125I]17-alpha-iodovinyl 11-beta-methoxyestradiol interaction in vivo with estrogen receptors in hormone-independent MCF-7 human breast cancer transfected with the v-rasH oncogene

[125I]17-alpha-Iodovinyl 11-beta-methoxyestradiol [( 125I]MIVE2) has been evaluated as a potential radiotracer for the diagnostic imaging of estrogen receptor (ER)-positive human breast cancer. In vivo distribution experiments with athymic ovariectomized nude mice bearing human breast tumors revealed an apparent correlation between uptake of 125I-labeled compound and estrogen receptor concentration in the tumors. At 4 h after i.v. injection of [125I]MIVE2, HS578T (ER negative), ZR75-B (intermediate ER), and MCF-7ras (high ER) tumors accumulated 0.320 +/- 0.186, 0.679 +/- 0.467, and 2.6163 +/- 1.0121% injected dose/g, respectively. With coinjection of unlabeled 17-beta-estradiol, levels of radioactivity in MCF-7ras tumors were decreased to 0.4859 +/- 0.1424% injected dose/g, indicating a receptor-mediated process. Peak activity of radioligand in MCF-7ras tumors and uteri was observed at 2 h and was retained for the 8-h time course. Blood and nontarget tissue, such as muscle, revealed a rapid clearance of 125I-labeled compound by 8 h. Eight hours after injection, uterus and tumor-to-blood ratios were calculated to be 225 and 21, respectively. Also, MCF-7ras tumors were shown to accumulate 6.5-fold more radioactivity than muscle. These data suggest that [125I]MIVE2 has the capability of interacting specifically and with high affinity with estrogen receptors in human breast tumors in nude mice and may possibly be used for imaging receptor-positive tumors in breast cancer patients with very low serum estrogen levels. Selective uptake of compound in MCF-7ras tumors emphasizes the usefulness of an estrogen receptor-positive tumor model which has a unique ability to grow in a host system without circulating estrogens.     

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.     

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.     

Biological response of the anti-estrogen ICI 164,384 in human hormone-dependent and hormone-independent mammary cancer cell lines

The biological response on proliferation and progesterone receptor (PR) of the anti-estrogen ICI 164,384 [N-n-butyl-N-methyl-11-(3,17 beta-dihydroxyestra-1,3,5(10-trien-7 alpha-yl))-undecanamide] was studied in different mammary cancer cell lines. In the hormone-dependent cancer cell lines (MCF-7 and T-47D) this anti-estrogen significantly decreased cell proliferation, but to reduce 50% of the growth in the MCF-7 cells a very low concentration (10(-9) M) is necessary. Similar effects in the T-47D cell are obtained with a dose of 100-1000 times (10(-6)-10(-7) M). The stimulatory effect in cell proliferation induced by estradiol is also inhibited by ICI 164,384 in both cell lines. This anti-estrogen has no effect on proliferation in the anti-estrogen resistant cell line LY-2, or in the hormone-independent cell line MDA-MB-436. Studies on thymidine incorporation correlate with the effect on cell proliferation. ICI 164,384 also blocks the stimulatory effect on progesterone receptor provoked by estradiol in MCF-7 cells and in T-47D cells which contain high concentration levels of progesterone receptor ICI 164,384 significantly decreases the PR concentrations in both the non-treated and estradiol-treated cells. It is concluded that ICI 164,384 is a full antagonist in the hormone-dependent breast cancer cells, but it has no effect in the anti-estrogen-resistant or in hormone-independent cell lines.     

Inhibition of human breast cancer cell proliferation in tissue culture by the neuroleptic agents pimozide and thioridazine

Permanent cell culture lines derived from human breast cancer tissue are important experimental models in the study of human breast cancer cell proliferation. In the present work, pimozide, thioridazine, W-13, and W-12 were shown to inhibit MCF-7 human breast cancer cell growth. The 50% inhibition concentration values determined in two proliferation assays, [3H]thymidine incorporation and cell number, were in close agreement for each compound tested. The order of potency for growth inhibition in the presence of 2% stripped calf serum was pimozide (Ki 2 microM) greater than thioridazine (Ki 5 microM) greater than W-13 (Ki 15 microM) greater than W-12 (Ki 39 microM). Similar concentrations of these compounds blocked estradiol-induced growth of MCF-7 cells, but estrogen receptor (ER) interactions do not seem to be involved. Pimozide and thioridazine had no effect on the estradiol binding properties of the MCF-7 ER, nor did pimozide interfere with the induction of progesterone receptors by estradiol. Furthermore, pimozide also inhibited incorporation of [3H]thymidine into MCF-7 cells stimulated by polypeptide hormones in serum-free medium. The Ki for pimozide in serum-free medium alone, 0.46 microM, was similar to that determined in the presence of insulin (0.42 microM), insulin-like growth factor I (0.54 microM), and epidermal growth factor (0.43 microM). The effects of pimozide on breast cancer cell growth were not limited to the MCF-7 cell line. Pimozide also blocked cell growth and [3H]thymidine incorporation into the ER-positive T47D and ZR75-1B human breast cancer cell lines and the ER-negative human breast cancer cell line, MDA-MB-231. Although numerous mechanisms of action of pimozide and thioridazine have been identified, both drugs are calmodulin antagonists at drug concentrations that inhibit breast cancer cell growth in vitro. Inhibition of MCF-7 cell growth by the selective calmodulin antagonists W-13 and W-12 is consistent with a role for calmodulin antagonism in the broad growth-inhibitory properties of pimozide. We conclude that pimozide and thioridazine may be useful in the control of estradiol- and polypeptide hormone-induced growth of ER-positive and ER-negative human breast tumors.     

New approach for visualizing estrogen receptors in target cells using inherently fluorescent ligands and image intensification

Four fluorescent estrogen ligands were investigated as agents for visualization of estrogen receptors in cells: 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid delta-lactone (coumestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5-(10),9(11)-estratetraene-3, 17 beta-diol] (12-oxoestradiol), which are inherently fluorescent compounds; and tamoxifen [Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene) and 4-hydroxytamoxifen [Z)-1-[4-(2-dimethylaminoethoxy) phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene), which become maximally fluorescent only after ultraviolet irradiation. By conventional fluorescence techniques, these agents can be detected down to 10(-8) M in water, but only to 10(-6) to 10(-7) M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 X 10(-10) M. Three of these compounds have good affinity for the estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Under conditions where autoradiographic controls indicate that most of the estrogen receptor of MCF-7 human breast cancer cells is in the nucleus, we could demonstrate nuclear fluorescence using 10(-9) M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethystilbestrol and could only be observed through a fluorescence microscope equipped with a microchannel image intensifier and a video camera detector that together provide a sensitivity enhancement of approximately 10(4). These studies indicate that the estrogen receptor in breast cancer cells can be visualized by fluorescence techniques, provided that the visualizing ligands have adequate affinity and specificity for the receptor and appropriate fluorescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.     

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.     

Antiestrogenic effects of Z-1, 1-dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane(5a) on human breast cancer cells in culture.

Compound 5a ([Z]-1, 1-Dichloro-2,3 diphenyl-2-(4-methoxyphenyl)cyclopropane) is a novel cyclopropyl compound which was shown to be a pure antiestrogen. In the present study, the antiproliferative activity of 5a was examined on estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 human breast cancer cells and A-549 human lung cancer cells using the hemocytometric trypan blue exclusion method. Compound 5a inhibited the growth of MCF-7 cells in a dose-related manner over a concentration range of 10(-9) to 10(-5) M, but did not alter the growth of MDA-MB-231 or A-549 cells. Co-administration of estradiol (10(-8) M) reversed the antiproliferative activity of 5a (10(-7) M) on MCF-7 cells. Further, an ER-dependent mechanism of action is supported by the specific ER binding of 5a in MCF-7 cells observed in this study. The influence of 5a on the cell surface morphology of MCF-7 and MDA-MB-231 cells was studied using scanning electron microscopy (SEM). Compound 5a at 10(-6) M reduced the length and density of microvilli (MV) on MCF-7 cells, which was reversed by co-administration of estradiol (10(-8) M). This compound did not alter the cell surface morphology of ER-negative MDA-MB-231 cells. In conclusion, 5a and tamoxifen inhibited the growth of ER-prositive MCF-7 cells in an estradiol-reversible manner, and had no effect on ER-negative MDA-MB-231 cells. The results of this study with human breast cancer cells suggest that 5a may be highly effective in the treatment of estrogen-dependent breast cancer and/or in the prophylactic treatment of women with a high risk of breast cancer development.     

Thyroid hormone receptors in human breast cancer

Optimum conditions were established for preparation of nuclei from human breast cancer biopsies. Incubation of nuclei with various concentrations of L-3,3',5-[125I]triiodothyronine showed the presence of three binding systems: a high-affinity binding system (type I) (KD approximately 0.5 micro M); an intermediate-affinity saturable system (type II) (Kd approximately 0.5 micro M)p and a nonsaturable nonspecific system. Salt at high concentrations (0.4 M-KCl) extracted only type I triiodothyronine-binding protein, thus simplifying its study and quantification. Type I binding protein was shown to have the affinity for triiodothyronine and the hormonal specificity usually ascribed to thyroid hormone receptors. Its sedimentation coefficient was 3.6S at 0.4 M KCl. Extractable triiodothyronine receptors was measured in 58 individual biopsies of primary and metastatic breast cancer. It was present in all tumors, but its concentration was highly variable (average, 0.20 pmol/mg DNA; range 0.044 to 0.702). Triiodothyronine receptor concentration was not correlated with age or endocrine status of the patient or with extension or histological grading of the tumor. Moreover, there was no correlation with estradiol and progesterone receptor concentration.     

Prolonged retention of estradiol by human breast cancer cells in tissue culture.

Conditions are described under which prolonged estradiol retention and estrogenic activity are observed in human breast cancer cells in tissue culture. The cells were incubated for three hr with a physiological concentration of [3H]estradiol (3 to 5 nM) and then were washed with 3 successive exchanges of medium 3, 17, and 24 or 48 hr following incubation with [3H]estradiol. The total wash period was 78 hr. The following parameters were monitored to assess the duration of estrogen action in MCF-7 human breast cancer cells in tissue culture; (a) the concentration of [3H]estradiol and [3H]estradiol metabolites in the media washes; (b) the intracellular concentration of [3H]estradiol and [3H]estradiol metabolites; and (c) the time course of estradiol-enhanced rates of radiolabeled thymidine incorporation. The [3H]estradiol concentration in the final medium wash was approximately 0.05 nM. The total intracellular concentration of tritium was about 50 nM prior to wash and 9 nM following 78 hr of wash. The intracellular concentration of specifically bound [3H]estradiol was initially 18 nM, and after 78 hr of wash, it was 2.8 nM. After 48 hr of wash, nearly all specifically bound [3H]estradiol was present in the nucleus. Following incubation of the cells with 5 nM estradiol and an identical wash procedure, estrogenic activity as measured by a stimulation of thymidine incorporation was observed throughout the 78 hr monitored. When 10(-6) M tamoxifen or 10(-7) M unlabeled estradiol was included in the medium washes, the washout of nonspecific binding was unaffected; however, specifically bound [3H]estradiol was essentially eliminated within 24 hr. When bovine serum albumin was included in the medium washes, total, nonspecific, and specific [3H]estradiol binding was reduced in a parallel and dose-dependent fashion. After 48 hr, cells washed with medium containing 3.5 or 7% bovine serum albumin contained one-tenth of the [3H]estradiol present in cells washed with medium alone. We conclude that medium exchanges alone do not effectively remove estradiol from MCF-7 cells, and suggest that estrogen retention by estrogen-responsive cells may mask in vitro assessments of such responsiveness in this and other systems. Inclusion of bovine serum albumin in the washes may alleviate this problem.     

Receptor characteristics of the rat mammary carcinoma cell line 64-24

Saturable binding of androgens, glucocorticoids, and triiodothyronine was found in the 64-24 hormone-responsive rat mammary carcinoma cell line. Androgen receptors had a dissociation content (Kd) for methyltrienolone of 3.4 X 10(-10) M and a binding capacity of approximately 10,000 sites/cell in whole cells. 5 alpha-[3H]dihydrotestosterone (DHT) was specifically taken up into approximately 2,150 nuclear sites with an affinity of 8.3 X 10(-10) M when nuclei were isolated from whole cells incubated with [3H]DHT. Sucrose gradient centrifugation of cytosol prepared from these cells revealed a displaceable [3H]DHT-binding component which migrated at 8S. Sedimentation analysis with high salt gradients of nuclear extracts from cells incubated with [3H]DHT revealed a peak of radioactivity in the 4S region which was abolished by coincubation of the cells with excess nonradioactive methyltrienolone. Receptors for [3H]dexamethasone were more abundant (approximately 50,000 sites/cell) in whole cells and had a Kd of 7.5 X 10(-9) M, but the number of nuclear binding sites was similar to that for androgens. Specificity studies using unlabeled steroids showed that each of the two classes of steroid receptors had greater affinities for their appropriate hormones. High affinity receptors for estrogens and progestins were not detectable in these cells. Triiodothyronine receptors were demonstrable but at a very low binding capacity (1,100 sites/cell). The Kd of these receptors was 0.6 X 10(-10) M. Cytogenetic studies revealed 44 chromosomes/mitosis with several unique markers. These receptor and karyotypic features suggest that the 64-24 cells may be useful in studying androgen action on breast cancer independently of estrogen or progestin influence, as well as the effects of thyroid hormone and glucocorticoids on breast cancer cells.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds on the occupied nuclear estrogen receptor in MCF-7 human breast cancer cells

Treatment of MCF-7 human breast cancer cells with 17 beta-[3H]estradiol resulted in a rapid accumulation of occupied nuclear estrogen receptor complex in which levels were maximized within 1 h and decreased after 3 h. Pretreatment of the cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 6 or 12 h prior to the addition of the radiolabeled hormone resulted in a 38 and 63% reduction in the levels of the occupied nuclear estrogen receptor, respectively, whereas addition of TCDD 1 h prior to the radioligand did not cause any significant change in the levels of the occupied nuclear receptor using velocity sedimentation analysis. Moreover, it was also shown with estrogen receptor antibodies that the TCDD-mediated dose-dependent decrease in occupied nuclear receptor levels was paralleled by a comparable decrease in immunoreactive protein at concentrations of 10(-8) to 10(-11) M TCDD. The reduction in levels of the occupied nuclear estrogen receptor was not due to increased estradiol metabolism since a significant reduction was observed at TCDD concentrations (10(-11) to 10(-13) M) which do not induce cytochrome P-450-dependent monooxygenase enzyme activities in MCF-7 cells. Treatment of the MCF-7 cells with actinomycin D or cycloheximide resulted in a greater than 2-fold increase in levels of the occupied nuclear estrogen receptor, and cotreatment of the cells with both TCDD and these inhibitors significantly decreased levels of the nuclear estrogen receptor complex. The structure-activity relationships for TCDD and several congeners were similar for both the reduction of occupied nuclear estrogen receptor levels and several aryl hydrocarbon (Ah) receptor agonist activities, and these results support a role of the Ah receptor in these processes.     

Growth inhibition of human breast cancer cells in vitro with an antibody against the type I somatomedin receptor

Insulin and insulin-like growth factors (IGFs) stimulate the growth of human breast cancer cells in vitro. The type I somatomedin receptor (SR), expressed in these cells, may mediate the mitogenic effects of these peptides. We have examined the effect of type I SR blockade on human breast cancer growth with a monoclonal antibody (alpha-IR3) that blocks the receptor binding domain. alpha-IR3 inhibited binding of 125I-IGF-I in all breast cancer cell lines tested. Binding affinity of alpha-IR3 was 2 to 5 times higher than that of IGF-I in MDA-231 (Kd 2.1 nM) and MCF-7 cells (Kd 0.6 nM), respectively. In the presence of 10% calf serum, the antibody inhibited anchorage-independent growth of six of seven breast cancer cell lines. This inhibition was reversible with excess IGF-I. In serum-free medium, alpha-IR3 blocked IGF-I-stimulated DNA synthesis in four of four breast cancer cell lines (MCF-7, ZR75-1, MDA-231, and HS578T). However, the antibody did not inhibit basal growth of any of the breast cancer cell lines in serum-free conditions. In three estrogen receptor-positive, estrogen-responsive breast cancer cell lines (MCF-7, ZR75-1, and T47D), type I SR blockade with alpha-IR3 failed to block estrogen-stimulated DNA synthesis or cell proliferation, indicating that secreted IGF activity is not the sole mediator of the growth effects of estrogen. In conclusion, antibody-mediated type I SR blockade does not inhibit basal growth of breast cancer cells under serum-free conditions, arguing against a critical autocrine role of endogenously secreted IGF activity in vitro. However, type I SR blockade inhibits breast cancer cell growth in the presence of serum, suggesting that serum IGFs might be critical endocrine or paracrine regulators of human breast cancer.     

Decrease in estradiol-stimulated progesterone receptor production in MCF-7 cells by epidermal growth factor and possible clinical implication for paracrine-regulated breast cancer growth

These studies have evaluated the modulation by epidermal growth factor (EGF) of estrogen receptor (ER) levels and estradiol-stimulated progesterone receptor (PgR) synthesis. Short-term culture of MCF-7 cells in an "estrogen (phenol red indicator)-free" environment caused a rise in ER concentration that is inhibited by EGF at 10(-8) M and 10(-7) M. Estradiol at 10(-10) M induced a 5-fold increase of PgR over a 5-day assay period. However, the rise in PgR was diminished or prevented by increasing concentrations of EGF (10(-9) M to 10(-7) M). Similarly, the concentration-related rise in PgR caused by estradiol (10(-13) M to 10(-9) M) was abolished after a 7-day pretreatment with EGF (10(-7) M). For both the ER and PgR receptor, EGF treatment caused a decrease in receptor number without an apparent change in receptor affinity. Thus, EGF appears to down regulate the ER by approximately 50% and to diminish the ability of estradiol to induce PgR. In addition, a survey of ER+PgR+ and ER+PgR- values of primary breast tumors from women between the ages of 55 and 70 demonstrated significantly less (50%) (85 to 39 fmol/mg of cytosol protein) ER in ER+PgR- tumors (P = 0.0005). The median PgR values for the PgR-positive tumors were 139 fmol/mg of cytosol protein. We propose that ER+ breast cancer that has changed to a paracrine growth factor-driven system (from stromal cells or ER- breast cancer cells) is less responsive to gonadal steroids. The loss of PgR in these ER+ carcinomas may be an indicator of this type of hormone independence.     

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)     

Influence of static magnetic field on the antiproliferative effects of vitamin D on human breast cancer cells

We describe the effect of a 0.2 tesla (T) static magnetic field generated by a magnetic resonance tomograph and of vitamin D treatment on a human breast cancer cell line (MCF-7). Cell damage and proliferation were monitored by measuring the incorporation of [3H]thymidine in duplicating DNA and by the clonogenic assay. [3H]Thymidine incorporation in MCF-7 was stimulated by vitamin D at low doses (10(-12)-10(-10) M), whereas it was inhibited at higher concentrations (10(-9)-10(-6) M). Magnetic field treatment (0.2 T) decreased [3H]thymidine incorporation in human breast cancer cells, eliminating the proproliferative effect of low doses of vitamin D, and enhanced the vitamin D antiproliferative effect, further reducing [3H]thymidine incorporation, from -12.5% (P < 0.05) to -66.7% (P < 0.001), over the range of 10(-9) to 10(-6) M. In the clonogenic assay, ability of MCF-7 to form colonies was inhibited by vitamin D 10(-9) M and above, whereas 3-h exposure to 0.2 T magnetic field had no effect on the number of cell colonies formed. In conclusion, vitamin D treatment yields a permanent antiproliferative effect, while magnetic field exposure only temporarily slows down cellular growth. These findings suggest that therapy with vitamin D may prove beneficial for chemoprevention or treatment of breast cancer. Static magnetic field, alone or in combination, does not appear to represent an effective candidate for breast cancer therapy, at least at the intensity used in the present study.     

Presence of steroid receptors in human soft tissue sarcomas of diverse histological origin

The incidence of cytosol receptors for androgen, estrogen, glucocorticoid, and progesterone was determined in a series of histologically diverse soft tissue sarcomas. Receptor binding was characterized in tumor specimens from 29 adult patients (14 female, 15 male) obtained during surgery. Nineteen (66%) tumor cytosols bound at least one steroid. Seven (24%) had more than one receptor. Ten of 23 (43%) cytosols assayed for androgen binding were positive (Kd 0.97 +/- 0.23 X 10(-9) M; 60.6 +/- 25.9 fmol/mg protein), 7 of 29 (24%) for estrogen (Kd 1.00 +/- 0.19 X 10(-9) M; 33.2 +/- 10.9 fmol/mg protein), 9 of 26 (35%) bound glucocorticoid (Kd 4.22 +/- 1.77 X 10(-9) M; 231.0 +/- 155.0 fmol/mg protein), and 1 of 28 (4%) bound progesterone (Kd 0.41 X 10(-9) M; 18.7 fmol/mg protein). Density gradient analysis suggested that androgen and estrogen binding was located predominantly in the 6S and 7 to 8S regions, respectively, whereas receptor for glucocorticoid sedimented at 8S. Steroid binding was not related to patient age. Estrogen-positive (71%) and glucocorticoid-positive (78%) cytosols appeared predominantly in tumors from female patients. Receptor distribution also appeared to be correlated to tumor histogenetic origin.     

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.     

Estradiol and progesterone receptors in malignant gastrointestinal tumors

Estradiol and progesterone receptors were assayed in tumors from 79 patients with primary colorectal and 56 patients with stomach adenocarcinomas. Eighteen of 79 colorectal cancers contained estradiol receptor, while 34 specimens were positive for progesterone receptor. In stomach cancer, the positive samples were 8 for estradiol and 14 for progesterone receptors. In both types of tumors, the Kd was in the range of 10(-10) M for estradiol and 10(-9) M for progesterone receptor, respectively. In colorectal adenocarcinomas, the presence of progesterone receptor seems to be partially correlated to the presence of estradiol receptor while, in stomach tumors, this correlation is lost. The positivity of at least one receptor in colorectal cancers is higher in the female sex. The contrary occurs for stomach cancer. Sucrose gradient centrifugation showed that cytoplasmic estradiol receptor of stomach cancer sedimented at 8S or 4 to 5S at low ionic strength. The isoelectric point of stomach cancer estradiol receptor is 6.5.