Human Estrogen Receptor Gene Research Articles

DNA Sequence-recognizing Properties of Minor Groove Alkylating Agents

The linkage of an heterocycle, like N-methylimidazole, to minor DNA groove binders containing two or three pyrroles lead to a new class of oligopeptides with reduced antitumor activity both in vitro and in vivo if compared to tallimustine (CAS 115308-98-0) and its tetrapyrrole homologue 9. In the present paper is reported the correlation between the cytoxicity of tallimustine and its derivatives 9-11 with their ability to inhibit polymerase chain reaction (PCR) amplification of oestrogen receptor and Ha-ras gene sequences, containing A + T rich and G + C rich regions, respectively. Tallimustine and its tetrapyrrole homologue 9 were found to have higher sequence selectivity for the human oestrogen receptor (ER) gene with respect to the relative imidazole-containing analogues.

ESR1 promoter polymorphism is not associated with nonsyndromic cryptorchidism

The ESR1 promoter microsatellite (TA)n was reported as a potential functional polymorphism. In a case-control study, we were unable to demonstrate any association between (TA)n and nonsyndromic cryptorchidism in Italian and Spanish study populations.

Bioassay of estrogenic compounds in transgenic Arabidopsis plants carrying a recombinant human estrogen receptor gene and a GFP reporter gene

Transgenic Arabidopsis plants carrying a recombinant human estrogen receptor gene and a green fluorescent protein reporter gene were used to bioassay estrogenic compounds. We constructed four recombinant human estrogen receptor genes by combining the DNA-binding domain of LexA, a synthetic nuclear localization signal, a ligand-binding domain of the human estrogen receptor, and a transactivation domain of VP16 in different orders; the XEV plants were the most sensitive, and were able to detect 0.001 ng ml(-1) of 17ss-estradiol (E(2)). The transgenic plants absorbed E(2) and 4-nonylphenol present in the nutrient solution, whereas most of the other compounds seemed to be retained in, or on, the roots. Estrone, methoxychlor, bisphenol A, 4-nonylphenol, and 4-t-octylphenol in the medium were clearly detected by RT-PCR and PCR of the genomic DNA. The transgenic Arabidopsis XEV plants thus have potential for the bioassay of estrogenic compounds.

Impact of activated sludge‐derived colloidal organic carbon on behavior of estrogenic agonist recombinant yeast bioassay

The impact of size-fractionated colloidal organic carbon (COC) originating from a biological wastewater treatment facility on the sensitivity of the yeast estrogen screen (YES) bioassay containing the human estrogen receptor (hER) gene was evaluated. Dose-response curves of serially diluted 17beta-estradiol (E2), both in the presence and absence of COC, were generated by the YES bioassay. The concentration of E2 leading to a 50% YES response (effective concentration 50%, or EC50) was used to evaluate quantitatively the estrogenic activity of the different COC-E2 mixtures. The EC50 values for all COC size fractions, including COC-free samples (<1 kD), were statistically greater than the controls using Milli-Q water. Normalized EC50 values significantly increased as a function of COC concentration for the larger size fractions (>0.22 microm), but were not significantly affected by smaller COC material at environmental levels (1-5 mg/L), while both colloidal polysaccharide concentrations and colloidal fluorophores (measured at an excitation/emission wavelength pair of 350 nm/450 nm) appear to have an important role in the sensitivity of the YES bioassay. Estimates of the colloid-associated E2 fraction did not predict accurately increases in EC50 values. Matrix effects of the specific environment being tested with the YES bioassay need to be evaluated closely due to the sensitivity of the hER and reporter plasmid.

Developing a biosensor for estrogens in water samples: Study of the real-time response of live cells of the estrogen-sensitive yeast strain RMY/ER-ERE using fluorescence microscopy

Using a fluorescein di-beta-D-galactopyranoside (FDG) substrate we show that in live cells of an estrogen-sensitive yeast strain RMY/ER-ERE with human estrogen receptor (ERalpha) gene and the lacZ gene which encodes beta-galactosidase, the uptake of 17beta-estradiol (E2) and the subsequent production of beta-galactosidase enzyme occur quite rapidly, with maximal enzyme-catalyzed product formation evident after about 30 min of exposure to E2. This finding which agrees with the well-known rates of enzyme-catalyzed reactions could have implications for shortening the duration of environmental sample screening and monitoring regimes using yeast-based estrogen assays, and the development of biosensors for environmental estrogens to complement quantification methods.

In vitro estrogenicity of polybrominated flame retardants

Estrogenicity of five brominated flame retardants (BFRs), namely BDE-47, BDE-99, BDE-205, PBB-153 and technical Firemaster BP-6™, were assessed by in vitro assays developed to detect chemicals with estrogenic properties. Recombinant yeast cells containing a human estrogen receptor gene failed to give any response to the chemicals tested. However, the positive control compound, estradiol-17β, showed that the yeast cell assays had worked properly. The freshly separated fish hepatocyte assay based on the synthesis and secretion of vitellogenin from the isolated liver cells produced a clear dose–response curve in the presence of all tested flame retardants except Firemaster BP-6™. The toxicity of the BFRs was detected by determining the cell ethoxyresorufin-O-deethylase activity (EROD). The BFRs tested induced hepatic EROD activity at low test concentrations, but started to inhibit activity at higher concentrations. The decreased detoxification capacity of the hepatocytes resulted in a decrease in the vitellogenin production of the cells. The capability of in vitro assays to detect estrogenic properties of chemicals seems to vary. Thus, further work is needed to understand the mechanisms responsible for these reactions.

Cis element ‘decoy’ against the upstream promoter of the human estrogen receptor gene

It is well known that breast carcinomas without estrogen receptor (ER) have a poor prognosis and do not respond to endocrine therapy. In analyzing the question of the lack of ER gene expression, we have considered the possibility that specific negative transcription factors are present in ER-negative breast cancers. Inside the P3 upstream promoter of human ER gene we identified a transcriptional regulatory sequence able to bind protein factors expressed in ER-negative MDA-MB-231 breast cancer cells. This sequence, lying between nucleotides −3258 to −3157, seems to be critical for inhibition of ER gene transcription. In fact, the selected sequence in the form of double-stranded DNA has been introduced into ER-negative breast cancer cells as ‘decoy’ cis elements showing the ability to remove the putative negative transcription factor(s) and to induce the reactivation of ER gene transcription. In addition, in transient transfection assays the selected sequence decreased the SV-40 promoted luciferase activity. Gel shift assays identified multiple DNA–protein interactions which specifically form in this region, and data from Southwestern experiments strongly suggested the presence of a specific protein expressed in MDA-MB-231 ER-negative, but not in MCF7 ER-positive cells.

Identification of an enhancer element in the estrogen receptor upstream region: implications for regulation of ER transcription in breast cancer

The estrogen receptor (ER) serves as a diagnostic marker for the treatment of breast cancer. Patients with ER-positive breast tumors are likely to respond to hormonal therapies, while ER-negative breast cancers are resistant to endocrine therapies. Most ER-negative tumors do not express detectable levels of ER transcript, highlighting the importance of transcriptional regulation. A novel regulatory element which resembles a steroid hormone response element has been identified in the 5′-flanking region of the human ER gene. We observed 3- to 5-fold higher specific binding to this element in nuclear extracts from ER-expressing MCF-7 breast cancer cells compared to ER-negative MDA-MB-231 breast tumor cells. We termed the factor(s) which bind to this cis-element estrogen receptor upstream binding factor-1 (ERUBF-1). In transient transfection assays in MCF-7 cells, the ERUBF-1 binding site elicited a 20-fold increase in luciferase activity over the ER P 1, promoter. This enhancer element was significantly more active in the ER-positive MCF-7 cell line compared to the ER-negative MDA-MB-231 cell line. These data indicate that ERUBF-1 plays an important role in the transcriptional regulation of the ER gene in breast cancer.

Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor α gene

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB−4 to −1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA)·poly(dT) tracts including the potential bend core sequence A 2N 8A 2N 8A 2 (A/A/A). Fine mapping of the ERB−2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB−2 revealed that the bend center was located 10–30 bp from the experimental and predicted nucleosome dyad axes.

Selective binding to human genomic sequences of two synthetic analogues structurally related to U-71184 and adozelesin

In this paper, we analyse the in vitro sequence-selectivity of two synthetic analogues of U-71184 and adozelesin by polymerase chain reaction (PCR) performed on human genomic DNA. In addition, Dnase footprinting and nucleotide sequence analysis on arrested-PCR products were performed to confirm sequence-selective binding. Finally, the antitumor effects were studied in vitro on human leukemic L1210 cells. The binding activity of the two newly synthesized compounds to human gene sequences was compared with the CC-1065 analogue U-71184, the A+T sequence-selective drug distamycin and the G+C sequence-selective drugs mithramycin and chromomycin. As molecular model systems for in vitro DNA-binding studies we used the human estrogen receptor gene and the Ha-ras oncogene. In some experiments the PCR approach was performed using as target DNA a portion of the long terminal repeat (LTR) of the human immunodeficiency type 1 virus (HIV-1). These genomic regions contain sequences that are different with respect to A+T/G+C ratios, being the upstream sequence of the human estrogen receptor gene A+T rich, while the Ha-ras and HIV-1 LTR sequences contain G+C–rich regions. The first conclusion that can be drawn from the experiments reported in our paper is that the two newly synthetized analogues of U-71184 and adozelesin inhibit PCR-mediated amplification of genomic regions in a sequence-dependent manner. A second conclusion of our experiments is that these compounds are active inhibitors of tumor cell growth in vitro. Drug Dev. Res. 46:96–106, 1999. © 1999 Wiley-Liss, Inc.

In vitro stability of polymerase chain reaction-generated DNA fragments in serum and cell extracts

The potential use of polymerase chain reaction (PCR)-generated DNA fragments (PCR-DNAs) as pharmaceutical agents has previously been suggested, with the demonstration of the in vitro cellular internalization and biologic activity of PCR-DNA decoy molecules targeted to human estrogen receptor gene. In order to provide information on the stability of these double-stranded DNA molecules, the nuclease resistance of PCR-DNAs of different sizes was studied in different conditions and experiments. Simulating in vitro and in vivo transfection protocol, we demonstrated that PCR-DNAs exhibited good stability toward fetal bovine serum (FBS) and adult human serum nuclease digestion. In addition, when the protective activity of liposome-based formulations toward nuclease digestion was tested, it was shown that the stability of PCR-DNAs could be further increased (up to 7 days) when a liposome-mediated delivery system was employed.

Modulation of estrogen receptor gene expression in human breast cancer cells: a decoy strategy with specific PCR-generated DNA fragments.

Transcriptional activity of human estrogen receptor (hER) gene was modulated by competition with double-stranded PCR-generated DNA fragments (decoys) that contain 5' upstream sequences of the hER gene. Two DNA fragments belonging to the P1 canonical promoter and the P3 distal promoter, 120 and 102 bp in size respectively, were produced by PCR and directly transfected in MCF7 breast cancer cells. After 24 hours transfection, RT-PCR analysis revealed that the 120 bp decoy significantly reduced the expression of the ER gene and estrogen responsive genes (PR and c-myc), whereas the 102 bp decoy increased the ER mRNA level. An ER unrelated PCR product, used as control, had no activity. The biological activity of these ds DNAs was related to their high stability, binding affinities, and lack of cytotoxicity. These findings suggest that such PCR product decoys may be a non-antisense tool to analyze putative regulatory sequences and to study the function of DNA-binding transcription factors.

The multiple untranslated first exons and promoters system of the oestrogen receptor gene in the brain and peripheral tissues of the rat and monkey and the developing rat cerebral cortex

Recent studies on the human oestrogen receptor (ER) gene have revealed the complex system with the multiple untranslated first exons and promoters in the ER gene expression. Little information is however available on the system in the ER gene of the rat or nonhuman primate. The rat genomic library was first screened by the rat ER cDNA (0–1) probe. One of the four positive clones ( λ rEgEl) was subcloned and sequenced. The nucleotide sequence was found to contain the exon 0, the intron 0, and the exon 1 with its 3′-ends. The novel untranslated first exons, the exon ON and the exon OS, were further identified. These results indicated the presence of at least four subtypes of the rat ER mRNAs; the messages transcribed from promoter P-0 (ER mRNA (0–1)), putative promoter P-1 (ER mRNA (1–1)), promoter P-ON (ER mRNA (ON-1)) and promoter P-OS (ER mRNA (OS-1)). The P-O- or P-1 driven message (0–1) or (1–1) appeared to be expressed most strongly in major oestrogen central- (anterior pituitary, AP, hypothalamus–preoptic area, HPOA, and amygdala, AMG) and peripheral targets (uterus and ovary). The message (ON-1) was strongly expressed in the liver and kidney, but not in the HPOA, AMG, cerebral cortex, CC, and cerebellum, Ce. The OS-1 message was expressed variably but generally in the tissues examined except for the CC and Ce. Thus, the region- and tissue specific expression of the rat ER gene is likely to be regulated by the multiple untranslated exons and promoters system. Furthermore, when the ER mRNA subtypes were examined in the rat neonatal CC where the ER protein level rose transiently, considered as a model for the development of the ER or progestin receptor A and B isoforms, the expression of the ER mRNAs seemed to be differential postnatally, implicating some stage dependent usage of the promoters in the development. In the monkey, we identified the untranslated first exon OS, the homologue of the rat exon OS. Interestingly, the exon C was found to consist of two different exons, the exon OK and the exon OG. By the alternative usage of the promoters and the alternative splicing, at least six ER mRNA subtypes, that is, ER mRNAs (0–1), (1–1), (OS-1), (OS-OG-1), (OK-1) and (OK-OG-1) were identified in the monkey tissues. These messages were also differentially distributed in the monkey brain and other tissues. It was noteworthy that the P-OK driven messages were expressed almost exclusively in the monkey liver. These results have suggested that the systems of the multiple untranslated first exons and promoters and the alternative splicing are involved in the regulation of the region- and tissue specific expression of the ER gene in the brain and peripheral tissues of the rat and monkey. Stage-related usage of the promoters was also suggested in the ER gene expression in the CC of the postnatal rat in development.

Estrogen receptor variants.

Estrogen receptor (ER)3 gene expression in breast epithelium is an intricately regulated event. The human ER gene is transcribed from at least three different promoters which are expressed in a cell- and tissue-specific manner, and result in mRNA isoforms with unique 5'-untranslated exons. The ER is overexpressed in about two thirds of breast tumors, and even in early premalignant breast lesions compared with adjacent normal breast epithelium. Furthermore, normal breast epithelium as well as breast cancer tissue contains alternatively spliced ER mRNA variants where single or multiple exons are skipped. It is still unclear if any or all of the ER mRNA splicing variants are translated in vivo, and if a change in the balance of ER variants could effect tumor development and progression to hormone-independent growth. Although infrequent in primary breast cancer, single amino acid changes within the ER in metastatic disease which might influence cell proliferation may also contribute to neoplastic progression of the mammary epithelium.

A point mutation in the human estrogen receptor gene is associated with the expression of an abnormal estrogen receptor mRNA containing a 69 novel nucleotide insertion.

A novel ER-like mRNA containing a 69 nucleotide insertion precisely between exon 5 and 6 sequences was previously identified in human breast cancer biopsy samples. Data are presented which suggest that the 69 nucleotide sequence is normally present in intron 5 of the human estrogen receptor gene. The region corresponding to and surrounding this 69 nucleotide sequence was cloned and the nucleotide sequence determined. Cloning and sequencing of the corresponding region in genomic DNA isolated from a breast tumor expressing the 69 nucleotide inserted ER mRNA, revealed an A-->G point mutation immediately 3' to the 69 nucleotide sequence. This point mutation resulted in the generation of a consensus splice donor site. A consensus splice acceptor site sequence is normally present immediately 5' to the 69 nucleotide sequence. These data are consistent with the 69 nucleotide sequence being recognized as an exon by the splicing machinery, and resulting in processing of a mature ER mRNA containing the 69 nucleotide insert.

Auto-regulation of the estrogen receptor promoter.

The presence or absence of estrogen receptor (ER) plays a key role in the diagnosis and treatment of breast tumors. It is known that patients with breast tumors classified as ER-positive have a better prognosis. Observations such as this have led us to explore the question of what makes some breast tumors overexpress ER whereas others express either very low levels or none at all. To begin a study of ER regulation, we first chose to examine a 200 bp region of the ER promoter located immediately upstream from the transcribed sequence of the human ER gene. We found that this region of the ER promoter contained basal activity when transiently transfected into ER-negative HeLa cells. ER promoter activity was further increased by co-transfection of a wild-type ER expression vector, and this increased activity was hormone-dependent. Several ER deletion mutant constructs were also able to increase the activity of the ER promoter fragment, but none could support equivalent activity as was seen with the full-length ER. Therefore, we conclude that the ER can contribute to its own expression, and we hypothesize that this auto-regulation may contribute to its overexpression in some breast tumors.

A transcriptional enhancer required for the differential expression of the human estrogen receptor in breast cancers.

Breast cancers lacking estrogen receptor (ER) expression have an adverse prognosis and fail to respond to endocrine therapy. We have identified a transcriptional enhancer in the human ER gene which is differentially active in ER-positive (ER+) and ER-negative (ER-) human breast cancer cell lines. Enhancer function was mapped to a 35-bp element located from -3778 to -3744 upstream of the major human ER mRNA start site, which we have termed ER-EH0 (for estrogen receptor enhancer). Gel retardation assays with ER+ and ER- cell lines identified multiple DNA-protein complexes which specifically form on this enhancer. One of these complexes could be supershifted by anti-Jun or anti-Fos antibodies, identifying it as an AP-1-containing complex. Methylation interference assays suggest binding of factors to both the AP-1 site and adjacent base pairs. Enhancer activity requires both the AP-1 site and these adjacent sequences. Mutations introduced into ER-EH0 and the recently described proximal promoter element ERF-1 in the context of the full-length promoter confirm ER-EH0 as the dominant cis-acting element involved in differential ER expression.

In vitro and in vivo binding of a CC-1065 analogue to human gene sequences: a polymerase-chain reaction study

In this paper we analyse the in vitro sequence selectivity of the CC-1065 analogue 2-[[5-[(1 H-indol-2-ylcarbonyl)-1 H-indol-2-yl]carbonyl]-7-methyl-1,2,8,8 a-tetrahydrocyclopropa[ c]-pyrrolo-[3,2- e]-indol-4-one (U-71184) employing the polymerase-chain reaction (PCR). In addition, we determined whether alteration of PCR by U-71184 is detected when DNA is isolated from cells cultured in the presence of this drug. As molecular model systems we employed the human estrogen receptor gene, the Ha-ras oncogene and the chromosome X-linked, (CGG)-rich fragile X mental retardation-1 gene. The first conclusion that can be drawn from the experiments reported in our paper is that U-71184 inhibits PCR in a sequence-dependent manner. A second conclusion of our experiments is that PCR performed on DNA from U-71184-treated cells is inhibited when the primers amplifying the estrogen receptor gene region are used. This approach might bring important information on both in vivo uptake of the drug by target cells and binding to DNA.

Determination of transcription start sites in the human estrogen receptor gene and identification of a novel, tissue-specific, estrogen receptor-mRNA isoform

The human estrogen receptor (ER) gene has previously been shown to be transcribed from two different promoters which are expressed in a cell- and tissue-specific manner. Whereas the transcriptional start sites of the proximal promoter are known, the transcriptional initiation site(s) of the distal promoter has not been identified. In this study, a PCR-based technique was used to isolate the 5′ ends of ER cDNAs from different human cell lines and tissues. Using this technique the positions of the transcription initiation sites in the distal promoter were determined. In addition a novel 5′ untranslated exon was isolated from human liver. These results demonstrate that the human ER gene contains a third, previously not described promoter which appears to be predominantly expressed in liver.

Kinetic analysis of the interaction of human estrogen receptor with an estrogen response element

The kinetics of the interaction between recombinant human estrogen receptor and chicken vitellogenin gene II estrogen response element (ERE) were determined by ERE-Sepharose chromatography. The association constant of the interaction between the ERE and the human estrogen receptor was dependent on receptor concentration, estradiol binding and temperature. The highest association constant (80−100 × 10 6 M −1) was measured for the estradiol-bound receptor prepared at 25°C and at concentrations higher than 7 nM. At high receptor concentrations (> 7 nM) the binding mechanism of estradiol to the receptor was positive cooperative, indicating receptor homodimerization. At lower concentrations the binding mechanism was partially cooperative and the association constant of the liganded receptor was significantly lower. The binding mechanism at 4°C was cooperative as well, and the association constants were similarly dependent upon receptor concentration, but were 50% lower than the receptor prepared at 25°C. The association constant of the unliganded receptor was 4- to 5-fold lower than that of the liganded receptor at 25°C. These data suggest that in addition to estradiol-induced conformational changes in the receptor, the receptor dimers are subjected to temperature-dependent changes, which further increase their affinity for an ERE.