Antiestrogen binding sites in microsomal fractions of malignant and nonmalignant human breast tissues

Antiestrogen binding sites (AEBS) were measured in microsomal fractions of 102 malignant breast tumors and 24 nonmalignant breast tissues. The number of AEBS was determined by Scatchard analysis. The cytosol contents of estrogen and progesterone receptors were also analyzed in these tissues. Overall, 50% of the malignant tumors and 33% of the nonmalignant normal breast tissues had detectable contents of AEBS. No correlation was observed between cytosol estrogen receptor (ER) content and microsomal AEBS in the tumors. Detailed data analysis in patients at Stage IV disease revealed that 60% of estrogen receptor positive tumors had no detectable microsomal AEBS contents whereas in the remaining 40% tumors, high affinity AEBS were observed. On the other hand, AEBS were also detected in 35% of ER-poor tumors. Antiestrogen binding sites were higher in tumors obtained from premenopausal women than in those of postmenopausal women. The incidence of AEBS-positive tumors or AEBS concentration was not influenced by either the patients' obesity or their disease stage.     

Resistance to tamoxifen with persisting sensitivity to estrogen: possible mediation by excessive antiestrogen binding site activity.

The growth of a large proportion of estrogen receptor-positive breast tumors is stimulated by estrogen and can often be controlled through antiestrogen therapy. Resistance to antiestrogen (AE) therapy can occur while tumors retain the expression of estrogen receptors (ERc) and remain functionally responsive to estrogens. The ability of specific antiestrogen binding sites (AEBS) to prevent AE from interacting with ERc has been examined as a possible mechanism through which this appropriation of AE could interfere with antiestrogen action. Comparisons were performed between uterine preparations where ERc activity exceeded AEBS binding and liver preparations where AEBS binding predominated. Identical estimates of ERc activity were obtained in uterine preparations using either [3H]estradiol or [3H]-4OH-tamoxifen and radioinert diethylstilbestrol (alpha,alpha'-diethyl-4,4'-stilbenediol) to estimate nonspecific binding. AEBS binding was observed only when [3H]-4OH-tamoxifen was used, while binding to Type II sites was resolved only with [3H]estradiol. When excess AEBS activity predominated, analyses with radiolabeled estrogen and antiestrogen present simultaneously showed that virtually all of the antagonist was bound to AEBS with little of the antagonist available to associate with ERc. In an effort to relate these observations to AE resistance per se, ERc and AEBS were measured in MCF-7 human breast cancer cells (ERc-positive, responsive to estrogens and antiestrogens) and in variant AE-insensitive LY-2 human breast cancer cells (ERc-positive, responsive only to estrogens). In AE-resistant LY-2 cells, the ratio of AEBS:ERc was approximately three times greater than in MCF-7 cells. Examination of 128 human breast carcinomas revealed that AEBS activity was present and could exceed ERc activity. Importantly, the partition of significant AE away from ERc was observed in human specimens. These observations identify a biochemical mechanism for antiestrogen resistance through which AE access to ERc can be totally incapacitated while sensitivity to estrogens continues. These observations indicate that AEBS activity, in addition to ERc activity, may provide helpful information for predicting the response of certain cancers to hormonal therapy.     

The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture.

We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites.     

Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short-term and long-term absence of estrogens

We have examined the effect of short-term and long-term growth in the absence of estrogens on the proliferation rate and estrogen and antiestrogen responsiveness of MCF-7 human breast cancer cells. The removal of phenol red, the pH indicator in tissue culture medium that is weakly estrogenic (Y. Berthois et al., Proc. Natl. Acad. Sci. USA, 83:2496-2500, 1986), immediately slows the cell proliferation rate, and MCF-7 cells grown in phenol red-free medium with charcoal dextran-treated serum for periods up to 1 mo maintain this reduced rate of cell proliferation. In these short-term phenol red-withdrawn cells, estradiol stimulates proliferation markedly and reproducibly, and antiestrogens inhibit estrogen-stimulated proliferation. Antiestrogens by themselves appear as partial agonists/antagonists; at low concentrations they stimulate proliferation weakly, but they show no stimulation at the high concentrations where they fully inhibit estrogen-stimulated proliferation. In contrast to the short-term phenol red-withdrawn cells, cells maintained for several months (5 to 6 mo) in the apparently complete absence of estrogens (no phenol red, with charcoal dextran-treated calf serum) show a markedly increased basal rate of cell proliferation; estradiol is unable to increase this rate of proliferation further, but antiestrogens are able to decrease proliferation. This change in growth pattern is associated with a 3-fold increase in cellular estrogen receptor levels. Despite their differing basal growth rates, cells grown in either the short-term (less than 1 mo) or long-term (greater than 6 mo) absence of estrogens both have progesterone receptor levels that are very low and, in both cases, estradiol increases progesterone receptor levels markedly. Thus, under long-term estrogen-free conditions, there is a dissociation between the stimulation of cell proliferation and of specific protein synthesis (progesterone receptor) by estrogen. The increase in the cell proliferation rate observed in cells grown in the long-term absence of estrogen may reflect altered regulation of growth factor production or altered sensitivity to growth factors in the medium or produced by the cells themselves. Hence, these breast cancer cells adapt significantly to long-term growth in estrogen-free conditions, an observation that may be relevant to understanding the growth of hormone-responsive human breast cancers in vivo.     

Compared effects of GnRH analogs and 4-hydroxytamoxifen on growth and steroid receptors in antiestrogen sensitive and resistant MCF-7 breast cancer cell sublines

Summary Antiestrogens, such as tamoxifen, have a direct antitumor action and are widely used in cancer therapy. The direct antitumor action of GnRH analogs has been documented in bothin vitro and clinical studies. GnRH analog direct action could therefore provide an alternative approach in postmenopausal patients developing antiestrogen resistance, a frequent cause of relapse during hormonal treatment of breast cancer. This study was carried out to compare the effects of two GnRH analogs (the agonist Decapeptyl and the antagonist BIM 21009C) and the antiestrogen 4-hydroxy-tamoxifen (OH-TAM) on proliferation in antiestrogen-responsive or antiestrogen-resistant human breast cancer cell lines (MCF-7, MCF-7 LY2). Ineffective when used without estrogen stimulation, both of the GnRH analogs and OH-TAM inhibit the 17-estradiol-stimulated growth of the estrogen-responsive cell line MCF-7. Compared with parental MCF-7 cell line responsiveness, the antiestrogen-resistant variant MCF-7 LY2 appears to be resistant to GnRH analogs. These findings indicate similarities between the two types of compounds with regard to their antiestrogenic effects on growth observedin vitro. However, since unlike OH-TAM, Decapeptyl displays no effect on steroid receptor levels of sensitive MCF-7 cells, the intracellular inhibitory mechanisms of these drugs are probably in part different. This study suggests that the direct effect of the studied GnRH analogs would not be a potent tool for treating antiestrogen-resistant breast tumors.     

Antiestrogen inhibition of prolactin-induced growth of the Nb2 rat lymphoma cell line

The rat lymphoma cell line Nb2 is highly prolactin responsive in terms of growth. Estrogens are without effect in these cells that lack the estrogen receptor. The growth stimulation by lactogenic hormones is effectively inhibited by antiestrogens, such as tamoxifen and nafoxidine, at concentrations as low as 10(-10) M. The growth inhibition is partially reversed by replacement of the antiestrogens by prolactin. Nb2 cells contain estrogen-noncompetitive specific antiestrogen binding sites on their membranes that bind tamoxifen with an average Kd of 3.1 X 10(-10) M. Addition of antiestrogens to the binding reaction inhibits lactogenic hormone binding to membrane-bound receptors. The order of affinities of various antiestrogens (tamoxifen, nafoxidine, 2-(4-tert-butyl-phenoxy)ethyl diethylamine hydrochloride, and LY117018) for the antiestrogen binding sites parallels the order of their potencies as growth and lactogen binding inhibitors. These data suggest that antiestrogens, possibly acting through the antiestrogen binding sites, may function as antilactogens.     

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.     

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)     

Activity of tamoxifen and its metabolites on endocrine-dependent and endocrine-independent breast cancer cells

To better understand the mechanism of action of antiestrogens, the growth-inhibitory effect of tamoxifen and its main metabolites N-desmethyltamoxifen and 4-hydroxytamoxifen, was studied in 6 breast cancer cell lines characterized by different steroid receptor contents. On the basis of the results, our cell lines could be classified into three groups: a first group, including 734B and ZR-75.1 cell lines, characterized by a clear endocrine-dependent behavior, in which cells were sensitive to antiestrogens although to different degrees; a second group, including MDA-MB 231 and BT20 cell lines, characterized by a clear endocrine-insensitive behavior, in which cells were affected only by the highest (10(-6) M) antiestrogen concentration; a third group, including MCF7 and T47D cell lines, characterized by a peculiar behavior. The T47D cell line displayed an increased growth rate after treatment with all three antiestrogens considered. Despite the positive receptor content in the MCF7 cell line, only 4-hydroxytamoxifen showed a clear antiestrogen dose-dependent effect, whereas tamoxifen decreased the cell growth rate only at lower concentrations (10(-8) and 10(-7) M). These results and the well-known heterogeneity of human breast tumors explain the failure of antiestrogen treatment in a certain percentage of patients with breast cancer with a positive estrogen receptor status.     

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.     

Antiestrogen regulation of cell cycle progression and cyclin D1 gene expression in MCF-7 human breast cancer cells

The molecular mechanisms by which antiestrogens inhibit breast cancer cell proliferation are not well understood. Using cultured breast cancer cell lines, we studied the effects of antiestrogens on proliferation and cell cycle progression and used this information to select candidate cell cycle regulatory genes that are potential targets for antiestrogens. Under estrogen- and serum-free conditions antiestrogens inhibited proliferation of MCF-7 cells stimulated with insulin. Cells were blocked at a point in G₁ phase. These effects are comparable with those in serum- and estrogen-containing medium and were also seen to a lesser degree in nude mice bearing MCF-7 tumors. Similar observations with other peptide mitogens suggest that the process inhibited by antiestrogens is common to estrogen and growth factor activated pathways. Other studies have identified G₁ cyclins as potential targets for growth factor and steroid hormone/steroid antagonist regulation of breast epithelial cell proliferation. In MCF-7 cells growing in the presence of fetal calf serum, cyclin D1 mRNA was rapidly down-regulated by steroidal and nonsteroidal antiestrogens by an apparently estrogen receptor mediated mechanism. Cyclin D1 gene expression was maximally inhibited before effects on entry into S phase and inhibition was therefore not merely a consequence of changes in cell cycle progression. Together with data on the effects of antiestrogens in serum-free conditions [1], these results suggest down-regulation of cyclin D1 by antiestrogens may be a general phenomenon in estrogen receptor-positive breast cancer cells, independent of culture conditions and class of antiestrogen. These observations are compatible with the hypothesis that reductions in cyclin D1 levels may mediate in part the action of antiestrogens in blocking entry of cells into S phase.     

Antiestrogenic properties of keoxifene,trans-4-hydroxytamoxifen,and ICI 164384, a new steroidal antiestrogen,in ZR-75-1 human breast cancer cells

The agonistic/antagonistic properties of two non-steroidal antiestrogens, namelytrans-4-monohydroxy-tamoxifen (OH-TAM) and keoxifene (LY156758), and the new steroidal antiestrogen ICI164384, a 7-alkylamide derivative of estradiol (E₂), were assessed by measuring their effect on the proliferation of ZR·75-1 cells, an estrogen-responsive human breast cancer cell line. While subnanomolar concentrations of both OH-TAM and LY156758 had significant estrogenic stimulatory activity on cell growth in the absence of estrogens and higher concentrations were inhibitory, ICI164384 behaved exclusively as a growth inhibitor and more potently so than the two other compounds. The three antiestrogens had similar potency to inhibit the mitogenic effect of E₂ and at 300 nM, all antiproliferative effects were completely reversible by the estrogen. ICI164384 was a weaker competitor of³H-labeled E₂ or R2858 (moxestrol) uptake in intact ZR-75-1 cells in a 1-hour assay, partly because of a slower intracellular access to estrogen specific binding sites. Moreover, ICI164384 interacted in a rapidly (~ 6 h) reversible manner with estrogen-specific binding sites, while the non-steroidal antiestrogens induced a longer-acting (> 24 h) down-regulation of specific [³H]R2858 uptake. The present data indicate that, among the antiestrogens studied, ICI164384 is the only compound acting as a pure antiestrogen in ZR-75-1 breast cancer cells, while LY156758 and OH-TAM behave as antiestrogens endowed with partial agonistic activity in this system.     

Effect of estrogens and antiestrogens on growth of human breast cancer cells in athymic nude mice

Endocrine therapy with estrogen deprivation or with antiestrogens results in tumor regression in a subset of patients with advanced breast cancer. To better understand the mechanisms by which estrogens and antiestrogens modulate breast cancer growth in vivo, we have studied the effects of endocrine manipulation on the development and growth of tumors derived from cultured human breast cancer cells in the athymic nude mouse. MCF-7 breast cancer cells were inoculated into 6-week-old female BALB/c athymic nude mice. Tumor growth did not occur in ovariectomized mice. Cells remained viable, however, since estrogen supplementation more than 30 days later resulted in tumor formation. Minimal tumor growth was observed in intact female nude mice which have low circulating estrogen levels. Tumor development and growth in ovariectomized or intact mice supplemented with 17 beta-estradiol in the form of a s.c. pellet were dose dependent; growth rates increased with estrogen doses ranging from 0.01 to 0.5 mg. Antiestrogen treatment with either tamoxifen or LY156758 caused transient stimulation of tumor growth, followed by a prolonged stationary phase. Growth resumed with estrogen supplementation. Treatment of mice bearing established MCF-7 tumors with estrogen withdrawal (removal of estrogen pellet) resulted in cessation of tumor growth, but not in tumor regression. Growth inhibition was also observed with antiestrogens and was dose dependent. However, tumor regression did not occur, even in mice treated with high doses of tamoxifen (serum concentration of 1.0 microM) for as long as 60 days. Tumor growth was restored in these mice with estrogen replenishment. Tumor cells also remained viable histologically despite prolonged (1 month) estrogen deprivation or antiestrogen therapy, although the mitotic index was markedly reduced. Similar observations were made with mice inoculated with the hormone-responsive ZR75-1 human breast cancer cells, but not with hormone-independent MDA-231 cells which were not influenced by estrogen or antiestrogen treatment. In summary, development and growth of MCF-7 and ZR75-1 tumors in nude mice are estrogen dependent. Endocrine therapy by estrogen deprivation or antiestrogen treatment inhibits tumor cell proliferation in nude mice, but does not cause tumor regression or loss of cell viability.     

Effects of tamoxifen and 4-hydroxytamoxifen on synchronized cultures of the human breast cancer cell line MCF-7

Summary The effect of antiestrogens on cultures of synchronized MCF-7 human breast cancer cells was studied. Cultures were synchronized by an eighteen hour block with 1.5mM hydroxyurea. Tamoxifen or 4-hydroxytamoxifen was added at various times following removal of the hydroxyurea block and the cell number and rate of incorporation of³H-thymidine by the cultures were followed for 48 hours through two waves of DNA synthesis. Antiestrogens added when hydroxyurea was removed inhibited only the second wave of DNA synthesis. Inhibition of the second wave only occurred if the antiestrogen was added before or at about the time of mitosis. To determine whether antiestrogen had to be present before mitosis in order to block the next round of DNA synthesis, cells in mitosis were selected by shaking them loose. The subsequent division of these cells was followed to determine the effect of treating them with antiestrogens before, after, or both before and after mitosis. The addition of antiestrogens at the time of mitosis proved sufficient to block the next round of cell division. Thus antiestrogens are cell cycle specific agents and appear to act at a point early in G₁ to block the next round of DNA synthesis and cell division.     

Binding Sites of Droloxifene in the Cytosol of 7,12-Dimethylbenz[α]anthracene-induced Rat Mammary Tumor Cells

The binding sites, other than the estrogen receptor (ER), of the antiestrogens droloxifene (DROL, (E)-α-[ p [2-(dimethylamino)ethoxy]-phenyl]-α'-ethyl-3-stilbenol) and tamoxifen (TAM), and estradiol-17β (E₂) in the cytosol of 7,12-dimethylbenz[α]anthracene-induced rat mammary ER-positive tumor cells were studied using a high-performance liquid chromatography (HPLC) gel filtration assay. The cytosol was incubated with ³H-labeled drug with or without unlabeled drug, and separated by HPLC gel filtration. ³H-E₂ produced two major peaks of radioactivity at fractions No. 40 and No. 70. The peak at fraction No. 70 was identified as the ER in an ER-enzyme-immuno assay. This peak was dose-dependently inhibited by unlabeled DROL or TAM, DROL being a more potent inhibitor than TAM. The peak at fraction No. 40 was also inhibited by co-incubation with unlabeled DROL or TAM. ³H-DROL or ³H-TAM provided only one peak at fraction No. 43. This peak was thought to be an antiestrogen binding site (AEBS), because it was inhibited by unlabeled antiestrogen hut not by E₂. The results suggest that the antiestrogens DROL and TAM have a higher affinity for the AEBS than for the ER in the absence of E₂, while in the presence of E₂ both have an affinity for the ER and inhibit E₂ binding to the ER.     

Sequential Versus Combined Treatment of Human Breast Cancer Cells with Antiestrogens and the Vitamin D Analogue EB1089 and Evaluation of Predictive Markers for Vitamin D Treatment

Development of resistance to antihormonal therapy is a major problem in the treatment of breast cancer patients. Metastatic tumors, which progress after a period of response to treatment, often respond to second line endocrine treatment, but eventually develop estrogen independent growth. Vitamin D analogues are promising new drugs, using alternative mechanisms to inhibit growth of breast cancer cells. The sensitivity to antiestrogens and vitamin D analogues has been proposed to be inverse, indicating that the sensitivity to vitamin D analogues might increase after development of antiestrogen resistance and vice versa. In this study, the inverse sensitivity between antiestrogens and the vitamin D analogue EB1089 was examined in antiestrogen and vitamin D resistant breast cancer cell lines. The majority of the investigated antiestrogen resistant cell lines had increased sensitivity to treatment with the vitamin D analogue EB1089. In addition, growth of a vitamin D resistant cell line was more inhibited by the pure antiestrogen ICI 182,780 than the growth of the parental cells, indicating that the compounds may be used sequentially. An association between the expression level of the vitamin D receptor (VDR) and EB1089 sensitivity was observed, suggesting that VDR is a possible predictive marker for response to vitamin D treatment. Valuation of Bcl-2 gene expression may be useful in combination with VDR to predict the outcome of treatment with EB1089. Furthermore, we observed a synergistic growth inhibition and an abrogated development of resistance upon combined treatment with ICI 182,780 and EB1089. Therefore, antiestrogens and vitamin D analogues may also be used as combined treatment for breast cancer patients in the future.     

Effect of estrogens and antiestrogens on growth-regulatory enzymes in human breast cancer cells in tissue culture

The effect of estrogens and antiestrogens is examined on three enzymes the activities of which are known to correlate with cell growth. Estrogen treatment increases thymidylate synthetase binding sites up to 4-fold over controls. The extent of induction is dependent on incubation time and the basal rate of cell growth in untreated cells. Amount of active enzyme generally shows a positive correlation with rates of DNA synthesis and cell growth. Thymidine kinase activity and the number of dihydrofolate reductase binding sites are similarly induced by estrogen treatment. Conversely, the effect of antiestrogens on MCF-7 cells is exceedingly complex in that responses in enzyme activities and several generally accepted indices of cell growth (cell number, protein content, rate of DNA synthesis) are dissimilar. Dose response, magnitude of response, and direction of response (increase or decrease) are distinct for each enzyme and for each measure of cell growth with each antiestrogen tested. These results suggest that specific cellular activities are modulated independently by estrogens and antiestrogens and that changes in ligand-receptor complex cannot be the sole explanation for the specificity of estrogen and antiestrogen action. Some degree of specificity and heterogeneity may reside at the level of receptor interaction with the various genes subject to estrogenic modulation.