Human Estrogen Receptor Gene Research Articles

Incidence of an estrogen receptor polymorphism in breast cancer patients

We previously identified a polymorphism in the human estrogen receptor (ER) gene, within the coding region for the protein's amino terminal B-domain. In estrogen receptor-positive (ER+) breast tumors, the variant allele was preferentially associated with lower levels of ER, and was clinically correlated with frequent spontaneous abortions. DNA sequencing revealed a point mutation that changes codon 86 from Ala to Val and a silent mutation in codon 87. Because we initially detected the variant allele by analyzing RNA, only those tissues in which the ER gene is actively expressed were suitable for genotype analysis. We now describe an assay that uses genomic DNA as the substrate for determining the ER B genotype, DNA containing the polymorphic region of the ER gene is amplified by the polymerase chain reaction, then the amplified DNA is hybridized with radiolabeled oligonucleotide probes complementary to the wild type and variant ER alleles. This method allowed us to determine the ER B genotype of women with ER+ and ER- tumors, starting with minute amounts of DNA from frozen or paraffin embedded tissues. ER B genotyping was also performed on women without breast cancer using DNA extracted from blood cells. The combined results from analyses of RNA and DNA from 300 breast cancer patients showed that 12% were heterozygotes. In the ER+ group (n = 183), 11.5% carried the variant gene compared to 12.8% in the ER-negative group (n = 117) (chi 2 = 0.11; df = 1; p greater than 0.25).(ABSTRACT TRUNCATED AT 250 WORDS)

Stable expression of the human estrogen receptor in HeLa cells by infection: Effect of estrogen on cell proliferation and c- myc expression

We have introduced the human estrogen receptor (ER) gene into HeLa cells, a human adenocarcinoma cell line of uterine origin, by infection. The ER cDNA was inserted into a retroviral vector (pMV7-ER) which also contains the neomycin resistance gene to allow for selection of stable infected clones. Northern analysis showed exogenous ER expression in stable clones. The ER protein expressed was about 66 kDa, similar to native MCF-7 ER, and binds with high affinity to estrogen (E 2). We have also observed that addition of E 2 at 10 −8 M inhibits the growth of the I-1 clone which expresses high levels of the ER (223 fmol/mg cytosol protein). The inhibitory effects of E 2 directly correlate with the quantity of ER in the cells. E 2-induced gene expression analysis showed that pS2 and progesterone receptor (PgR), genes induced in MCF-7 cells by E 2, are not induced in the ER + HeLa clones. However, c- myc expression was found to be decreased and may be responsible for the observed growth inhibition by E 2.

EVIDENCE FOR A PREVIOUSLY UNIDENTIFIED UPSTREAM EXON IN THE HUMAN OESTROGEN RECEPTOR GENE.

The presence of a previously unidentified exon upstream of the originally described human oestrogen receptor (hOR) gene is demonstrated. This is shown to be spliced to the 5' untranslated region of the previously designated exon I. The resulting genomic structure of the human gene is thus in agreement with the structure of the mouse OR gene and highlights the conservation of an 18 amino acid upstream open-reading frame formed from the above splicing event. Taken in conjunction with previous publications this would suggest that the hOR gene is a complex transcriptional unit that contains two promoters.

Estradiol Regulation of Estrogen Receptor Messenger Ribonucleic Acid in Rat Mediobasal Hypothalamus: An in situ Hybridization Study

Abstract We have used in situ hybridization to investigate estradiol regulation of estrogen receptor (ER) mRNA in regions of the mediobasal hypothalamus which contain ER and are related to specific neuroendocrine functions. Ovariectomized rats were treated with oil or 10 mug estradiol benzoate for 2, 4, 18 or 24 h. Brains were sectioned and hybridized with a [(3) H]single-stranded DNA probe prepared from the pORF cDNA of the human ER gene and exposed to autoradiographic emulsion for 4 months. Specificity of labeling was determined by counting the number of grains over cells in hypothalamic regions known to bind estradiol, compared to cells in the thalamus and cortex, and by comparing with sections pretreated with ribonuclease or hybridized with a [(3) H]single-stranded message-sense (control) probe. Labeling for ER mRNA was distributed differently than glucocorticoid and thyroid hormone receptor mRNAs, and was regulated by estrogen differently than progestin receptor mRNA. These differences indicated specific hybridization for ER mRNA. ER-expressing cells constituted 11.5% of the cells in the dorsomedial nucleus, 30% of the cells in the arcuate nucleus and 43% in the ventromedial nucleus, in close accordance with previous studies of ER autoradiography and binding. Quantitative analysis showed that the highest level of ER mRNA was present in the ovariectomized controls. ER mRNA levels fell 42% (ventromedial), 64% (arcuate), or remained unchanged (dorsomedial) 18 h following estradiol benzoate treatment. The pattern of decrease was similar for cells in the ventromedial nucleus and arcuate nucleus. These data show that estrogen regulation of ER mRNA in brain parallels that reported for MCF-7 cells and rat uterus. These results also demonstrate that in situ hybridization can be used to detect and measure the relative level of a low abundance mRNA in a heterogeneous tissue in which only 12% to 40% of the cells in limited regions express the message.

Oestrogen receptor B-region polymorphism and spontaneous abortion in women with breast cancer

To examine whether a variant human oestrogen receptor gene, which differs from the wild-type gene in the B region, was associated with spontaneous abortion, obstetric histories of breast cancer patients with this variant were compared with those of breast cancer patients with the wild-type gene. In women with the B-variant, 50% of pregnancies ended in spontaneous abortion, compared with 10% for women homozygous for the wild-type gene. B-variant women also had a higher proportion of spontaneous abortions and a higher number of spontaneous abortions per woman than did those with the wild-type gene.

Different methylation of oestrogen receptor DNA in human breast carcinomas with and without oestrogen receptor

The methylation of the human oestrogen receptor (ER) gene was analysed by restriction enzymes in normal and neoplastic human breast tissues and cell lines. CCGG sequences in regions inside the gene, which are methylated both in normal breast and in tissues that are not the target of the oestrogen, are hypomethylated in 30% of tumours, both ER+ and ER- carcinomas. Moreover, 5' sequences of the gene, which are hypomethylated in normal breast and not in tissues not the target of oestrogen, are methylated to a lower degree in ER+ carcinomas, whereas they are methylated to a greater degree in ER- carcinomas. However, the same region is equally hypomethylated in both ER+ and ER- cancer cell lines. Our results indicate that in breast carcinomas ER DNA methylation is deranged, and in cancer cell lines is different from that observed in primary tumours. Furthermore, the abnormal methylation in the 5' end seems to be related to abnormal expression, namely diffuse hypomethylation in carcinomas with high ER content and hypermethylation in carcinomas without ER. These findings support our previous hypothesis that DNA methylation could be involved in the control of ER gene expression and demonstrate that abnormal ER gene methylation is a typical feature of breast cancers.

Detection of two high molecular weight hydrophobic forms of the human estrogen receptor

The human estrogen receptor gene encodes a single protein of molecular weight 65,000 daltons. However, using a sensitive and rapid technique of high-performance hydrophobic interaction chromatography we have detected two distinct estrogen receptor species both of which are high molecular weight proteins ( ca. 60A) as determined by high-performance size-exclusion chromatography. These are detected either in the presence or absence of sodium molybdate; rechromatography of individual isoform indicates that the two protein complexes have independent hydrophobic contact points. Consistent elution patterns of the two receptor species indicates they are formed selectively. We conclude that different post-translational modifications of the estrogen receptor protein could allow their specific interaction with non-receptor components resulting in the formation of two distinct high molecular weight complexes which would be rapidly resolved by high-performance hydrophobic interaction chromatography.

Chimaeras of myc oncoprotein and steroid receptors cause hormone-dependent transformation of cells.

The human proto-oncogene myc encodes a nuclear phosphoprotein whose primary biochemical function is still unknown. To facilitate further study of that function, we have created conditional alleles of myc by fusing the hormone-binding domain of the human oestrogen receptor gene to the 5' or the 3' end of human myc. The two chimaeric genes, designated mycer and ermyc, encode proteins that bind oestrogen with high affinity. Expression of one of the genes, mycer, transforms a rat fibroblast cell line in a tightly oestrogen-dependent manner. Transformation is dependent on the presence of a functional myc gene in the chimaera and is reversible upon removal of the hormone. The chimaeric genes will be useful tools to study the mechanisms by which Myc affects cellular phenotype. Recently, chimaeras between the adenovirus E1A protein and the hormone binding domain of the rat glucocorticoid receptor were shown to activate transcription in a manner characteristic for E1A, but in a hormone regulated manner. We therefore asked whether the same strategy could be applied to the product of myc.

An analysis by restriction enzymes of the genomic structure of the 3′ untranslated region of the human estrogen receptor gene

The estrogen receptor gene has a very long 3′ untranslated region. As a first step towards the analysis of this structural feature for any functional role, we have cloned the human genomic estrogen receptor gene. Extensive restriction enzyme analysis of this DNA and comparison of the sizes of the DNA fragments obtained with those predicted from published cDNA sequences indicate that the 3′ exon extends for at least 4304 bases from base number 2018 in the cDNA to the end of the cDNA. The data also show that the most 3′ intron in this gene occurs between bases 1902 and 2018 of the cDNA.

An analysis of the genomic structure of the 3′ untranslated region of the human oestrogen receptor gene

An analysis of the genomic structure of the 3′ untranslated region of the human oestrogen receptor gene

Hypomethylation of the human estrogen receptor gene in primary human breast cancers.

Hypomethylation of the human estrogen receptor gene in primary human breast cancers.

Localization of the human oestrogen receptor gene to chromosome 6q24→q27 by in situ hybridization

The oestrogen receptor gene (ER) was mapped by in situ hybridization. Using a human cDNA probe containing the coding sequence for the oestrogen receptor, the gene was localized to 6q24----q27.

A strategy for the cloning of the human oestrogen-receptor gene: optimization of an immunodetection system for use in the screening of a cDNA library

A strategy for the cloning of the human oestrogen-receptor gene: optimization of an immunodetection system for use in the screening of a cDNA library