Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Mechanism of estrogen action on cellular proliferation: evidence for indirect and negative control on cloned breast tumor cells

Human MCF7 breast tumor cells grew as estrogen-dependent tumors in nude mice. In contrast, they were not estrogen-dependent for proliferation in serumless culture media. Charcoal-dextran stripped female human serum supplemented media (5% to 40%) inhibited their proliferation in a dose dependent pattern. Estrogens reversed this inhibition. Concentrations of 2% of this serum allowed for maximal yield regardless of the presence of estrogens. Charcoal-dextran stripped fetal bovine serum was also inhibitory but less potent than the human serum. Non-estrogenic steroids, insulin, epidermal growth factor and transferrin failed to overcome the inhibitory effect of human serum. These results suggest that 1) human and bovine sera contain an inhibitor of the proliferation of estrogen-sensitive cells, and 2) estrogens promote cell proliferation by neutralizing this serum-borne inhibitor.     

Endogenous aromatization of testosterone results in growth stimulation of the human MCF-7 breast cancer cell line

Estrogens produced within breast tumors may play a pivotal role in growth stimulation of the breast cancer cells. However, it is elusive whether the epithelial breast cancer cells themselves synthesize estrogens, or whether the surrounding tumor stromal cells synthesize and supply the cancer cells with estrogen. The aromatase enzyme catalyzes the estrogen production, aromatizing circulating androgens into estrogens. The aim of this study was to investigate aromatase expression and function in a model system of human breast cancer, using the estrogen responsive human MCF-7 breast cancer cell line. Cells were cultured in a low estrogen milieu and treated with estrogens, aromatizable androgens or non-aromatizable androgens. Cell proliferation, expression of estrogen-regulated proteins and aromatase activity were investigated. The MCF-7 cell line was observed to express sufficient aromatase enzyme activity in order to aromatize the androgen testosterone, resulting in a significant cell growth stimulation. The testosterone-mediated growth effect was completely inhibited by the aromatase inhibitors letrozole and 4-hydroxy-androstenedione. Expression studies of estrogen-regulated proteins confirmed that testosterone was aromatized to estrogen in the MCF-7 cells. Thus, the results indicate that epithelial breast cancer cells possess the ability to aromatize circulating androgens to estrogens.     

Differentiation in human endometrial cells in monolayer culture: Dependence on a factor in fetal bovine serum

Human epithelial cells of the Ishikawa endometrial line can be stimulated to differentiate and form multicellular structures in 4–5 day-old monolayer cultures by the addition of a protein factor from fetal bovine serum. Multicellular structures become obvious over an 18–30-h period as the cells enlarge, separate from the dish, and form domes. These structures are similar to those that result from polarization in other epithelial cell lines. Ishikawa dome formation appears to be a multistage process. The appearance of enlarged differentiated cells is detected within hours of adding fetal bovine serum; these enlarged cells lift off the surface of the dish within 6–8 more hours. Domes are observed about 24 h after the addition of fetal bovine serum. Sometimes dome cells migrate into a “bud-like” structure that extends out from the dome. Differentiation of the domes is dependent on a factor from fetal calf serum that behaves similarly to a very large protein or complex of proteins, greater than 300 kd. Progesterone appears to enhance the formation of domes but does not elicit dome formation in the absence of serum factor.     

Expression of transforming growth factor alpha and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.     

Ribozyme targeting demonstrates that the nuclear receptor coactivator AIB1 is a rate-limiting factor for estrogen-dependent growth of human MCF-7 breast cancer cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified in breast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor alpha or progesterone receptor beta on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.     

Product of aromatase activity in intact LNCaP and MCF-7 human cancer cells

We investigated conversion rates of androgens to estrogens in cultured, hormone-responsive prostate (LNCaP) and breast (MCF-7) human cancer cells. For this purpose, we adopted an intact cell analysis, whereby cells were incubated for different incubation times in the presence of close-to-physiological (1 nM) or supraphysiological (1 μM) concentrations of labelled androgen precursors, i.e. testosterone (T) and androstenedione (Δ⁴Ad). The aromatase activity, as measured by estrogen formation, was detected in LNCaP cells (0.5 pmol/ml), even though to a significantly lower extent than in MCF-7 cells (5.4 pmol/ml), using 1 μM T after 72 h incubation. Surprisingly, LNCaP cells displayed a much higher aromatase activity when T was used as a substrate with respect to Δ⁴Ad. In either cell line, T transformation to Δ⁴Ad was relatively low, attaining only 2.8% in LNCaP and 7.5% MCF-7 cells. However, T was mostly converted to conjugates (over 95%), glucuronides and some sulphates, in LNCaP cells, whereas it was only partly converted to sulphates (<10%) in MCF-7 cells. Aromatase activity seems to be inconsistent in LNCaP cells, being strongly affected by culture conditions, especially by fetal calf serum (FCS). Further studies should assess the regulation of aromatase expression by serum or growth factors in different human cancer cells, also using anti-aromatase and/or anti-estrogen compounds, in different culture conditions.     

Stimulation of growth of human breast cancer cell lines in culture by linoleic acid

Linoleic acid, an omega-6 unsaturated fatty acid, stimulated growth of the MDA-MB-231 and MCF-7 human breast cancer cell lines in culture. Responses of the estrogen-independent MDA-MB-231 cells both in serum-free medium and with 1% fetal bovine serum added were positively correlated with linoleic acid concentration over the entire range examined (5-750 ng/ml). Growth stimulation of the estrogen-responsive MCF-7 cell line was maximal at a LA concentration of 500 ng/ml when cultured in 1% fetal bovine serum-containing medium with added estradiol. Linoleic acid had no mitogenic effect on three human cancer cell lines derived from sites other than breast, or on untransformed 3T3 cells.     

Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal

Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR- hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E(2)) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E(2) on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10(-9)M E(2) caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E(2) and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E(2)-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1x10(7)cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E(2). Overall, these results show that low concentrations (>10(-11)M) of E(2) are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.     

A multidrug-resistant MCF-7 human breast cancer cell line which exhibits cross-resistance to antiestrogens and hormone-independent tumor growth in vivo

MCF-7 human breast cancer cells provide a useful in vitro model system to study hormone-responsive breast cancer as they contain receptors for estrogen and progesterone, and estrogen both induces the synthesis of specific proteins in these cells and increases their rate of proliferation. An MCF-7 cell line which was selected for resistance to adriamycin (MCF-7/AdrR) exhibits the phenotype of multidrug resistance (MDR), and displays multiple biochemical changes. MDR in MCF-7/AdrR is also associated with a loss of mitogenic response to estrogen and the development of cross-resistance to the antiestrogen 4-hydroxytamoxifen. In addition, while the parental MCF-7 cell line responds to estrogen with increased levels of progesterone receptors and the secretion of specific proteins, these estrogen responses are lost in MCF-7/AdrR. Furthermore, while the formation of tumors in nude mice by wild-type MCF-7 cells is dependent upon the presence of estrogen, MCF-7/AdrR cells form tumors in the absence of exogenous estrogen administration. These changes in hormonal sensitivity and estrogen-independent tumorigenicity of the multidrug-resistant MCF-7 cell line are associated with a loss of the estrogen receptor and a concomitant increase in the level of receptors for epidermal growth factor. Thus, in MCF-7/AdrR cells, the development of MDR is associated with alterations in the expression of both cytosolic and membrane receptors, resulting in resistance to hormonal agents and the expression of hormone-independent tumor formation.     

Mitogenic effect of estradiol on MCF-7 human breast cancer cells can be modulated by serum

The MCF-7 human breast cancer cell line responds to estradiol stimulation in vitro by increased proliferation only if prolonged subcultures in dextran-coated charcoal-treated fetal calf serum have been made previously. This growth stimulation is not obtained when cells are grown in medium containing 5% untreated fetal calf serum. We describe here the culture conditions under which we obtain a reproducible estradiol effect on cell growth.     

Correlation between the activation of Neu tyrosine kinase and promotion of foci formation induced by selected organochlorine compounds in the MCF-7 model system.

Several studies have shown that some organochlorine compounds act like estrogen in certain animals and in vitro cell culture systems, and therefore, there is a possibility that they could promote the process of tumorigenesis in breast cancer cells. In our previous study, two representative organochlorines, 1,1,1-trichloro 2-o-chlorophenyl-2'-p-chlorophenyl ethane (o,p'-DDT) and beta-1,2,3,4,5,6-hexachlorocyclohexane (beta HCH), were found to directly activate the protein tyrosine kinase of Neu (c-erbB-2 proto-oncogene product) immunoprecipitates isolated from MCF-7 breast cancer cells. In the current study, we also found that 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) at 1 nM and alpha-HCH isomers at 100 nM could also significantly activate protein tyrosine kinase of Neu immunoprecipitates in a cell-free system. We also found that organochlorines result in an increase of Neu protein tyrosine kinase after intact cell treatment in estrogen-depleted medium. This Neu kinase activation by beta-HCH (100 nM) was blocked when the cells were pretreated with Neu mRNA antisense oligonucleotide (p < 0.07, Student's t-test). Endogenously added alpha-, beta-, and gamma-HCH, o,p'-DDT, 2,2'-dichlorobiphenyl (2,2'-PCB), and 2,4,5-T at 100 nM were found to promote foci formation in postconfluent cultures of this cell line. This stimulatory effect caused by 17beta-estradiol, o,p'-DDT, and beta-HCH on foci formation was inhibited by coincubation with Neu monoclonal antibody (p < 0.05). Those two events induced by organochlorines (i.e., Neu kinase activation and foci formation) seemed causally correlated.     

Estrogen stimulates cell proliferation and the increase of a 52,000 dalton glycoprotein in human breast cancer cells

In an estrogen supersensitive variant of the MCF-7 cell line, CG-5, estrogen was found to stimulate the labelling of a glycoprotein released into the culture medium which has the same electrophoretic migration pattern as that previously reported in MCF-7 cells (Biochem. Biophys. Res. Commun., 90: 410-416, 1979). To test the possibility that the 52 K is a marker of estrogen-dependent breast cancer cell proliferation, we have correlated the effect of estrogen and antiestrogen on protein labelling and cell proliferation under different experimental conditions. In cells cultured in the presence of 5% charcoal-treated fetal calf serum, physiological concentrations (0.1-1 nM) of estradiol stimulated in a dose- and time-related fashion both 52 K labelling and cell proliferation. However at high concentrations (10-100 nM) estrogen decreased 52 K labelling while it still stimulated cell proliferation. Concentrations of the tamoxifen derivative, 4-hydroxytamoxifen, which effectively prevented estrogen-stimulated cell proliferation also blocked estrogen-stimulated increase of 52 K labelling. Time-course experiments suggest that the estrogen-stimulated increase of 52 K labelling (detectable after 22 h of hormone exposure) precedes the effect of cell proliferation (detectable after 3 days of hormone exposure). In cells cultured under serum-free conditions there was no effect of estradiol at any of the concentrations and times used on either 52 K labelling or cell proliferation.     

Estrogen and progesterone interactive effects in postconfluent MCF-7 cell culture

17β-Estradiol (E2) stimulates morphological differentiation of an MCF-7 human mammary carcinoma cell line resulting in the development of multicellular rounded nodules (foci) above the epithelial monolayer. Examining the combined effect of progesterone (P4) and E2 on foci formation we detected P4-dependent foci enlargement and phenotypic modification. Notably, P4 dose-dependently potentiated lower dose E2-induced increases in foci numbers. We detected P4-dependent changes in cytoskeleton protein expression levels and accelerated cell division. P4 alone or in combination with E2 additively modified the expression of adhesion proteins and stimulated expression of tropomyosin (Tm). Antiprogestin and antiestrogen pretreatment abrogated P4-dependent increases in foci number and stimulation of Tm expression, indicating involvement of both E2 and P4 receptor signaling. Novel aspects of endocrine-regulated changes in microfilament and adhesion protein composition are discussed in association with tumorigenesis and metastatic capability in breast carcinoma cells.     

The role of estrogens on the proliferation of human breast tumor cells (MCF-7)

The cloned human breast tumor cell line C7MCF7-173 behaved as an estrogen-dependent tumor in the nude mice. In contrast, E2 added to serum-less media did not increase the multiplication rate of these cells over the values obtained in the control cultures. Media supplemented with charcoal-dextran stripped (CD) human female serum (FHS) resulted in inhibition of cell proliferation in a concentration-dependent pattern (40% = 20% greater than 10% greater than 5% greater than 2.5%). E2 addition to all but the 2.5% CDFHS significantly increased the proliferation rate of these cells. The E2 concentration required to attain maximal proliferation rate increased as the serum concentration of the medium increased (e.g. 3 X 10(-11)M for 10% CDFHS, 3 X 10(-10)M for 40% CDFHS). E2 concentrations higher than the one needed to achieve maximal proliferation rate resulted in decreased cell yields (shut-off mechanism). Similar effects were obtained with synthetic and other natural estrogens. CD fetal bovine serum (FBS) also inhibited the proliferation of C7MCF7-173 cells; however, at similar concentration the inhibitory effect of CDFHS was more potent than the one obtained with CDFBS. The addition of "growth factors" (insulin, Epidermal Growth Factor and transferrin) and non-estrogenic steroids to 10% CDFHS failed to overcome the inhibitory effect of this serum. These results suggest that: (1) human and fetal bovine sera contain a specific inhibitor of the proliferation of E2-sensitive cells (estrocolyones), and (2) E2 promotes cell proliferation by neutralizing this inhibitor.     

Cripto-1 overexpression leads to enhanced invasiveness and resistance to anoikis in human MCF-7 breast cancer cells

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-CFC protein that has been shown to signal through nodal/Alk-4, PI3K/Akt, and/or ras/raf/MEK/MAPK pathways in mammalian cells, and that is frequently expressed in human primary breast carcinomas. In the present study, the human estrogen receptor positive, MCF-7 breast cancer cell line, that expresses low levels of endogenous CR-1, was transfected with a CR-1 expression vector. MCF-7 CR-1 cells expressed high levels of a 25 kDa recombinant CR-1 protein that was not detected in MCF-7 cells transfected with a control vector (MCF-7 neo). Overexpression of CR-1 did not induce an estrogen independent phenotype in MCF-7 cells. In fact, MCF-7 CR-1 cells showed a response to exogenous estrogens that was similar to MCF-7 neo cells, and failed to grow in immunosuppressed mice in absence of estrogen stimulation. However, MCF-7 CR-1 cells showed a rate of proliferation in serum free conditions, and an ability to form colonies in soft-agar that were higher as compared with MCF-7 neo cells. More importantly, overexpression of CR-1 enhanced the resistance to anoikis and the invasion ability of MCF-7 cells. MCF-7 CR-1 cells showed levels of activation of both Akt and Smad-2 that were significantly higher as compared with MCF-7 neo. These findings suggest that CR-1 overexpression might be associated with the progression towards a more aggressive phenotype in breast carcinoma, through the activation of both Akt and Smad-2 signalling pathways.     

Characterization of a fetal urogenital sinus mesenchymal cell line U4F: Secretion of a negative growth regulatory activity

Summary Mesenchymal cell lines derived from fetal rat urogenital sinus organ cultures have been characterized to establish an in vitro system for addressing growth and differentiation regulatory factors involved in mesenchymal-epithelial interactions during prostate morphogenesis. A continuous cell line was developed and designated U4F. Immunocytochemical analysis showed vimentin intermediate filament content confirming a mesenchymal origin. Previous studies with urogenital sinus organ cultures have reported the expression of a negative growth activity, which is stimulatory to protein synthesis and secretion and alters phenotypic morphology of NBT-II bladder epithelial cells. Subconfluent and confluent U4F monolayers did not produce this growth inhibitory activity. Foci of stacked cells were observed 3 wk postconfluency, which evolved into multicellular spheroids. The negative growth activity was expressed in the conditioned medium coordinate with spheroid formation. Transplanted spheroids continued to express the growth inhibitory activity. Morphologic analysis of spheroids showed a cellular capsule and a core of extracellular matrix. A continuous cell strain (U4F1) with altered phenotypic properties, arose spontaneously from long-term U4F cultures. The U4F1 cell strain did not form spheroids, yet expressed the negative growth activity constitutively in monolayer culture. Analyses of physicochemical, immunological, and biological properties showed the activity is identical in conditioned media from urogenital sinus organ cultures, U4F spheroids, and U4F1 monolayers. Based on the combined properties, this activity cannot be ascribed to previously characterized negative growth factors. The establishment of this mesenchymal cell culture system will aid in the further identification of paracrine-acting growth and differentiation regulatory factors secreted by fetal mesenchyme.     

Effect of nomegestrol acetate on estrogen biosynthesis and transformation in MCF-7 and T47-D breast cancer cells

Although ovaries serve as the primary source of estrogen for pre-menopausal women, after menopause estrogen biosynthesis from circulating precursors occurs in peripheral tissues by the action of several enzymes, 17beta-hydroxysteroid dehydrogenase 1 (17beta-HSD1), aromatase and estrogen sulfatase. In the breast, both normal and tumoral tissues have been shown to be capable of synthesizing estrogens, and this local estrogen production can be implicated in the development of breast tumors. In these tissues, estradiol (E(2)) can be synthesized by three pathways: (1) estrone sulfatase transforms estrogen sulfates into bioactive estrogens, (2) 17beta-HSD1 converts estrone (E(1)) into E(2), (3) aromatase which converts androgens into estrogens is also present and contributes to the in situ synthesis of active estrogens but to a far lesser extent than estrone sulfatase. Quantitative assessment of E(2) formation in human breast tumors indicates that metabolism of estrone sulfate (E(1)S) via the sulfatase pathway produces 100-500 times more E(2) than androgen aromatization. Breast tissue also possesses the estrogen sulfotransferase involved in the conversion of estrogens into their sulfates that are biologically inactive. In the present review, we summarized the action of the 19-nor-progestin nomegestrol acetate (NOMAC) on the sulfatase, 17beta-HSD1 and sulfotransferase activities in the hormone-dependent MCF-7 and T47-D human breast cancer cell lines. Using physiological doses of substrates NOMAC blocks very significantly the conversion of E(1)S to E(2). It inhibits the transformation of E(1) to E(2). NOMAC has a stimulatory effect on sulfotransferase activity in both cell lines, with a strong stimulating effect at low doses but only a weak effect at high concentrations. The effects on the three enzymes are always stronger in the progesterone-receptor rich T47-D cell line as compared with the MCF-7 cell line. Besides, no effect is found for NOMAC on the transformation of androstenedione to E(1) in the aromatase-rich choriocarcinoma cell line JEG-3. In conclusion, the inhibitory effect provoked by NOMAC on the enzymes involved in the biosynthesis of E(2) (sulfatase and 17HSD pathways) in estrogen-dependent breast cancer, as well as the stimulatory effect on the formation of the inactive E(1)S, can open attractive perspectives for future clinical trials.     

The promoter of the rat 3-hydroxy-3-methylglutaryl coenzyme A reductase gene contains a tissue-specific estrogen-responsive region.

The isoprenoid metabolic pathway is mainly regulated at the level of conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonate, catalyzed by HMG CoA reductase. As estrogens are known to influence cholesterol metabolism, we have explored the potential regulation of the HMG CoA reductase gene promoter by estrogens. The promoter contains an estrogen-responsive element-like sequence at position -93 (termed Red-ERE), which differs from the ERE consensus by one mismatch in each half of the palindrome. A Red-ERE oligonucleotide specifically bound estrogen receptor in vitro and conferred receptor-dependent estrogen responsiveness to a heterologous promoter in all cell lines tested. However, expression of a reporter driven by the rat HMG CoA reductase promoter was induced by estrogen treatment after transient transfection into the breast cancer cell line MCF-7 cells but not in hepatic cell lines expressing estrogen receptor. Estrogen induction in MCF-7 cells was dependent on the Red-ERE and was strongly inhibited by the antiestrogen ICI 164,384. A functional cAMP-responsive element is located immediately upstream of the Red-ERE, but cAMP and estrogens inhibit each other in terms of transactivation of the promoter. Similarly, induction by estrogens was inhibited by micromolar concentrations of cholesterol, likely acting via changes in occupancy of the sterol-responsive element located 70 bp upstream of the Red-ERE. Thus, within its natural context, Red-ERE is able to mediate hormonal regulation of the HMG CoA reductase gene in tissues that respond to estrogens with enhanced cell proliferation, while it is not operative in liver cells. We postulate that this tissue-specific regulation of HMG CoA reductase by estrogens could partially explain the protective effect of estrogens against heart disease.