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)     

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.     

Counteraction of estradiol-induced activation of tissue-type plasminogen activator in a human breast cancer cell line by an anti-estrogen, LY117018.

LY117018 is a non-steroid anti-estrogen which exhibits about 100 times higher affinity for estrogen receptor than tamoxifen, another anti-estrogen. The cell line ES-1, which was isolated from human breast cancer MCF-7 cells, was highly sensitive to the cytocidal action of estradiol. Growth of ES-1 cells was inhibited by 10(-8)M 17 beta-estradiol, a concentration that stimulated the growth of parental MCF-7 cells. The estradiol-induced growth inhibition of ES-1 cells was almost completely reversed by treatment with LY117018, but not by treatment with tamoxifen. The relative binding affinity of LY117018 for estradiol receptor was equal to that of estradiol in both MCF-7 and ES-1 cells. Treatment of ES-1 cells with estradiol specifically induced tissue-type plasminogen activator (t-PA), whereas such estradiol-induced activation was not observed in parental MCF-7 cells. Quantitative immunoreactive assays and Northern blot analysis showed that estradiol-induced expression of t-PA was blocked by LY117018 in ES-1 cells. The inhibitory effect of tamoxifen was about 100 times lower than that of LY117018. The inhibition of t-PA gene expression by LY117018 might be due to competitive inhibition with estradiol in estradiol receptor binding.     

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

Summary 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 × 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 metabolismin 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.Our findings raise serious doubts about the role of the AEBS in mediating directly the growth suppressive actions of antiestrogens, and suggest that interaction with the estrogen receptor is most likely the mechanism underlying the growth-inhibitory effects of antiestrogens. At present, the role of the AEBS in the actions of antiestrogens or in possible antiestrogen metabolism remains unclear.     

Histamine and growth: interaction of antiestrogen binding site ligands with a novel histamine site that may be associated with calcium channels

N,N-Diethyl-2-[(4-phenylmethyl)-phenoxy]ethanamine hydrochloride (DPPE) is a novel paradiphenylmethane derivative with antiproliferative and antiestrogenic properties. Like tamoxifen (TAM), DPPE binds to the microsomal antiestrogen binding site with high affinity (Kd approximately 50 nM), but, conversely, not to estrogen receptor or calmodulin. We now demonstrate that DPPE competes for [3H]histamine binding in rat cerebral cortex with an affinity (Ki = 4.5 +/- 2.6 X 10(-6) M) significantly greater than that of the H1 antagonist pyrilamine (Ki = 7.2 +/- 2.2 X 10(-5) M), despite the previous demonstration that pyrilamine is up to 1000 times more potent than DPPE in antagonizing histamine-induced contraction in canine tracheal smooth muscle. DPPE demonstrates antiproliferative activity against MCF-7 cells at concentrations between 1 X 10(-7) and 1 X 10(-5) M; the IC50 value of DPPE for growth inhibition at 7 days in this assay is 5 X 10(-6) M, a value equivalent to its Ki value for histamine binding. DPPE also competes for [3H]verapamil binding in membranes from whole rat brain with an affinity equal to that for verapamil (Kd = 4.0 +/- 1.8 X 10(-7) M); however, verapamil competes for [3H]DPPE binding in brain membranes and rat liver microsomes with an affinity markedly lower (Ki approximately 1 X 10(-4) M) than that of DPPE, suggesting allosteric interactions between the verapamil and DPPE sites. Unlike DPPE, verapamil is not antiproliferative in vitro against MCF-7 cells at concentrations up 1 X 10(-5) M, but, like DPPE, is cytotoxic at concentrations of 1 X 10(-4) M. In immature oophorectomized rats, verapamil or DPPE alone is antiuterotropic; however, verapamil shows no antagonism of exogenous estradiol on uterine growth, as opposed to DPPE which is a partial antagonist. Thus, the antiproliferative and antiestrogenic properties of DPPE either are not associated with calcium channel antagonism, or result from a qualitatively different effect on channels than verapamil. The in vitro antiproliferative effect of DPPE (7.5 X 10(-6) M) on MCF-7 cells at 72 h is significantly reversed by 10 mM L-histidine (70.2 +/- 12.6% reversal) and L-methionine (92.4 +/- 11.1% reversal), but not by L-ornithine, L-arginine, L-phenylalanine, or exogenous histamine. At lower concentrations of TAM (0.75 X 10(-6) M), where growth inhibition is estrogen-reversible, L-ornithine, but not L-histidine or L-methionine, causes significant reversal of growth inhibition (66.8 +/- 13.3%; p less than 0.001).(ABSTRACT TRUNCATED AT 400 WORDS)     

Antitumor mechanism of action of a cyclopropyl antiestrogen (compound 7b) on human breast cancer cells in culture

 Cyclopropyl compound 7b [(Z)-1,1-dichloro2-[4-[2-(dimethylamino)ethoxy] phenyl]-2-(4-methoxy-phenyl)-3-cyclopropane] has been shown to be a pure antiestrogen in mouse uterine tissue. Antitumor activity was examined by evaluating the influence of 7b on the proliferation, estrogen receptor (ER) affinity and cell-surface morphology of ER-positive and ER-negative human breast cancer cells in culture. The antiproliferative potency of 7b was found to be equal to tamoxifen in ER-positive MCF-7 human breast cancer cells. Further, the antiproliferative activities of 7b and tamoxifen were reversed by coadministration of estradiol. Accordingly, the antiproliferative activity of compound 7b appears to be estrogen-mediated since it did not influence the growth of either ER-negative MDA-MB-231 human breast cells or A-549 human lung cancer cells in culture. An ER-dependent mechanism of action is also supported by the specific binding affinity of 7b for ER in MCF-7 cells. Further, a study of cell surface morphology using scanning electron microscopy (SEM) revealed that 7b reduced the density and distribution of microvilli (MV) on MCF-7 cells, which was reversed by coadministration of estradiol. Compound 7b did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 7b inhibited the growth of ER-positive MCF-7 cells in an estradiol-reversible manner, and had no effect on either ER-negative MDA-MB-231 cells or A-549 lung cancer cells. The results of this study confirm an antiestrogenic mechanism of action for 7b as previously observed in vivo and suggest that 7b would be effective in the treatment of estrogen-dependent breast cancer or as a prophylactic treatment for women with a high risk of breast cancer development. Received: 6 January 1995/Accepted: 9 October 1995     

Regulation of human estrogen receptor gene, epidermal growth factor receptor gene, and oncogenes by estrogen and antiestrogen in MCF-7 breast cancer cells

It is generally believed that estrogen may act either as an initiator or as a promoter in carcinogenesis of human breast cancer. This estrogenic action is generally dependent on the estrogen receptor. In the human estrogen receptor, cDNA has a homology to V-erb-A oncogene. Experiments using MCF-7 human breast cancer cells were carried out to study the regulatory effect of estrogen and antiestrogen on RNA activities of oncogenes, estrogen receptor gene, and epidermal growth factor (EGF) receptor gene. The effect of estradiol on activation of estrogen and EGF receptor genes and myc, ras, and fos oncogenes was positive in relation to the concentrations of supplemented estradiol. In addition, the effects of antiestrogen (tamoxifen) were investigated. Tamoxifen suppressed MCF-7 cell growth, and spot hybridization of the RNA of MCF-7 cells revealed that RNA activities of estrogen and EGF receptor genes and myc, ras, and fos oncogenes were suppressed by tamoxifen. These results suggest that the three oncogenes and two receptor genes are partly regulated by estrogen and antiestrogen (tamoxifen) in MCF-7 human breast cancer cells. This regulatory system may have a role in carcinogenesis and in the treatment of human breast cancer.     

Estrogen receptor and hormone responsiveness of medullary thyroid carcinoma cells in continuous culture

We have examined the estrogen responsiveness and estrogen receptor in medullary thyroid carcinoma using a model of an established human cell line, TT. TT cells bind [3H]estradiol with high affinity. Scatchard analysis reveals a single class of binding site with a concentration of 173 fmol/10(6) cells and a dissociation constant of 2.1 x 10(-9) M, values which are comparable to those of a well established model cell line for estrogen responsiveness, MCF-7 human breast cancer cell line. Estradiol in physiological concentrations moderately stimulated TT cell proliferation, whereas in pharmacological concentrations it markedly inhibited cell growth. [3H]Thymidine incorporation into acid-insoluble material was also stimulated following a 5-day treatment with 5 x 10(-9) M estradiol. Tamoxifen at a concentration of 1 microM reduced cell proliferation by 43-48% after 5-7 days of treatment. The growth suppression induced by tamoxifen was reversed by addition of 10 nM estradiol. This is the first report of estrogen growth stimulation and tamoxifen growth inhibition of a tumor cell line derived from human medullary thyroid carcinoma.     

Nitrosourea and nitrosocarbamate derivatives of the antiestrogen tamoxifen as potential estrogen receptor-mediated cytotoxic agents in human breast cancer cells

Summary We have prepared two analogs of the antiestrogen tamoxifen that incorporate known DNA-crosslinking functions, a chloroethyl nitrosourea and a nitrosocarbamate moiety, and we have tested their bioactivities in cultures of human breast cancer cells. Both compounds bind to the estrogen receptor from MCF-7 cells, with relative binding affinities of 0.18% for the nitrosocarbamate derivative and 0.35% for the nitrosourea derivative, while the affinity of tamoxifen is 1.8%, and that of estradiol is set at 100%. The tamoxifen-nitrosocarbamate compound demonstrated a dose-related cytotoxicity by the colony formation and cell proliferation assays that was not blocked by estradiol in either estrogen receptor-positive MCF-7 cells or estrogen receptor-negative MDA-MB-231 cells, and thus, was not studied further. Tamoxifen-nitrosourea (TAM-NU) showed dose-related cytotoxicity in MCF-7 cells that was blocked by estradiol, whereas its activity in MDA-MB-231 cells was unaffected by estradiol. N-2-(4-t-butylphenoxy)ethyl-N-chloroethyl-N-nitrosourea (BPE-NU), a control compound which contains the nitrosourea moiety but does not bind to the estrogen receptor, had no effect on cell proliferation or colony formation in MCF-7 cells, but was very inhibitory in the receptor-negative MDA-MB-231 cells. In contrast, TAM-NU was more active in the receptor-positive MCF-7 cells than in the MDA-MB-231 line. Thus, because TAM-NU appears to be active selectively against the receptor-positive cell line, and because this activity is suppressible by estradiol, its cytotoxic effect seems to be mediated via the estrogen receptor. However, since TAM-NU is active only in prolonged treatment protocols, it appears likely that its cytotoxic activity results from the hormone antagonistic effect of the hydrolysis product of TAM-NU (bis-desmethyltamoxifen), rather than from a direct receptor-mediated, DNA-directed cytotoxic action of TAM-NU itself. This study stresses the need for the use of appropriate control compounds and cell systems in order to assess whether the toxic activity displayed by hormone-cytotoxic conjugates is mediated by receptor interactions and whether it operates through the intended toxic mechanism.     

Endocrine-responsive pancreatic carcinoma: steroid binding and cytotoxicity studies in human tumor cell lines

We have begun to investigate the steroid responsiveness of pancreatic cancer by comparing human (MiaPaCa, Colo-357, RWP-1, RWP-2) and rodent (AR42j) pancreatic tumor cell lines with cultured estrogen receptor-positive breast cancer cells (MCF-7, T47-D). The four human pancreatic tumors contain measurable levels of specific estradiol binding sites with dissociation constants (Kd) that range from 1 to 9 nM, in contrast to the higher-affinity binding sites measured in the breast cancer cells (Kd less than or equal to 1 nM). Growth of one pancreatic tumor line (MiaPaCa) is stimulated 40% above control by exposure to nanomolar concentrations of estradiol, suggesting that the estrogen receptor in these cells is functioning like that in MCF-7 and T47-D cells. Glucocorticoids (dexamethasone, hydrocortisone) and androgen (fluoxymesterone) stimulate proliferation of Colo-357 cells by as much as 30%. Paradoxically, glucocorticoids inhibit AR42j cells to less than 50% of control growth. Micromolar exposures of estrogen (17 beta-estradiol), antiestrogen (tamoxifen), antiandrogen (dehydroxyflutamide), progestins (progesterone, R5020, medroxyprogesterone acetate), and inhibitors of steroid-metabolizing enzymes (17 beta-N,N-diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, danazol) impair growth of these pancreatic tumors to varying degrees, and with little relationship to estrogen receptor content. In general, progestins are slightly more growth inhibiting to these pancreatic tumor lines than the other endocrine agents tested, including tamoxifen. Only the RWP-2 cells appear completely resistant to steroidal therapy, showing less than 25% growth inhibition with exposure to therapeutic concentrations (less than or equal to 2.5 microM) of these agents. Colo-357, MiaPaCa, and AR42j cells are most responsive to these endocrine agents, and their overall pattern of sensitivity suggests that the steroid-dependent growth-inhibitory mechanisms of some pancreatic carcinomas may involve both receptor antagonism and direct inhibition of steroidal oxidoreductases. 17 beta-N,N-Diethylcarbamyl-4-methyl-4-aza-5 alpha-androstan-3-one, a potent inhibitor of 5 alpha-reductase with minimal affinity for androgen receptor, inhibits growth of Colo-357 cells to less than 40% of control and also inhibits AR42j and MiaPaCa cells. Dehydroxyflutamide, a potent androgen receptor antagonist with no direct influence on 5 alpha-reductase activity, inhibits growth of MiaPaCa and AR42j cells but has no affect on Colo-357 growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

An antiestrogenic action of interferons in human breast cancer cells

The antiproliferative and antiestrogenic effects of human interferon on estrogen-dependent CG-5 human breast cancer cells in vitro are reported. Fibroblast beta - interferon as well as recombinant alpha - and gamma -interferon inhibited proliferation of CG-5 cells in a dose and time - dependent fashion. In addition, they completely suppressed the two-fold increase in cell number induced by estradiol and showed an additive antiproliferative effect when used in combination with the antiestrogen tamoxifen. These antiestrogenic effects of interferons were accompanied by a reduced receptor binding of estradiol to the cells.     

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.     

Antiestrogenic action of 3-hydroxytamoxifen in the human breast cancer cell line MCF-7

The antiestrogenic action of 3-hydroxytamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1-(3-hydroxyphenyl)-2 -phenylbut-1-ene] was characterized in vitro and compared with that of tamoxifen [trans-1-(4-beta-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene]. The relative binding affinities of 3-hydroxytamoxifen to estrogen receptor were 3.3% in cytosol of MCF-7 cells and 1.5% in human mammary carcinoma cytosol compared to values of 0.2 and 0.3% for tamoxifen (the affinity of 17 beta-estradiol considered to be 100%). The concentration of 3-hydroxytamoxifen necessary to suppress the 17 beta-estradiol-induced growth stimulation of MCF-7 cells was about tenfold lower than that for tamoxifen. The induction of progesterone receptor in MCF-7 cells by 17 beta-estradiol was inhibited by 3-hydroxytamoxifen. In the absence of 17 beta-estradiol, 3-hydroxytamoxifen gave rise to a moderate increase in the progesterone receptor levels, which demonstrates the partially estrogenic character of hydroxytamoxifen.     

Distribution of antiestrogen-specific binding sites in normal and neoplastic mammary gland

Recently, several investigators have demonstrated the presence of triphenylethylene antiestrogen binding sites in the cytoplasm of many tissues, which specifically bind to radioactive tamoxifen with high affinity. Although mammary gland is one of the principal target organs for antiestrogen action, the characterization of antiestrogen binding in mammary tissues has not been reported. We have studied the antiestrogen binding properties of [3H]tamoxifen in the mammary glands of virgin, pregnant and lactating rats as well as in the N-methyl-N-nitrosourea-induced mammary tumors. Tritium labeled tamoxifen bound specifically and with high affinity (Kd = 10(-9) M) to components present in 25,000 g supernatant. Unlabeled estradiol or DES did not compete for these sites, whereas unlabeled tamoxifen showed competitive inhibition. The mammary glands contained threefold higher levels of cytoplasmic binding sites as compared to the mammary tumors. Mammary glands from the pregnant rats bound tamoxifen to a greater extent than that of either virgin or lactating rats. The functional relevance of these binding sites is still unknown.     

Interaction of [3H] estradiol — and [3H] monohydroxytamoxifen-estrogen receptor complexes with a monoclonal antibody

The aim of this study was to compare and contrast the interaction of estrogen [( 3H]17 beta-estradiol)- or antiestrogen [( 3H]monohydroxytamoxifen)-receptor complexes from human breast tumor cytosols with monoclonal antibodies raised to the human breast tumor estrogen receptor. Breast tumor cytosols containing estrogen receptor which sedimented as radiolabeled peaks in either the 8S, 8S and 4S, or 4S regions of sucrose density gradients, interacted with the monoclonal antibody D547 to produce a broad 9-10S peak, a broad 8S-10S peak, or a more discrete 8S peak, respectively. On high salt (0.4M KC1) sucrose density gradients the 4S ligand-receptor complex plus antibody produced a binding peak at approximately the 8S region of the gradient. These sedimentation studies with the monoclonal antibody D547, and similar studies with the monoclonal antibody D58, could detect no differences in the cytosolic estrogen receptor whether complexed with [3H]estradiol or with [3H]monohydroxytamoxifen. These observations were confirmed by Scatchard equilibrium saturation analysis and sucrose density gradient analysis of cytosols from the MCF-7 human breast cancer cell line. The antibody D547 interacted with 8S ER from these cytosols to produce a broad 8S-10S peak, but the antibody produced no change in the affinity or number of binding sites present in these cytosols. It seems, therefore, that the antigenic determinants recognized by these particular antibodies on the breast tumor cytosolic receptor are not significantly altered by the binding of either an estrogen or an antiestrogen to the receptor.     

Effects of estrogen and tamoxifen on the regulation of dihydrofolate reductase gene expression in a human breast cancer cell line

We have studied the effects of estrogen and the antiestrogen tamoxifen on the regulation of dihydrofolate reductase (DHFR) gene expression in a methotrexate-resistant (MTXR) human breast cancer cell line MCF-7, which contains a 50-fold increase in the level of DHFR enzyme and amplified DHFR gene sequences. Despite their selection for methotrexate resistance, the MTXR cells have retained many characteristics of the parental MCF-7 cell line. Concentrations of estrogen receptors as well as their binding affinity to estradiol are identical in both cell lines. MTXR MCF-7 cells remain sensitive to estrogen and respond to estradiol with an induction of progesterone receptors, as well as increases in the rate of DNA synthesis and cell growth. Incubation of MTXR MCF-7 cells with estradiol results in an additional 1.5- to 3.0-fold increase in their already elevated level of DHFR. The hormone-induced increases in DNA synthesis and DHFR levels are similar both with respect to the time course of inductions, as well as their dose response to estradiol. However, these two estrogen-induced effects are not coupled, since the induction of DHFR occurs even in the absence of concomitant DNA synthesis. Estradiol has no effect on DHFR enzyme stability; thus, the entire effect of estrogen on DHFR levels results from the increased synthesis of this housekeeping enzyme. In contrast, treatment of MTXR MCF-7 cells with the antiestrogen tamoxifen reduces the rate of DHFR enzyme synthesis, resulting in lower cellular levels of DHFR. These MTXR MCF-7 cells represent a useful model in which to study the mechanisms involved in the modulation of DHFR gene expression by estrogen and tamoxifen. Since the level of DHFR is a critical determinant of methotrexate cytotoxicity understanding, the regulation of DHFR gene expression may have clinical implications for the use of hormonal therapy in combination with chemotherapy for the treatment of breast cancer.     

Differential regulation of growth and invasiveness of MCF-7 breast cancer cells by antiestrogens

Estrogen increases the ability of the estrogen-dependent MCF-7 human breast cancer cell line to both proliferate and invade through an artificial basement membrane. In studying the response of MCF-7 cells to various antiestrogens, we found that 4-hydroxytamoxifen and tamoxifen inhibited cell proliferation but increased their invasiveness. In contrast, the structurally unrelated benzothiophene antiestrogens, LY117018 and LY156758, were potent antiproliferative agents which did not stimulate invasiveness. The differential effects of these antiestrogenic agents on invasion correlated with changes in production of collagenase IV, while no significant change was seen in the chemotactic activity of the cells. Invasiveness was increased by 17 beta-estradiol or 4-hydroxytamoxifen after a few hours of treatment and was rapidly lost when 17 beta-estradiol was withdrawn. Stimulation of invasiveness with 17 beta-estradiol was blocked by the antiestrogen, LY117018. Cells from the MDA-MB-231 line which lacks estrogen receptors were not affected by estrogen or antiestrogen in terms of proliferation or invasion. These studies indicate that the invasiveness of MCF-7 cells is regulated by antiestrogens through the estrogen receptor and may be mediated by collagenase IV activity. Antiestrogens which reduce both the proliferation and invasiveness of these cells may be interesting new candidates for clinical application.     

Opposite effects of tamoxifen on in vitro protein kinase C activity and endogenous protein phosphorylation in intact MCF-7 cells

We investigated the effects of the antiestrogen tamoxifen on MCF-7 cell protein kinase C either by using the in vitro histone kinase assay or by studying the phosphorylation of its endogenous Mr 28,000 protein substrate in intact cells. In the in vitro assay, tamoxifen inhibited the enzyme competitively with respect to phospholipid, whereas estradiol and morpholinobenzyl phenoxy ethanamine, a specific ligand for antiestrogen binding sites, were considerably less efficient. In contrast, tamoxifen did not affect phosphorylation of the Mr 28,000 protein induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in intact MCF-7 cells. Estradiol and morpholinobenzyl phenoxy ethanamine also had no effect. At high concentration (100 microM), tamoxifen itself stimulated specific phosphorylation of this Mr 28,000 protein. Estradiol and morpholinobenzyl phenoxy ethanamine neither mimicked nor interfered with this effect. Our data suggest that the effect of tamoxifen on protein kinase C activity depends on the phospholipid environment of the enzyme, and opposite effects may be observed in intact cells to those seen in disrupted cells. The action of tamoxifen on endogenous protein phosphorylation was thought to be due to direct interaction with the phospholipid binding domain of the enzyme rather than by interaction with the estrogen receptor or the antiestrogen binding site. Nevertheless, our results do not rule out a possible activation by tamoxifen of specific protein kinase(s) and phosphatase(s). In any case, the antiproliferative activity of tamoxifen on MCF-7 cells cannot be attributed to its effects on protein kinase C.     

Binding characteristics and biological activity of 17 alpha-[125I]iodovinyl-11 beta-methoxyestradiol, an estrogen receptor-binding radiopharmaceutical, in human breast cancer cells (MCF-7)

17 alpha-[125I]Iodovinyl-11 beta-methoxyestradiol ([125I]MIVE2), a gamma-emitting analogue of estradiol, previously shown to bind to rat uterine estradiol receptor, was studied to determine the binding characteristics and biological activity in human breast cancer cells. In vitro determination of receptor binding by dextran-coated charcoal assays indicates that [125I]-MIVE2 binds specifically and with a high affinity to cytosolic estrogen receptors in the human breast cancer cell line, MCF-7. [3H]Estradiol binds to the receptor with approximately four times the affinity of [125I]-MIVE2 (Kd = 2.55 X 10(-9) M for [125I]MIVE2; Kd = 6.4 X 10(-10) M for [3H]estradiol). Unlabeled MIVE2 produces estrogenic effects similar to those of estradiol such as progesterone receptor induction and increases in thymidine incorporation in MCF-7 cells in culture. Cytosolic progesterone receptor levels were elevated 2.8-fold over control levels by 6 X 10(-9) M MIVE2. Stimulation of thymidine incorporation (approximately 300% above control levels) was observed after exposure to 1 X 10(-9) M MIVE2. Preliminary data show receptor-mediated uptake by the uterus in biodistribution studies in athymic nude mice given injections of [125I] MIVE2 (32-34 microCi). At 4 h, uterus:blood ratios are 20.5 and target tissue:nontarget tissue ratios are 12.9. In light of the fact that this compound can be prepared with a high specific activity, [125I]MIVE2 may have potential as a radiotracer for imaging estrogen receptor-positive breast tumors or metastatic lesions in human breast cancer patients.     

Effects of a new clinically relevant antiestrogen (GW5638) related to tamoxifen on breast and endometrial cancer growth in vivo.

PURPOSE: Cross-resistance is an important issue for the evaluation of new antiestrogens to treat advanced breast cancer patients who have failed tamoxifen therapy. In addition, postmenopausal patients treated with long-term adjuvant tamoxifen show a 3-4-fold increase in the risk of developing endometrial cancer. Consequently, a new second line agent should be more antiestrogenic and less estrogen-like on the uterus, and be effective at controlling the growth of breast cancer after exposure to tamoxifen. The purpose was to evaluate the effects of the new tamoxifen analogue GW5638 on breast and endometrial cancer growth. EXPERIMENTAL DESIGN: Athymic mice were transplanted with an endometrial tumor model (ECC-1 E2) that is responsive to estrogen and has never been exposed to antiestrogen. In addition, we used three breast tumor models: a tamoxifen-na?ve tumor (T47D-E2) and two tamoxifen-stimulated tumors (MT2 TAM and MCF-7 TAM LT). The antiestrogen GW5638 (1.5 mg daily), tamoxifen (0.5 mg or 1.5 mg daily), and raloxifene (1.5 mg daily) were given p.o. The pure antiestrogen ICI182,780 (5 mg once a week) was given s.c. Western blots from MCF-7 TAM breast tumors were performed to demonstrate the regulation of estrogen receptor alpha expression by different ligands. RESULTS: Estradiol and GW5638 down-regulated the receptor compared with control. ICI182,780 completely degraded the receptor but tamoxifen had no effect. GW5638 did not promote tumor growth, and was effective in blocking the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve breast and endometrial tumors. However, raloxifene did not completely block the effects of postmenopausal estradiol on the growth of tamoxifen-na?ve endometrial tumor after 14 weeks. GW5638 and ICI182,780 but not raloxifene were also effective in blocking the tamoxifen-stimulated breast tumor growth in athymic mice. CONCLUSIONS: GW5638 is more effective than raloxifene in blocking the effect of estrogen on tamoxifen-na?ve endometrial cancer. More importantly, GW5638, like the pure antiestrogen ICI182,780, is able to block the growth of breast cancer stimulated by tamoxifen differently from raloxifene. GW5638 down-regulates estrogen receptor but does not completely destroy the receptor. Therefore, based on our findings, GW5638 could be developed as a second line agent for advanced breast cancer patients and an important first line agent to evaluate as an adjuvant treatment or chemopreventive.