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.     

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.     

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.     

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.     

Two new estrogen-supersensitive variants of the MCF-7 human breast cancer cell line

Two new estrogen-sensitive variants of MCF-7 human breast cancer cells, CG-4 and CG-5, are described in this report. These cells were derived from a casual contamination by MCF-7 cells of primary cultures from a human adenocarcinoma of the breast and a pleural effusion of a patient with advanced breast cancer, respectively. Careful characterization of these variants revealed chromosomal properties highly similar to and alloenzyme phenotypes identical to those of MCF-7 cells which were simultaneously cultured in the laboratory. MCF-7, CG-4 and CG-5 cells were tested for estrogen responsiveness under identical growth conditions, that is in the presence of culture medium supplemented with 5% charcoal-treated serum. While the number of MCF-7 cells increases by about 40% over the controls in the presence of physiological concentrations of estradiol, the number of CG-4 cells doubles and the number of CG-5 cells triples. All these cell lines have approximately the same number of estrogen, androgen, glucocorticoid, and progesterone receptor sites/cell. Since several difficulties arise in demonstrating the estrogen responsive growth stimulation of currently available human breast cancer cells, these two new variants, characterized by a high and reproducible estrogen responsiveness, afford a new model for studying the mechanisms by which estrogen regulates cell proliferation. The problems related to the careful characterization of every established cell line are discussed.     

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.     

Membrane-associated binding sites for estrogen contribute to growth regulation of human breast cancer cells

Membrane-associated binding sites for estrogen may mediate rapid effects of estradiol-17beta that contribute to proliferation of human breast cancers. After controlled homogenization and fractionation of MCF-7 breast cancer cells, the bulk of specific estradiol binding is found in nuclear fractions. However, a significant portion of specific, high-affinity estradiol-17beta binding-sites are also enriched in plasma membranes. These estradiol binding-sites co-purify with 5'-nucleotidase, a plasma membrane-marker enzyme, and are free from major contamination by cytosol or nuclei. Electrophoresis of membrane fractions allowed detection of a primary 67-kDa protein and a secondary 46-kDa protein recognized by estradiol-17beta and by a monoclonal antibody directed to the ligand-binding domain of the nuclear form of estrogen receptor. Estrogen-induced growth of MCF-7 breast cancer cells in vitro was blocked by treatment with the antibody to estrogen receptor and correlated closely with acute hormonal activation of mitogen-activated protein kinase and Akt kinase signaling. Estrogen-promoted growth of human breast cancer xenografts in nude mice was also significantly reduced by treatment in vivo with the estrogen receptor antibody. Thus, membrane-associated forms of estrogen receptor may play a role in promoting intracellular signaling for hormone-mediated proliferation and survival of breast cancers and offer a new target for antitumor therapy.     

Long-term cultivation of a human breast cancer cell line, MCF-7, in a chemically defined medium. Effect of estradiol

The human breast cancer cell line, MCF-7, has been adapted to long-term growth in chemically defined medium without loss of estrogen and progesterone receptors or tumorigenicity in athymic mice. An estrogen reversible inhibition of cell proliferation is exerted by newborn calf serum (NCS) 10%, athymic mouse serum (AMS) 2% or tamoxifen 0.1 to 1.0 microM. The mamma-related hormones, hydrocortisone, progesterone, prolactin, and insulin could not mimic this growth inhibitory effect, and estradiol alone or combined with these hormones did not stimulate cell proliferation in chemically defined medium.     

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.     

Estrogen responsive proliferation of clonal human breast carcinoma cells in athymic mice

A clone of MCF-7 (MCF-7ED), a cell in continuous in vitro cultivation derived from an estrogen-responsive human breast carcinoma, requires estrogen supplementation for progressive exponential (doubling time: 50–85 h) tumor growth in the mammary fat of athymic mice. The plasma concentration of estradiol which stimulated exponential growth of MCF-7ED corresponded to physiologic premenopausal levels in women. The tumors were carcinomas with murine and MCF-7ED cells intermixed. MCF-7ED cells could be repurified in subcultures of mixed tumors. Comparative studies of breast and non-breast cell lines showed concordance between the presence of estradiol receptor, sensitivity to the anti-estrogen tamoxifen for growth in vitro, and estradiol dependence for tumorigenic growth in athymic mice. Progesterone alone did not stimulate MCF-7ED growth, but acted synergistically with estrogen. Progesterone's action was to decrease tumor latent period, not to increase final tumor incidence or growth rate. Under estrogen-deficient conditions, conditions approximating postmeno-pausal status in women, (10^?10 M in plasma), a dormant state was established between MCF-7ED cells and murine mammary stroma which could be maintained several months. The dormant state could be broken by introduction of estradiol, but not progesterone. This system should be useful for defining host and cancer cell determinants in estrogen-responsive breast cancer growth.     

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.     

Effect of estrogen and antiestrogen on the human breast cancer cell line MCF-7 adapted to growth at low serum concentration

The human breast cancer cell line MCF-7 has been adapted to long-term growth at 0.5 and 0.05% fetal bovine serum. Free cytoplasmic and filled nuclear estrogen receptors were found in cells grown at 5, 0.5, and 0.05% fetal bovine serum. Cells grown in medium with 0.05% dextran-charcoal-treated serum contained cytoplasmic receptors but no filled nuclear receptors, indicating that this medium did not contain biologically active concentrations of estrogen. Addition of estradiol to the medium translocated the cytoplasmic receptor to the nucleus and stimulated progesterone receptor synthesis but did not increase the growth rate. The antiestrogen tamoxifen (TAM) inhibited cell growth at all serum concentrations investigated, at least in part by a reduction of the growth fraction. The sensitivity to TAM was highest at low serum concentrations. The effect of TAM could be reversed by estradiol at TAM concentrations of 10(-6) M or lower. It is concluded that estradiol does not have a direct growth-stimulatory effect on our MCF-7 cells in monolayer cultures although the cells contain functional estrogen receptors and growth of the cells in athymic mice requires estrogens. TAM has an estrogen-competitive, inhibitory effect on the growth of MCF-7 cells at concentrations lower than 10(-6) M. At higher concentrations, the growth inhibition is unspecific and noncompetitive by estradiol.     

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.     

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.     

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.     

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.     

Inhibition of breast cancer cell growth in vitro by a tyrosine kinase inhibitor.

Human breast cancer cell proliferation is regulated by growth factors that bind to receptors with intrinsic tyrosine kinase (TK) activity, including the epidermal growth factor (EGF) receptor. To determine whether inhibition of receptor TK activity inhibits tumor growth, we studied the effects of a tyrosine kinase inhibitor, RG-13022, on cultured human breast cancer cells. RG-13022 represents a class of compounds which have been shown to inhibit preferentially the TK activity of the EGF receptor in a cell-free system and also to inhibit EGF-stimulated growth of cultured cells. RG-13022 significantly inhibited EGF-stimulated autophosphorylation of its receptor in two breast cancer cell lines that have abundant, although not amplified, EGF receptor content (MDA-231 and T47D). RG-13022 also inhibited EGF-stimulated DNA synthesis and proliferation of T47D and MCF-7 breast cancer cells in a reversible and dose-dependent manner. Inhibition was observed at 0.1 microM, and it was maximal at 10 microM. The effect was rapid (within 3 h), persisted for 18 h, and was partially reversed by 24 h at 1 microM. At 5 microM, inhibition persisted for more than 50 h. Inhibitory effects were also observed in a panel of estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines. RG-13022 inhibited not only EGF-induced growth but also growth stimulated by insulin, insulin-like growth factor I, insulin-like growth factor II, or transforming growth factor alpha. RG-13022 also totally blocked estrogen-stimulated phosphorylation of the EGF receptor, as well as estrogen-induced cell proliferation, suggesting that functioning TK pathways are required for estrogen action. The TK inhibitor RG-13022 is a potent inhibitor of hormonally regulated growth of human breast cancer. Tyrosine kinase inhibitors have the potential of providing a new strategy for the "endocrine therapy" of 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.