Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method for estrogen receptor (ER) mRNA was performed on 33 human breast tumors, using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay by the method of Fuqua et al. In a preliminary experiment using the MCF-7 breast tumor cell line, ER/beta-actin ratio was almost same. ER protein was estimated by a dextran coated charcoal (DCC) assay and by an ER-immunocytochemical (ER-ICA) assay using a specific monoclonal antibody. We found RT-PCR assay correlates with ER-ICA assay (r = 0.664, p less than 0.01), whereas no significant correlation was seen between RT-PCR assay and DCC assay. These results suggests that RT-PCR assay is suitable for detection of ER from small amounts of tissue.
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PMID:[Detection of estrogen receptor (ER) mRNA by use of reverse transcriptase-polymerase chain reaction (RT-PCR) assay; comparison with dextran coated charcoal (DCC) assay and immunocytochemical assay]. 137 12

The expression and quantitation of the estrogen receptor (ER) in human thyroid tumors were examined by biochemical, immunohistochemical, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. For this study, neoplasms, adenomatous goiters and adjacent normal thyroid tissues were obtained from 35 patients which included 10 cases of papillary carcinomas, 17 cases of adenomas and 8 cases of adenomatous goiters. Regardless of the histopathological subtype, ER was detected in 19% (5/27) of the neoplastic tissues with the mean value of ER content of 5.0 +/- 1.3 fmol/mg protein and the mean Kd value of 0.38 +/- 0.28 nM. ER was also detected, but at a lower concentration (2.8 +/- 1.6 fmol/mg protein), in the surrounding normal tissues. There was no significant difference between the neoplasms and adenomatous goiters with respect to the incidence of ER positivity and ER content. Furthermore, ER-positive specimens, as determined by both biochemical and immunohistochemical techniques, also showed the expression of ER mRNA detected by RT-PCR method. These results demonstrate that both ER mRNA as well as ER protein are expressed in thyroid neoplasms. This suggests the possibility that estrogen may affect the tumorigenesis or the progression of some thyroid neoplasms.
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PMID:Expression of the estrogen receptor in human thyroid neoplasms. 752 Dec 73

Approximately one third of breast cancers grow independently of estrogen, lack detectable estrogen receptor (ER) protein, and rarely respond to hormonal treatment. Previous studies correlated the lack of ER gene expression in ER-negative breast tumor cells with hypermethylation of a CpG island in the 5' region of the ER gene. In order to determine whether demethylation of the ER gene in the ER-negative human breast cancer cell line MDA-MB-231 could affect ER transcription, cells were treated with two inhibitors of DNA methylation, 5-azacytidine or 5-aza-2'-deoxycytidine. DNA from cells treated with either drug became partially demethylated at several methylation-sensitive restriction enzyme sites, including HhaI, NotI, and SacII, within the ER CpG island. This demethylation correlated with reexpression of the ER gene as detected by reverse transcriptase-PCR and production of ER protein as detected by Western blot analysis. ER produced in drug-treated cells was functionally active as demonstrated by its ability to activate transcription of estrogen-responsive genes. These results suggest that DNA methylation of the ER CpG island may play a role in suppression of ER gene expression in ER-negative breast cancer cells.
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PMID:Demethylation of the estrogen receptor gene in estrogen receptor-negative breast cancer cells can reactivate estrogen receptor gene expression. 753

Recent studies failed to detect estrogen receptors in primate follicles. This study was initiated to determine whether estrogen receptor (ER) messenger ribonucleic acid (mRNA) is present in human granulosa cells and, further, if functional ER proteins are present. To evaluate the presence of ER, RNA from human granulosa cells obtained at the time of oocyte retrieval for assisted reproduction was extracted, and complementary DNA synthesis was performed by the reverse transcriptase-polymerase chain reaction. Oligonucleotide primers were used to amplify basepairs 570-852 in the B- and C- domains of the ER mRNA. Southern blotting was performed and confirmed that the amplified DNA fragment identified in granulosa cells represented ER. By reverse transcriptase-polymerase chain reaction, mRNA for the ER is clearly identified in primary human granulosa cells obtained at the time of oocyte retrieval. To expand these studies and determine whether functional ER were present in human granulosa cells in culture, a simian virus-40-transformed human granulosa cell line was studied. Cells were transfected with a plasmid containing as estrogen response element up-stream from the bacterial reporter gene chloramphenicol acetyltransferase (CAT). In transfected cells, CAT activity is inducible by estradiol if endogenous functional ER are present. In these studies, the transfection analysis confirmed that functional, transcriptionally competent ER are present in a human granulosa cell line, with a 4- to 5-fold enhancement of CAT activity demonstrated after the addition of estradiol compared to that in nonhormone-treated cells. In conclusion, ER mRNA is present in human granulosa cells. Functional ER are also demonstrated in a transformed human granulosa cell line. We hypothesize that low, but biologically significant, amounts of ER protein are present in human granulosa cells, which are not routinely detectable by standard assays.
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PMID:Estrogen receptors are present in human granulosa cells. 782 17

A variant form of the human oestrogen receptor (ER) mRNA lacking sequences encoded within exon 5 has been described (Fuqua SAW, Fitzgerald SD, Chamness GC, Tandon AK, McDonnell DP, Nawaz Z, O'Malloy BW, McGuire WL 1991, Cancer Res 51: 105-109). We have examined the expression of the exon 5-deleted ER (HE delta5) mRNA variant in breast biopsies using reverse transcriptase polymerase chain reaction (RT - PCR). HE delta5 mRNA was present in only 13% of non-malignant breast tissues compared with 32% of carcinomas (95% CI, P=0.05). Presence of the HE delta5 mRNA was associated with the presence of immunohistochemically detected ER (P=0.015) and progesterone receptor (PR) (P=0.02). There was a positive correlation between the presence of HE delta5 and disease-free survival (P=0.05), suggesting that the presence of HE delta5 may be an indicator of better prognosis. We have raised a monoclonal antibody specific to the C-terminal amino acids of HE delta5. This antibody recognized the variant but not the wild-type ER protein. We show that HE delta5 protein is present in breast cancer using immunohistochemical techniques. We also analysed trans-activation by HE delta5 in mammalian cells and showed that, in MCF-7 cells, HE delta5 competes with wild-type ER to inhibit ERE-dependent trans-activation. Our results indicate that this variant is unlikely to be responsible for endocrine resistance of breast cancer, but its presence at both the mRNA and protein level suggest that it may, nevertheless, be involved in regulating the expression of oestrogen-responsive genes in breast cancer.
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PMID:Presence of exon 5-deleted oestrogen receptor in human breast cancer: functional analysis and clinical significance. 909 67

Osteoblasts are established targets of estrogen action in bone. We screened 66 conditionally immortalized clonal human osteoblast cell lines for estrogen receptors (ERs) using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for ER alpha mRNA and transactivation of adenovirus-estrogen response element (ERE)-tk-luciferase by 17 beta-estradiol (17 beta-E2) for functional ER protein. One of these cell lines, termed HOB-03-CE6, was chosen for further characterization. The cells, which were conditionally immortalized with a temperature-sensitive SV40 large T antigen, proliferated at the permissive temperature (34 degrees C) but stopped dividing at the nonpermissive temperature (> or = 39 degrees C). Alkaline phosphatase activity and osteocalcin secretion were upregulated by 1 alpha, 25-dihydroxyvitamin D3 in a dose-dependent manner. The cells also expressed type I collagen and other bone matrix proteins, secreted a variety of growth factors and cytokines, formed mineralized nodules based on alizarin red-S and von Kossa histochemical staining, and responded to dexamethasone, all-trans retinoic acid, and transforming growth factor-beta 1. This cell line expressed 42-fold less ER message than MCF-7 human breast cancer cells, as determined by quantitative RT-PCR. However, adenovirus-ERE-tk-luciferase activity was upregulated three- to fivefold in these cells by 17 beta-E2 with an EC50 of 64 pM. Furthermore, this upregulation was suppressed by co-treatment with the anti-estrogen ICI-182, 780. Cytosolic extracts of these cells specifically bound [125I]-17 beta-E2 in a concentration-dependent manner with a Bmax of 2.7 fmoles/mg protein (approximately 1,200 ERs/cell) and a Kd of 0.2 nM. DNA gel-shift analysis using a [32P]-ERE demonstrated the presence of ERs in nuclear extracts of these cells. Moreover, binding of the extracts to this ERE was blocked by a monoclonal antibody to the human ER DNA-binding domain. We evaluated these cells for 14 of 20 reported endogenous responses to 17 beta-E2 in osteoblasts. Although most of these responses appeared to be unaffected by the steroid, 17 beta-E2 suppressed parathyroid hormone-induced cAMP production, as well as basal interleukin-6 mRNA expression; conversely, the steroid upregulated the steady-state expression of alkaline phosphatase message in these cells. In summary, we have identified a clonal, conditionally phenotypic, human osteoblast cell line that expresses functional ERs and exhibits endogenous responses to 17 beta-E2. This cell line will be a valuable in vitro model for exploring some of the molecular mechanisms of estrogen action in bone.
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PMID:Functional properties of a conditionally phenotypic, estrogen-responsive, human osteoblast cell line. 913 93

In a study involving 50 breast cancer tumours, we compared two oestrogen receptor (ER) detection methods developed by us--a microplate immunoenzymometric assay (EIA96) and an immunohistochemistry kit (HistoCIS-ER)--with the radioligand assay (RLA), the Abbott immunoenzymometric assay ER-EIA and the reverse transcriptase polymerase chain reaction technique (RT-PCR). Among the three ER protein cytosolic assays (EIA96, ER-EIA and RLA), the two EIAs showed the best agreement (y = 1.086x - 7.840; r2 = 0.876). At the calculated optimal cut-off values (8 and 14 fmol mg(-1) protein for EIA96 and RLA respectively), EIA96 was more sensitive than RLA (0.94 for EIA96, 0.88 for RLA), but slightly less specific (0.82 for EIA96, 0.94 for RLA). The Cox logistical regression model applied to EIA96, RLA and RT-PCR showed that EIA96 discriminated the best between ER-EIA+ and ER-EIA- samples. The RT-PCR technique and HistoCIS-ER both had a positivity-negativity concordance of 86% with EIA96.
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PMID:Comparison of a new microplate oestrogen receptor (ER) enzyme immunoassay with other ER detection methods. 927 30

The oestrogen receptor (ER) is widely used to predict response to tamoxifen in patients with breast cancer. Recently a new form of ER known as ER-beta was discovered, the original ER is now designated ER-alpha. In this investigation, ER-alpha and ER-beta were measured in 107 breast carcinomas and 22 fibroadenomas. Using reverse transcriptase-polymerase chain reaction (RT-PCR), ER-beta mRNA, but not ER-alpha mRNA was expressed more frequently in fibroadenomas than carcinomas. In the carcinomas, ER-beta mRNA was present in a greater proportion of samples positive for ER-alpha mRNA than in those lacking this form of the receptor. ER-alpha, but not ER-beta mRNA, was significantly associated with ER protein-positivity in the cancers. ER-alpha mRNA was also positively related to progesterone receptors (PR), but ER-beta mRNA showed an inverse relationship with PR. We conclude that the presently used enzyme-linked immunosorbent assay (ELISA) for ER appears to be mostly measuring ER-alpha and is unlikely to be detecting ER-beta.
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PMID:Studies on oestrogen receptor-alpha and -beta mRNA in breast cancer. 1137 42

Estrogen induction of progesterone receptor (PR) expression may be important to bone physiology because progesterone has been implicated in the control of bone formation and resorption. Although PR gene expression can be induced in osteoblasts by estrogen signaling through the estrogen receptor (ER) a isoform, it is unknown whether the ER-beta isoform is involved in this regulation. The effect of estrogen on PR expression was examined in human fetal osteoblast (hFOB) cell lines stably transfected with either ER-alpha or ER-beta. Estrogen treatment of hFOB/ER-a cells induced PR messenger RNA (mRNA) steady-state levels after 24 h and protein levels after 48 h, as established by competitive reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Interestingly, no induction of PR expression was observed in the hFOB/ER-beta cells during this period. However, PR mRNA was induced progressively after 48 h of treatment with estrogen with maximum levels achieved at 12 days posttreatment. ER protein also was increased after 12 days of treatment. Both A and B isoforms of PR (PRA and PRB) were induced by estrogen in the hFOB/ER-a cells as well as much later in hFOB/ER-beta cells. The pure antiestrogen ICI 182,780 prevented PR induction by estrogen in both cell lines. An ER-beta-selective antagonist R, R-tetrahydrochrysene (THC) abolished the induction of PR mRNA in hFOB/ER-beta but not in hFOB/ER-a cells, verifying that the response in the former cell line was ER-beta-mediated. Transient cotransfection of hFOB cells with ER-a or ER-beta together with either a human PRA or PRB promoter linked to a reporter plasmid revealed that although the PRB promoter was stimulated equally by estrogen activation of either ER isoform, PRA was activated preferentially by ER-alpha. Together, these results show that although estrogen can up-regulate endogenous PR gene expression in osteoblasts via both ER isoforms, ER-alpha is the predominant inducer.
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PMID:Estrogen receptor isoform-specific induction of progesterone receptors in human osteoblasts. 1191 16

Previously, we demonstrated that enforced activation of signal transducer and activator of transcription 5 (STAT5A) in human cord blood (CB)-derived stem/progenitor cells results in enhanced self-renewal and impaired myelopoiesis. The present study identifies C/EBPalpha as a critical component that is down-regulated by STAT5. Microarray and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis on STAT5A(1*6)-transduced CD34(+) cells identified C/EBPalpha as the most prominently down-regulated gene. To determine the cell-biological relevance of these observations, a 4-OHT-inducible C/EBPalpha-ER protein was co-expressed with the STAT5A(1*6) mutant in CB CD34(+) cells using a retroviral approach. Re-expression of C/EBPalpha in STAT5A(1*6) cells resulted in a marked restoration of myelopoiesis. The proliferative advantage imposed on CD34(+) cells by STAT5A(1*6) depended on the down-modulation of C/EBPalpha, as reintroduction of C/EBPalpha induced a quick cell-cycle arrest and the onset of myeloid differentiation. Long-term culture-initiating cell (LTC-IC) frequencies were elevated from 0.8% +/- 0.6% to 7.8% +/- 1.9% by STAT5A(1*6) as compared with controls, but these elevated LTC-IC frequencies were strongly reduced upon re-introduction of C/EBPalpha in STAT5A(1*6) cells, and no second cobble-stone area-forming cells (CAFCs) could be generated from double-transduced cells. Enumeration of progenitors revealed that the number of colony-forming cells (CFCs) was reduced more than 20-fold when C/EBPalpha was co-expressed in STAT5A(1*6) cells. Our data indicate that down-modulation of C/EBPalpha is a prerequisite for STAT5-induced effects on self-renewal and myelopoiesis.
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PMID:STAT5-induced self-renewal and impaired myelopoiesis of human hematopoietic stem/progenitor cells involves down-modulation of C/EBPalpha. 1645 47


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