharply sedimenting species. The time course of this shift is the same as that seen for the 4S urea-dissociated nuclear receptor form (t1/2 approximately 4h), suggesting that the 5.4S nuclear receptor is composed of two species which turn over at the same rate. We conclude from these cross-linking and density shift experiments that the nuclear 5S receptor complex consists of two similarly sized units, which turn over with similar half-lives. These data provide strong evidence that the 5S nuclear receptor complex is a homodimer of two 4S, 65,000 mol wt monomers.

Amino Acids↗
PubMed1985

An evaluation of the role of antiestrogen-binding sites in mediating the growth modulatory effects of antiestrogens: studies using t-butylphenoxyethyl diethylamine, a compound lacking affinity for the estrogen receptor.

Tert-butylphenoxyethyl diethylamine (BPEA), a compound synthesized by us, was designed to incorporate features important in binding to antiestrogen-binding sites (AEBS) while lacking features important in binding to the estrogen receptor (ER). With this compound, we have addressed the question of the role of AEBS in mediating the growth modulatory effects of antiestrogens. BPEA has an affinity for AEBS 6% that of tamoxifen and an affinity for ER less than 0.0003% that of estradiol. BPEA (10(-11)-10(-6) M) had no effect on the growth of MCF-7 breast cancer cells and no effect on inhibition of the growth of MCF-7 cells by different concentrations of the antiestrogen tamoxifen. In addition, BPEA (even at doses of 1 mg/day X 50 g rat) exhibited no uterotropic or antiuterotropic activity in immature rats and had no influence on the agonistic or antagonistic activity of varying concentrations of tamoxifen on uterine weight. Hence, we conclude that occupancy of AEBS, at least by BPEA, does not modulate growth of the uterus or breast cancer cells and does not influence the potency of tamoxifen as an antiestrogen. These findings raise serious doubts about the role of the AEBS in mediating directly the growth modulatory effects of antiestrogens.

Binding, Competitive↗
PubMed1985

Antiestrogenic potency and binding characteristics of the triphenylethylene H1285 in MCF-7 human breast cancer cells.

The antiestrogenic character and potency of 4-(N,N-diethylaminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl)-alpha' -ethylstilbene (H1285) and its binding to estrogen receptor and to estrogen-noncompetible antiestrogen binding sites have been studied in MCF-7 human breast cancer cells. H1285 has an affinity for the estrogen receptor (Kd 0.23 nM) which is comparable to that of estradiol (Kd 0.25 nM), and the binding of these two compounds to estrogen receptor is mutually competitive. On high salt sucrose gradients, the sedimentation profiles of nuclear receptor complexes with H1285 and estradiol are different. While the sedimentation profile of the complex with estradiol varies with the buffer composition, being 4.1S in phosphate:thioglycerol: glycerol and predominantly 5.5S in Tris:EDTA buffered gradients, the H1285 receptor complex shows the same sedimentation (5.5S) regardless of the buffer composition. H1285 also binds to estrogen-noncompetable antiestrogen binding sites that are distinct from the estrogen receptor with a low affinity, only 15% that of the antiestrogen tamoxifen. The biological character and potency of H1285 were examined by determining its effects on cell proliferation, cellular progesterone receptor levels, and plasminogen activator activity. In MCF-7 cells, H1285 was a 30- to 100-fold more potent inhibitor of cell proliferation than was the antiestrogen tamoxifen, and it was approximately equipotent with the higher affinity antiestrogen trans-hydroxytamoxifen. H1285 evoked very minimal increases in cellular progesterone receptor levels, and no increase in plasminogen activator activity over a broad range of concentrations (10(-10)-10(-6)M), and it suppressed plasminogen activator activity stimulated by estradiol. Therefore, by the criteria we have used, we conclude that H1285 is a potent and very effective antiestrogen in MCF-7 cells. The ability of estradiol to reverse the suppression of cell proliferation by H1285, and the high affinity of H1285 for estrogen receptor and its low affinity for estrogen-noncompetible antiestrogen binding sites suggest that H1285 exerts its antiestrogenic effects via interaction with the estrogen receptor of these breast cancer cells.

Binding Sites↗
PubMed1984

Dissociated regulation of growth and ornithine decarboxylase activity by estrogen in the rat uterus.

To evaluate the role of polyamines in mediating the effect of estrogen on growth of the rat uterus, we examined the effect of an irreversible inhibitor of ornithine decarboxylase (ODC), 5 alpha-difluoromethylornithine (DFMO), on estrogen-stimulated uterine growth. Estradiol alone evoked two major surges of ODC activity at 5 h and at 16 h, and 2-fold increases in the concentration of spermidine in the uterus. When DFMO was administered to either intact or ovariectomized immature (20 day-old) rats for 2 or 6 days prior to and then during the 1 or 3 days of estradiol treatment, uterine ODC activity was completely suppressed as was the associated increase in spermidine concentration, but the magnitude of uterine growth stimulated by estradiol was equally great in the presence or absence of DFMO. These observations suggest that although uterine growth stimulation by estrogen is normally accompanied by marked increases in ODC activity and spermidine accumulation, these changes are not essential for hormonally regulated uterine cell proliferation in vivo.

Aging↗
PubMed1984

Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo.

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17 alpha-ethynyl estriol, estriol-3-cyclopentyl ether, and 17 alpha-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E3 has 12% the affinity of estradiol (E2) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E3 (10(-10) M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E3 to be only slightly less effective than E2. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E3 while E2 is metabolized to estrone. Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.

Animals↗
PubMed1984

Relationships among uterine growth, ornithine decarboxylase activity and polyamine levels: studies with estradiol and antiestrogens.

We have examined the effects of estradiol (E2) and antiestrogen (AE) on ornithine decarboxylase (ODC) activity and uterine polyamine concentrations as an aid to understanding the dissimilar effects of E2 and AE on uterine growth. ODC activity rises rapidly from very low levels in immature (day 20-23) rat uteri, showing two markedly (approximately 500-fold) increased peaks of activity at 5 h and 16 h after E2 (2 micrograms) injection. After AE (50 micrograms CI628 or U23469) treatment, the temporal increase in ODC activity is slow and only one major peak is found at 24 h. Daily injections of E2 over a 3-day period evoke continued stimulation of uterine ODC activity; however, the same daily treatment regimen with AE results in stimulation of ODC activity only during the first day. After pretreatment with AE, E2 can still elicit the early (5 h) peak of ODC activity, but the later (16 h) peak of ODC activity is blocked. Quantitation of uterine polyamines by high performance liquid chromatography reveals a 1.5-2-fold increase in the concentration of spermidine after 1 day of E2 or AE exposure, with little change in putrescine or spermine levels. After 3 days of E2, AE, or E2 plus AE treatment, spermidine concentrations are elevated 2-2.5-fold and spermine concentrations are elevated 1.5-fold. Putrescine concentrations are unchanged. Hence, regardless of the different degree to which the uterus is stimulated to grow in response to E2 and/or AE, there are corresponding changes in uterine ODC activity, such that polyamine concentrations are elevated to a similar extent in the E2- or AE-treated uterus.

Animals↗
PubMed1984

Estrogen receptor transformation in MCF-7 breast cancer cells: characterization by immunochemical and sedimentation analyses.

We have examined the sedimentation properties of MCF-7 cell cytosol estrogen receptors (ERc) and salt-extracted nuclear estrogen receptors (ERn), and the interaction of these receptors with a monoclonal antibody, as assessed on 0.4 M KC1 sucrose gradients prepared in different buffers. ERc labeled with [3H]estradiol (E2) sediments as a 4 S species in all gradient buffers tested, whereas ERn extracted from cells incubated with [3H]E2 sediments as a 4 S species in sucrose gradients prepared in phosphate-thioglycerol-glycerol buffer, but as a 5.4 S species in Tris-EDTA buffered gradients. Interaction of ERc with antibody D547Sp gamma results in formation of a 7.2 S complex, whereas the antibody reacts with ERn to form complexes that are 8.5 S in sucrose gradients prepared in either buffer. These findings are consistent with the hypothesis that estrogen receptors from MCF-7 cells undergo a 4 S to 5.4 S transformation during nuclear interaction, but that the 5.4 S ERn is converted to a 4 S form during centrifugation in phosphate-buffered gradients. Antibody interaction blocks conversion of receptor to the 4 S form, since only 8.5 S complexes are observed, regardless of the gradient buffer composition. These results highlight that the pattern of receptor transformation by E2 in these cancer cells need not differ from that seen in normal estrogen target tissues such as uterus.

Antibodies↗
PubMed1984

Estrogen receptor synthesis and turnover in MCF-7 breast cancer cells measured by a density shift technique.

The level of estrogen receptor (ER) is a major factor regulating cell sensitivity to estrogen. We have determined the rates of ER synthesis and turnover in MCF-7 breast cancer cells by incubating cells in medium supplemented with 13C15N2H-amino acids (dense amino acids) and monitoring the shift from "old-light" (preexisting) to "new-dense" (newly synthesized) receptors by velocity sedimentation on 0.4-M KCl, 5-20% sucrose gradients prepared in buffered deuterium oxide. Cytosol or unoccupied nuclear ER prepared from control cells grown in the presence of 12C14N1H-amino acids (normal amino acids) and labeled in vitro with [125I]iodoestradiol sediments as a single, normal density peak. When cells were incubated in medium supplemented with dense amino acids for various periods of time, the [125I]iodoestradiol-labeled receptor showed a progressive shift with time to a denser species; by 3 h, a more rapidly sedimenting shoulder was observed on the normal density peak; by 6 h, 40% of the receptor sedimented at the normal density rate, with 60% at a more rapid rate; by 8 h, the predominant peak (70%) was at the more dense position, and at 15 h, all of the sites sedimented as the denser form. Thus, the magnitude of the receptor peak of normal density as a function of time in dense medium indicates that the receptor in control cells has a half-life of 4.0-4.5 h. To determine whether estradiol or the antiestrogens nafoxidine (U11,100A; (1-(2-[p-(3,4-dihydro-6-methoxy-2-phenyl-1-naphthyl)phenoxy] ethyl)pyrrolidine hydrochloride) or CI628 affected the turnover rate of the receptor, measurements were performed on cells grown in the continuous presence of estradiol or antiestrogen (steady state conditions). Exposure of cells to 10 nM estradiol was found to increase the turnover rate of the nuclear receptor, while exposure to 200 nM nafoxidine or CI628 (alpha-(p-[2-(1-pyrrolidino)ethoxy]phenyl)4-methoxy-alpha'- nitrostillbene) did not substantially alter the turnover rate of the nuclear receptor. The half-lives of receptor were found to be 4.02 +/- 0.23 and 4.47 +/- 0.26 h for control unoccupied cytosol and nuclear receptors, 3.00 +/- 0.38 h for estradiol-occupied nuclear receptors, 4.90 +/- 0.66 h for CI628-occupied nuclear receptors, and 3.43 +/- 0.37 h for nafoxidine-occupied nuclear receptors. These results indicate that ER turns over rapidly, with a half-life of 3-5 h, in the presence or absence of estradiol or antiestrogen, and that receptor synthesis is also rapid, with the rate of appearance of newly synthesized receptor being 0.3-0.5 pmol/mg DNA . h. These rates provide the cell with the capacity for dynamic and rapid regulation of its ER levels.

Breast Neoplasms↗
PubMed1984

Characterization of the estrogen receptor and its dynamics in MCF-7 human breast cancer cells using a covalently attaching antiestrogen.

We have used a covalently attaching antiestrogen, tamoxifen aziridine [TA; (Z)-(1-[4-(2-[N-aziridinyl] ethoxy)phenyl])1,2-diphenyl-1-butene], to analyze the structure and dynamics of the estrogen receptor in MCF-7 human breast cancer cells. The labeling of receptor with [3H]TA is specific, being blocked only by estrogens and antiestrogens, and the labeling is very efficient in that TA labels covalently the same number of receptors that are labeled reversibly by estradiol. In cells exposed to [3H]TA for 1 h, most of the covalently associated radioactivity is found in the 0.6 M KCl extract of the nuclear fraction; this receptor has an apparent mol wt of 63,000 +/- 2000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.7 by gel isoelectric focusing in the presence of 8 M urea. The mol wt and pI of cytosol receptor labeled with [3H] TA are identical. In cells labeled with [3H]TA (20 nM) for 1 h and then exposed to a chase of 10(-6) M estradiol, [3H]TA-labeled nuclear receptor disappears with a half-life of 4 h. Analysis of nuclear receptor by sodium dodecyl sulfate-gels during the chase period reveals that this loss reflects a decrease in the 63,000 mol wt species; no significant quantities of lower mol wt TA-labeled fragments are observed in the nuclear, cytosol, or membrane fractions. Affinity labeled receptor interacts with several monoclonal antibodies to MCF-7 estrogen receptor, and it can be purified extensively by immunoadsorbent chromatography. TA has a low affinity (8% that of tamoxifen) for microsomal antiestrogen-binding sites that are distinct from the estrogen receptor, but TA reacts reversibly, rather than covalently, with these sites. The findings of similar mol wt and isoelectric points for soluble cytosol and nuclear extracted receptors under strongly denaturing and disaggregating conditions reveal that nuclear localization of receptor after ligand binding is not associated with major structural alterations in the receptor component labeled by TA. In addition, the receptor, even when occupied by a covalently attached ligand, is rapidly turned over in these cells.

Affinity Labels↗
PubMed1984

Antiestrogen binding in antiestrogen growth-resistant estrogen-responsive clonal variants of MCF-7 human breast cancer cells.

Although antiestrogen therapy is effective in the treatment of hormone-responsive breast tumors, approximately 40% of the patients with estrogen receptor-positive tumors fail to respond to antiestrogens. To better understand the mechanisms by which antiestrogens inhibit the growth of hormone-dependent breast cancers, we have investigated the physicochemical properties and binding characteristics of the estrogen receptors with estradiol and antiestrogens and the occurrence of estrogen-noncompetible antiestrogen binding sites in two estrogen-sensitive but tamoxifen-growth-resistant estrogen receptor-positive MCF-7 cell variant clones, R3-98 and R27. In the variant cells, estradiol (10(-8) M) significantly stimulates cell proliferation as in the parent MCF-7 cells, but the antiestrogen tamoxifen (10(-6) M) has no significant effect on growth of the variant cells, whereas antiestrogen strongly inhibits proliferation of the parent MCF-7 cells. All three cell types contain high concentrations of estrogen receptor (150 to 250 fmol/mg protein), and competition binding analysis shows that the relative binding affinity of a series of compounds for estrogen receptor is similar among the three cell types with the affinity of trans-hydroxytamoxifen greater than estradiol greater than alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrostilben e greater than tamoxifen. Salt-extracted nuclear receptor complexes prepared from the three cell types showed similar sedimentation behavior on 0.4 M KCl-containing sucrose gradients with [3H]estradiol-labeled receptor complexes sedimenting at 4.2S, whereas receptors complexed with either of the antiestrogens trans-[3H]-hydroxytamoxifen or [3H]alpha-[4-pyrrolidinoethoxy]phenyl-4-hydroxy-alpha'-nitrosti lbene sediment at 5.5S. In all 3 cell types, the nuclear receptor forms react with an estrogen receptor monoclonal antibody, D547Sp gamma, to form complexes which sediment at 8.5S. The nuclear estrogen receptors from the parental MCF-7 and the two variant cells, when covalently labeled with [3H]-tamoxifen aziridine in intact cells and then salt extracted have identical molecular weights of approximately 62,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The covalently labeled nuclear and cytosol receptors in these 3 cell lines also show identical migration in 8 M urea polyacrylamide isoelectric focusing gels consistent with a predominant receptor species of isoelectric point approximately 5.7.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1984

Bioactivities, estrogen receptor interactions, and plasminogen activator-inducing activities of tamoxifen and hydroxy-tamoxifen isomers in MCF-7 human breast cancer cells.

Tamoxifen is used widely in the treatment of endocrine-responsive breast cancers in humans. Studies were undertaken to examine the biological character (estrogenic-antiestrogenic properties) and estrogen receptor (ER) interaction of the cis- and trans-isomers of tamoxifen and hydroxytamoxifen in MCF-7 human breast cancer cells. For each compound, the following parameters were monitored: affinity for ER and effects on cellular ER levels; stimulation-inhibition of cell growth, plasminogen activator activity, and cellular progesterone receptor levels; and isomer interconversion and metabolism in vitro. The relative binding affinities of the compounds cis-tamoxifen, trans-tamoxifen, cis-hydroxytamoxifen, and trans-hydroxytamoxifen for cytosol ER were 0.3, 2.5, 1.8, and 310%, respectively, in which the affinity of estradiol is considered 100%. cis-Tamoxifen behaved as a weak estrogen agonist in all assays, while trans-tamoxifen was an effective estrogen antagonist. cis-Tamoxifen behaved like estradiol in stimulating MCF-7 cell growth and increasing plasminogen activator activity and cellular progesterone receptor content, although very much higher concentrations of cis-tamoxifen (10(-6) M) were needed to achieve the levels of stimulation observed with 10(-10) M estradiol. trans-Tamoxifen and trans-hydroxytamoxifen suppressed cell growth, inhibited plasminogen activator activity of control cells, and suppressed estradiol-stimulation of plasminogen activator activity, and they evoked minimal increases in cellular progesterone receptor levels. trans-Hydroxytamoxifen had a 100-fold increased affinity for ER and was approximately 100-times more potent than was trans-tamoxifen in suppressing cell growth and plasminogen activator activity. cis-Hydroxytamoxifen behaved as an estrogen antagonist, suppressing cell growth and plasminogen activator activity, and it elicited submaximal increases in progesterone receptor levels. This apparently paradoxical behavior of cis-hydroxytamoxifen was shown to be due to the fact that the cis- and trans-hydroxytamoxifens readily undergo isomeric interconversion upon exposure to our cell culture conditions, resulting in substantial accumulation of the higher-affinity trans-hydroxytamoxifen in the nuclear ER fraction of cells. In contrast to the facile interconversion of the hydroxytamoxifen isomers, there is no metabolism or interconversion of the parent compounds cis- and trans-tamoxifen in vitro. Hence, by the criteria we have used, the biological characters of trans-tamoxifen and trans-hydroxytamoxifen are similar, the major difference being the approximately 100-fold enhanced potency of the hydroxylated form. In contrast, cis-t

Breast Neoplasms↗