Tamoxifen inhibition of prolactin action in the mouse mammary gland.

Binding of lactogenic hormones to particulate and solubilized microsomal membranes isolated from mammary glands of lactating mice is inhibited by direct addition of 10(-10) M or greater concentrations of triphenylethylene antiestrogens [i.e. tamoxifen (TAM), 4-hydroxy-tamoxifen, and Nafoxidine] to the binding assays. Estradiol and other antiestrogens such as BPEA (-2-(4-tert-butyl-phenoxy) ethyl diethylamine hydrochloride, LY117018, and LY 156758 do not have this effect. The triphenylethylene antiestrogens bind to the membrane-associated antiestrogen binding sites (AEBS). Effectiveness of binding to the AEBS parallels the effectiveness of inhibition of the lactogen binding. The effect is selective in that binding of epidermal growth factor and insulin to these same membranes is unaffected by the antiestrogens. Binding of PRL to membranes prepared from the livers of the lactating mice is also unaffected. Both the PRL receptor and AEBS are primarily localized to the microsomal membrane fraction of cells. Maximal inhibition of PRL binding by TAM is observed in the light microsomes that contain plasma membranes. In addition to inhibition of PRL binding, TAM also prevents the PRL-induced accumulation of caseins by cultured mouse mammary explants. Thus it appears that the triphenylethylene antiestrogens, acting through the AEBS, act as antilactogens in the normal mammary gland.     

An estrogen receptor positive MCF-7 clone that is resistant to antiestrogens and estradiol.

The antiestrogen tamoxifen has been successfully used to control estrogen receptor (ER) and progesterone receptor positive breast cancer. However, the development of antiestrogen resistance is frequently observed in patients following long term treatment. We have studied the development of antiestrogen resistance in vitro and established an antiestrogen resistant variant of MCF-7 cells (clone 5C) after long term culture in estrogen free medium. The growth of clone 5C cells was not altered by either estradiol-17 beta or the antiestrogens 4-hydroxytamoxifen and ICI 164,384. Estrogen-stimulated progesterone receptor and reporter gene expression were markedly reduced in 5C cells compared to wild type MCF-7 cells. Only minor alteration in the levels of ER and no alteration in the affinity of ER for ligand were found in 5C cells. No mutation of ER cDNA in 5C cells was detected by polymerase chain reaction and DNA sequencing. This study demonstrates that change(s) in ER-mediated gene expression rather than the amino acid sequence of the ER itself may be associated with the development of at least one form of antiestrogen resistance.     

Insulin-like growth factor binding protein 2 is a marker for antiestrogen resistant human breast cancer cell lines but is not a major growth regulator.

Antiestrogens target the estrogen receptor and counteract the growth stimulatory action of estrogen on human breast cancer. However, acquired resistance to antiestrogens is a major clinical problem in endocrine treatment of breast cancer patients. To mimic acquired resistance, we have used a model system with the antiestrogen sensitive human breast cancer cell line MCF-7 and several antiestrogen resistant cell lines derived from the parental MCF-7 cell line. This model system was used to study the expression and possible involvement in resistant cell growth of insulin-like growth factor binding protein 2 (IGFBP-2). By an oligonucleotide based microarray, we compared the expression of mRNAs encoding insulin-like growth factor binding protein 1,2,3,4,5 and 6 (IGFBP-1 to -6) in the parental MCF-7 cell line to three human breast cancer cell lines, resistant to the antiestrogen ICI 182,780 (Faslodex/Fulvestrant). Only IGFBP-2 mRNA was overexpressed in all three resistant cell lines. Thus, we compared the IGFBP-2 protein expression in MCF-7 cells to nine antiestrogen resistant breast cancer cell lines, resistant to either ICI 182,780 or tamoxifen or RU 58,668 and found that IGFBP-2 was overexpressed in all nine resistant cell lines. Three of the resistant cell lines, resistant to different antiestrogens, were selected for further studies and IGFBP-2 overexpression was demonstrated at the mRNA level as well as the intra- and extracellular protein level. The objective of this study was to examine if IGFBP-2 is involved in growth of antiestrogen resistant human breast cancer cells. Therefore, IGFBP-2 expression was inhibited by antisense oligonucletides and siRNA. Specific inhibition of IGFBP-2 protein expression was achieved in MCF-7 and the three selected antiestrogen resistant cell lines, but no effect on resistant cell growth was observed. Thus, we were able to establish IGFBP-2 as a marker for antiestrogen resistant breast cancer cell lines, although IGFBP-2 was not a major contributor to the resistant cell growth.     

Physicochemical and genetic evidence for specific antiestrogen binding sites.

In rat uterus and human breast cancer MCF-7 cell cytosol, the antiestrogens tamoxifen (Tam) and 4-hydroxytamoxifen (OH-Tam) bind to "antiestrogen binding sites" (ABS), which do not bind estradiol (E). Demonstrated in total cytosol by binding studies with radioactive antiestrogens in the presence of a large concentration of E, ABS can be physically separated from E-binding estrogen receptor (ER) by removing the latter with an E-containing bioaffinity adsorbent or with heparin-Sepharose gel. ABS concentration is 10-20% of that of ER; the Kd for Tam and OH-Tam is 1-2 x 10(-9) M, whereas the Kd of OH-Tam binding by ER (approximately equal to 1 x 10(-10) M) is approximately equal to 1/50 that of Tam. Other triphenylethylene antiestrogens compete against Tam for binding to ABS, contrary to steroid hormones. Sucrose gradient ultracentrifugation analyses of total cytosol and of affinity gel effluents show a heterogenous pattern of ABS from 10 to 40 S, unchanged by 0.4 M KCl and limited trypsinization (which however provoke transitions of ER from 8S to 4S forms) and by 20 mM molybdate (which stabilizes the 8S form of ER and prevents large aggregates). Preliminary results suggest that ABS may be associated with particulate components of the cell. RTx6 cells of a clone selected from MCF-7 cells for resistance to the antigrowth effect of Tam have ER in the same concentration and have similar affinity for E and antiestrogens as do unselected MCF-7 cells. However, RTx6 cells have virtually no ABS detectable by binding and gradient ultracentrifugation studies. It is proposed that the double binding of Tam and OH-Tam to ER and ABS in estrogen target cells may be related to the complex double series of estrogenic and "antiestrogenic" activities displayed by nonsteroidal triphenylethylene derivatives.     

The effect of the new SERM arzoxifene on growth and gene expression in MCF-7 breast cancer cells

The benzothiophene arzoxifene is a new 3rd generation selective estrogen receptor (ER) modulator (SERM). We have investigated the effect of arzoxifene on growth and gene expression in the estrogen receptor alpha (ERalpha) positive human breast cancer cell line MCF-7. Arzoxifene inhibits cell growth as effectively as the antiestrogen tamoxifen. Northern analysis revealed that arzoxifene exerts a statistically significant inhibition of pS2 and progesterone receptor B mRNA expression. Significant agonistic effect was observed on the antitrypsin mRNA expression. In contrast to estradiol and tamoxifen, arzoxifene does not upregulate cathepsin D mRNA and protein expression. The metabolite of arzoxifene (ARZm) is a more potent growth inhibitor of MCF-7 cells than arzoxifene. A tamoxifen resistant MCF-7 subline displayed a significant dose-dependent growth inhibition to ARZm, whereas an ICI 182,780 resistant cell line only responded to high concentration. Our results indicate that arzoxifene and especially ARZm are efficient growth inhibitors of ER positive human breast cancer cells, including tamoxifen resistant cells. Moreover, arzoxifene displays less estrogen agonistic effects in MCF-7 cells than tamoxifen.     

Differential effects of tamoxifen and analogs with nonbasic side chains on cell proliferation in vitro

Structural analogs of the nonsteroidal antiestrogen tamoxifen, in which the basic dimethylaminoethoxy side chain was either absent or replaced with a variety of nonbasic side chains, were examined for their ability to inhibit the proliferation of a hormonally responsive cell line, MCF 7 human breast cancer. The degree of inhibition was compared with relative binding affinities for the estrogen receptor (RE) and a microsomal antiestrogen binding site (AEBS). All modifications resulted in loss of detectable affinity for AEBS. Replacement of the basic side chain of tamoxifen with a series of nonbasic side chains reduced affinity for RE by 78-93% except in the case of 1-(4-(1,2-diphenylbut-1-enyl)phenyl)-2,3-butanediol (ICI 145-680) where affinity was unchanged. When the basic side chain of tamoxifen was replaced by a hydroxyl group, to form the estrogenic analog ICI 141389 (Metabolite E), affinity for RE was reduced by 39%. ICI 141389 was a very weak inhibitor of MCF 7 cell growth, showing no significant growth inhibition at concentrations less than 10 microM. Despite the fact that ICI 145680 and tamoxifen had identical affinities for RE, ICI 145680 was a significantly weaker growth inhibitor than tamoxifen over the concentration range studied, i.e. 0.1-20 microM. Differences in potency were greatest at concentrations greater than 7.5 microM where the effects were not reversed by estradiol and where cytotoxicity played a major role in the decrease in cell numbers induced by tamoxifen. Like tamoxifen, ICI 145680 demonstrated both estrogen-reversible (at concentrations between 0.5-7.5 microM) and estrogen-irreversible (10-20 microM) inhibition of MCF 7 cell proliferation which was associated with a concentration-dependent accumulation of cells in the G0/G1 phase of the cell cycle. In contrast to tamoxifen, however, ICI 145680 appeared not to possess cytotoxic activity. Whereas ICI 145680 was without effect on proliferation of the RE negative human breast cancer cell line, MDA-MB-330, at doses less than 20 microM, tamoxifen inhibited growth at concentrations greater than 5 microM, but with changes in cell cycle kinetic parameters that were markedly different from those seen in RE positive cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resistance to different antiestrogens is caused by different multi-factorial changes and is associated with reduced expression of IGF receptor Ialpha

Development of antiestrogen resistance is a major clinical problem, and therefore it is crucial to elucidate the mechanisms involved. To investigate whether gain-of-function or loss-of-function mechanisms was most likely to be involved, cell fusion between the antiestrogen-sensitive MCF-7 and the ICI 164384- and ICI 182780-resistant MCF-7/164(R)-5 cell lines was performed. Furthermore, a fusion cell line between the tamoxifen-resistant MCF-7/TAM(R)-1 and the MCF-7/164(R)-5 cell line was established. A thorough investigation of growth parameters and expression of selected proteins (estrogen receptor-alpha (ERalpha), progesterone receptor (PR), Bcl-2, IGF-binding protein-2 (IGFBP2) and IGF receptor Ialpha (IGF-IRalpha)) in the fusion partners and fusion cells revealed that both gain- and loss-of-function changes occurred, and that the mechanisms resulting in resistance to the two antiestrogens were different. This multi-factoriality of antiestrogen resistance is promising in relation to sequential treatment of breast cancer patients with different types of endocrine therapy. Furthermore, we found an association between antiestrogen resistance and reduced IGF-IRalpha expression. Overall, the data presented in this report support the usefulness of cell fusion to clarify the mechanisms involved in development of resistance to the pure antiestrogens ICI 182780 and ICI 164384 and the selective ER modulator tamoxifen and suggest IGF-IRalpha as a new sensitive marker for response to antiestrogen treatment.     

Molecular mechanism of action at estrogen receptor alpha of a new clinically relevant antiestrogen (GW7604) related to tamoxifen

Tamoxifen is the endocrine treatment of choice for all stages of estrogen receptor (ER)-positive breast cancer, and it is the first drug approved to reduce the incidence of breast cancer in high-risk women. Unfortunately, tamoxifen also possesses some estrogen-like effects in the uterus that cause a modest increase in the risk of endometrial cancer. GW5638 is a tamoxifen derivative with a novel carboxylic acid side chain with no uterotropic activity in the rat (Willson et al., J Med Chem, 1994, 37:1550-1552). We have compared and contrasted the actions of 4-hydroxytamoxifen (4-OHT, the active metabolite of tamoxifen) with GW7604 [the presumed metabolite of GW5638 in breast (MCF-7) and endometrial (ECC-1) cell lines in vitro]. GW7604 did not cause the growth of ECC-1 cells at any concentration (10(-11)-10(-6) M), but 4-OHT was weakly estrogen-like at low concentrations (10(-11)-10(-10) M). Compounds (10(-7) M) blocked the growth promoting action of estradiol (10(-10) M) in both ECC-1 and MCF-7 cells. Western blotting was used to show that GW7604 and raloxifene did not affect ER levels significantly, compared with controls, in MCF-7 cells; whereas the pure antiestrogen ICI182,780 decreased ER levels (P < 0.05). An assay system was used that can classify compounds into tamoxifen-like, raloxifene-like, or pure antiestrogens. The assay depends on the activation of the transforming growth factor alpha (TGFalpha) gene in situ by wild-type or D351Y mutant ER stably transfected into MDA-MB-231 cells (MacGregor-Schafer et al., Cancer Res, 1999, 59:4308-4313). GW7604 inhibited both estradiol (10(-9) M) and 4-OHT (10(-8), 10(-7) M) induction of TGFalpha in a concentration related manner (10(-9)-10(-6) M). GW7604 and raloxifene stimulated TGFalpha with the D351Y ER. In contrast, ICI 182,780 (10(-6) M) did not initiate TGFalpha and blocked the induction of TGFalpha with GW7604, raloxifene, and 4-OHT in D351Y-transfected cells. Using computer-assisted molecular models of ER complexes, we found that the antiestrogenic side chain of 4-OHT weakly interacted with the surface amino acid 351 (aspartate), but the carboxylic acid of GW7604 caused a strong repulsion of aspartate 351. We propose that GW7604 is less estrogen-like than 4-OHT, because it disrupts the surface charge around aa351 required for coactivator docking in the 4-OHT:ER complex. This charge is restored in the D351Y ER, thus converting GW7604 from an antiestrogen to an estrogen-like molecule.     

Aromatase inhibitors and their antitumor effects in model systems

The potential of aromatase (estrogen synthetase) within the breast to provide a significant source of estrogen mediating tumor proliferation is suggested by studies reporting 4- to 6-fold higher estrogen levels in tumors than in plasma of postmenopausal patients with breast cancer. Recent studies in our laboratory have identified aromatase and its mRNA in tumor epithelial cells using immunocytochemistry and in situ hybridization. In addition, significant aromatase activity, which was stimulated 7-fold by dexamethasone, was measured in metastatic cells isolated from a breast cancer patient. Increase in proliferation, as measured by proliferating cell nuclear antigen immunostaining in tumor sections and by thymidine incorporation into DNA in response to testosterone, was observed in histocultures of breast cancer samples. This latter effect could be inhibited by 4-hydroxyandrostenedione. These results imply that intratumoral aromatase has functional significance and may be an important target for successful inhibitor treatment of breast cancer patients. To investigate treatment strategies with aromatase inhibitors and antiestrogens, we developed an intratumoral aromatase model to simulate the hormone responsive postmenopausal breast cancer patient. Tumors of estrogen receptor positive human breast carcinoma cells (MCF-7) transfected with the human aromatase gene are grown in ovariectomized nude mice. These cells synthesize sufficient estrogen to stimulate tumor formation. We have utilized this model to investigate the effects on tumor growth of the antiestrogens, tamoxifen and ICI 182780, and the aromatase inhibitors, letrozole and anastrozole (arimidex), alone and in combination. Both the aromatase inhibitors and the antiestrogens were effective in suppressing tumor growth. However, letrozole was significantly more effective than the antiestrogens. When the aromatase inhibitors were combined with the antiestrogen, tamoxifen, tumor growth was suppressed to about the same extent as with the aromatase inhibitors alone. Furthermore, the results do not suggest any benefit from combining tamoxifen with the pure antiestrogen, ICI 182780. Thus sequential use of these agents is likely to be more advantageous to the patient in terms of longer duration of effective treatment.     

Growth inhibitory effects of flavonoids in human thyroid cancer cell lines.

Previous studies have indicated that flavonoids exhibit antiproliferative properties on some hormone-dependent cancer cell lines, such as breast and prostate cancer. In the present study, the effects of some selected flavonoids, genistein, apigenin, luteolin, chrysin, kaempferol, and biochanin A on human thyroid carcinoma cell lines, UCLA NPA-87-1 (NPA) (papillary carcinoma), UCLA RO-82W-1 (WRO) (follicular carcinoma), and UCLA RO-81A-1 (ARO) (anaplastic carcinoma) have been examined. Among the flavonoids tested, apigenin and luteolin are the most potent inhibitors of these cell lines with IC50 (concentration at which cell proliferation was inhibited by 50%) values ranging from 21.7 microM to 32.1 microM. The cells were viable at these concentrations. Using NPA cells known to be estrogen receptor positive (ER+), it was shown that no significant [3H]-E2 displacement occurred with these flavonoids at the IC50 concentration. In WRO cells that are known to have an antiestrogen binding site (AEBS), biochanin A caused a stronger inhibitory growth effect (IC50 = 64.1 microM) than in NPA and ARO cells. In addition, it was observed that biochanin A has an appreciable binding affinity for the AEBS as indicated by the displacement of [3H]-tamoxifen from the WRO cells. In summary, flavonoids have potent antiproliferative activity in vitro against various human thyroid cancer cell lines. The inhibitory activity of certain flavonoid compounds may be mediated via the AEBS and/or type II EBS. The observation that ARO cells that lack both the AEBS and the ER are effectively inhibited by apigenin and luteolin suggest that other mechanisms of action are operative as well. The present study suggests that flavonoids may represent a new class of therapeutic agents in the management of thyroid cancer.     

Target size analysis of estrogen receptor in cultured intact cells: change in receptor structure between subconfluency and superconfluency in culture

MCF-7 human breast cancer cells were submitted to the tritiated antiestrogen tamoxifen aziridine, frozen at -170 degrees C, stored and irradiated at -78 degrees C in a calibrated Gammacell 60Co irradiator. A three-step protein extraction procedure provided protein samples for the determination of the target size (TS) of the covalently labelled estrogen receptor (ER). From the TS it is shown that ER bound to an antiestrogen was, in whole cells, part of a 265 kDa polypeptide structure if measured in MCF-7 cells at subconfluency, or of a 360 kDa species in superconfluent cells.     

A new MCF-7 breast cancer cell line resistant to the arzoxifene metabolite desmethylarzoxifene

The development of resistance in tamoxifen-treated breast cancer patients and the estrogenic side effects of tamoxifen have lead to the design of many new drugs. The new SERM arzoxifene and its active metabolite desmethylarzoxifene (ARZm) inhibits growth of breast cancer cells and has less estrogenic effects than tamoxifen on gene expression.

A cell line with acquired resistance to ARZm (MCF-7/ARZm^R-1) was established from MCF-7 cells. MCF-7/ARZm^R-1 cells responded to treatment with tamoxifen and the pure antiestrogen ICI 182,7870. The estrogen receptor (ER) level in MCF-7/ARZm^R-1 cells was lower than in MCF-7 cells due to a destabilization of the receptor by ARZm. A significant reduction in the mRNA and protein level of some estrogen-regulated genes was observed in MCF-7/ARZm^R-1 compared to MCF-7. However, both the level of the ER and several ER-regulated gene products increased towards parental MCF-7 level upon withdrawal from ARZm, concomitant with an increase in the sensitivity of MCF-7/ARZm^R-1 cells to ARZm treatment. These data show that ARZm resistant cells remain sensitive to treatment with both tamoxifen and to ICI 182,780. Furthermore, the partial reversion to ARZm sensitivity upon withdrawal of the SERM suggests that patients may benefit from a rechallenge with ARZm.     

Selection and characterization of a breast cancer cell line resistant to the antiestrogen LY 117018

We have selected and cloned a stable variant of the MCF-7 human breast cancer cell line (LY 2) that is resistant to LY 117018 (LY), a potent antiestrogen that inhibits cell growth at concentrations as low as 10(-10) M. The cell line was selected by increasing the concentration of LY in the growth medium in a stepwise manner from 10(-8) to 10(-6) M as the cells become resistant. LY2 has been cloned in soft agar and carried for over 50 passages with no change in resistance. Other antiestrogens, such as tamoxifen and 40-hydroxytamoxifen no longer inhibit cell proliferation of LY 2. The cell line is still responsive to estrogen in a cell proliferation assay, but contains somewhat less estrogen receptors than MCF-7. The cytosolic estrogen receptor sediments to a 4S position on high salt sucrose density gradient centrifugation and is completely shifted to a denser gradient region when the receptor is incubated with a monoclonal antiestrophilin. The nuclear estrogen receptor when covalently labeled with [3H]tamoxifen aziridine has the same mol wt (62,000) in both MCF-7 and LY2 cells, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In a competitive binding assay, LY 117018 competes for [3H]estradiol binding to its cytosol receptor with the same Ki in both MCF-7 and LY2 cells. When the induction of estrogen-specific proteins was examined, no detectable progesterone receptor could be detected in either estrogen-induced or control LY2 cells, in contrast to MCF-7 cells. However, both 52,000- and 160,000-dalton proteins were estrogen inducible in the medium of LY2 and MCF-7 cells, as measured by labeling with [35S]methionine. The phenotypic stability of the antiestrogen resistance in LY2 cells coupled with the cross-resistance the antiestrogens of widely different structures make this cell line an ideal model system for the study of hormone resistance in human breast cancer. In addition, while the mechanism of resistance is currently not elucidated, the selective loss of estrogen-inducible functions in this cell line may provide powerful clues for future study.     

Interactions of antiestrogens with human breast cancer in long-term tissue culture.

A variety of antiestrogens can be shown to antagonize estrogen action in animal model systems. Several of these compounds are useful in the management of metastatic human breast cancer. To further elucidate their mechanism of action, we studied several of these compounds using human breast cancer cell lines maintained in long-term tissue culture as a model system. Antiestrogens including tamoxifen (NSC-180973; ICI-46474), nafoxidine. CI-628, and clomiphene citrate inhibit macromolecular synthesis below control levels in two human breast cell lines. This effect is limited to cell lines which contain estrogen receptors. Simultaneous addition of as little as 1000-fold less estradiol prevents antiestrogen effects. Sequential addition of estrogen for up to 48 hours to cells incubated in antiestrogen reverses inhibition. If cells are continued in antiestrogen alone for more than about 3 days, inhibitory effects become irreversible. The cells detach from the surface of the culture vessel and are no longer viable. Tamoxifen competes with 3H-estradiol for specific receptor sites but with about a 100-fold lower apparent affinity. Direct binding of 3H-tamoxifen and 3H-estradiol to duplicate cytoplasmic extracts reveals equivalent numbers of binding sites but a 20-fold lower affinity for the antiestrogen. There is reasonable agreement between concentrations of tamoxifen which bind to receptor and concentrations which inhibit cells.     

Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.

Although much attention has been paid to the removal of hormones from sera and to the development of serum-free media for studies on hormone-responsive cells in culture, little consideration has been given to the possibility that the media components themselves may have hormonal activity. We have found that phenol red, which bears a structural resemblance to some nonsteroidal estrogens and which is used ubiquitously as a pH indicator in tissue culture media, has significant estrogenic activity at the concentrations (15-45 microM) at which it is found in tissue culture media. Phenol red binds to the estrogen receptor of MCF-7 human breast cancer cells with an affinity 0.001% that of estradiol (Kd = 2 X 10(-5) M). It stimulates the proliferation of estrogen receptor-positive MCF-7 breast cancer cells in a dose-dependent manner but has no effect on the growth of estrogen receptor-negative MDA-MB-231 breast cancer cells. At the concentrations present in tissue culture media, phenol red causes partial estrogenic stimulation, increasing cell number to 200% and progesterone receptor content to 300% of that found for cells grown in phenol red-free media, thereby reducing the degree to which exogenous estrogen is able to stimulate responses. The antiestrogens tamoxifen and hydroxytamoxifen inhibit cell proliferation below the control level only when cells are grown in the presence of phenol red; in the absence of phenol red, the antiestrogens do not suppress growth. The estrogenic activity of phenol red should be considered in any studies that utilize estrogen-responsive cells in culture.     

Acceptor sites on chromatin for receptor bound by estrogen versus antiestrogen in antiestrogen-sensitive and -resistant MCF-7 cells

We examined the chromatin binding characteristics of estrogen receptor from MCF-7 cells when bound by [3H] estradiol vs. the high affinity antiestrogen [3H]H1285 [4-(N,N-diethylaminoethoxy) 4'-methoxy-alpha-(p-hydroxyphenyl)alpha-ethylstilbene]. Two sublines of MCF-7 cells were used: E-3, which is sensitive to antiestrogens, and RR, which is antiestrogen resistant and was selected for its ability to grow in the presence of tamoxifen. Chromatin was prepared from both E-3 and RR cells, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine hydrochloride (Gdn X HCl). The chromatin acceptor activity unmasked by Gdn X HCl was determined using partially purified (30-fold) activated [3H]estradiol- or [3H] H1285-receptor complexes obtained by KCl step elution from DEAE-cellulose columns. With chromatin from E-3 cells, maximal binding (acceptor activity) for [3H]estradiol-receptor complexes prepared from either type of MCF-7 cells (E-3 or RR) was unmasked by 1 and 6 M Gdn X HCl, whereas [3H]H1285-receptor complexes exhibited maximal binding to 1 and 4 M Gdn X HCl-extracted chromatin subfractions. Chromatin prepared from RR cells was similar to that from E-3 cells in its binding activity for [3H]estradiol-receptor complexes. It differed, however, in that [3H]H1285-receptor complexes showed less chromatin acceptor site binding in general to 1-8 M Gdn X HCl-deproteinized RR chromatin, and the binding peak unmasked by 4 M Gdn X HCl was absent in chromatin from these cells. Receptor binding to chromatin was stable and was competitively inhibited by radioinert estradiol- or H1285-receptor complexes (but not by denatured receptors), demonstrating the saturability and specificity of these acceptor sites. Thus, estrogen receptor binds differently to chromatin depending on whether estradiol or an antiestrogen is bound to it. In addition, the acquisition of antiestrogen resistance by the RR subline of MCF-7 cells appears to result from alterations in the state of its chromatin rather than changes in the receptor itself. Finally, the observation that the chromatin from the resistant cells differs from that of the sensitive cells suggests that antiestrogens may be able to inhibit the growth of MCF-7 and other antiestrogen-sensitive cells not only by antagonizing the stimulatory effect of estrogens, but also by exerting some separate effect of their own.     

Laminin inhibits estrogen action in human breast cancer cells

Breast tumors that lack estrogen responsiveness have a poor prognosis. Despite the critical importance to breast cancer treatment, little is known about the loss of estrogen responsiveness and the development of antiestrogen resistance. We have examined the regulation of estrogen-induced proliferation, estrogen regulation of progesterone receptor (PR) expression, and estrogen signaling pathways in estrogen receptor (ER) positive (MCF-7 and T47D) breast cancer cell lines by specific extracellular matrix proteins (ECM) under serum-free conditions. Estrogen, supplemented with submaximal concentrations of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF), stimulated DNA synthesis of MCF-7 cells 7- to 10-fold and T47D cells 2-fold on collagen I or fibronectin. However, estrogen-induced proliferation was greatly reduced on laminin. In contrast, IGF-I or EGF, alone, stimulated proliferation of MCF-7 and T47D cells on all ECM. Thus, ER+ breast cancer cells were not refractory to mitogens when cultured on laminin. Similarly, estrogen induction of PR occurred on fibronectin or collagen I, but not on laminin. While ER content was similar on all ECM, estrogen stimulation of estrogen response element (ERE)-luciferase activity was significantly lower in MCF-7 cells cultured on laminin. Therefore, changes in ECM composition that occur in breast cancer may alter estrogen-responsiveness and the effectiveness of antiestrogen therapies in ER+ breast cancer cells.