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

Breast tumor cell lines have been shown to secrete at least five distinct insulin-like growth factor (IGF) binding proteins (IGFBP), the secretion being related to the estrogen receptor (ER) content. In this study we investigated IGFBP mRNA expression and IGFBP content in relation to ER content in human breast tumors. Tissue specimens from 47 breast cancers were studied. In five cases the adjacent histologically normal tissue was also analyzed. IGFBP content in tissue homogenates was studied by Western ligand blot analysis, using [125I] IGF-I as a label, and IGFBP mRNA expression by reverse transcriptase polymerase chain reaction. The results show that human breast tumors express mRNAs encoding IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5. The pattern of IGFBPs in different tumors varies. No correlation exists between ER content and IGFBPs with molecular weights 24,000 Mr, 28,000 Mr, 34,000 Mr or 43,000 Mr, whereas the 49,000 Mr IGFBP was more abundant in ER negative tumors (P less than 0.05). The IGFBP content was significantly (P less than 0.05) higher in five tumors than in their adjacent normal tissues suggesting that increased content of IGFBPs is a feature typically associated with the malignant transformation of breast tissue.
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PMID:Insulin-like growth factor binding proteins in human breast cancer tissue. 138 38

Estrogen is required for oocyte maturation and embryonic development in vivo; however, the mechanism involved is not clear. Since the effect of estrogen is mediated through the estrogen receptor (ER), we examined the ontogeny and expression of the ER gene in mouse oocytes and embryos of various gestational stages using the highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) technique. Total RNA, extracted from 40 ovulated oocytes, 2-cell embryos, morulae, and blastocysts, was reverse transcribed into cDNA. A pair of primers flanking the 453-bp region encoding the hormone-binding domain of ER was used for 30 cycles of PCR. The identity of the amplified product was confirmed by sizing and Southern blot hybridization. The results indicated that ER gene is expressed in unfertilized oocytes and cumulus-oocyte complexes. The amount of ER mRNA decreases in 2-cell embryos, coincident with degradation of maternal mRNA. No ER transcript can be detected in the morulae or blastocyst stage when the embryonic genome has been activated. Postimplantation embryos do not contain detectable ER mRNA until gestation day 8. The levels of ER mRNA increase from day 10 to day 18 of gestation. These data suggest that estrogen, secreted by granulosa cells, may directly influence oocyte growth and maturation in vivo. Since estrogen is known to stimulate the production of growth factors in mouse uteri, the absence of ER mRNA in periimplantation embryos suggests that the effects of estrogen on early embryogenesis may be indirect, i.e., through estrogen-regulated growth-promoting factors produced by the reproductive tract. In mid- and late-post-implantation embryos which contain ER mRNA, estrogen may affect embryonic development through the receptor-mediated mechanisms.
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PMID:Expression of estrogen receptor gene in mouse oocyte and during embryogenesis. 147 72

The expression of a mouse mammary tumor virus is inducible by hormones, and the virus contains a hormone-responsive element. Viral particles and RNA-directed DNA polymerase (RDDP, EC 2.7.7.7; reverse transcriptase) are both detectable in human breast tumors but the frequency and significance of these findings are unknown. Breast tumor biopsy specimens (from either the primary site or a metastasis), frozen in liquid nitrogen at the time of surgery, were routinely obtained to determine estrogen receptor (ERP) concentration. A sample of the tissue was pulverized, homogenized and centrifuged at low speed to remove nuclei and mitochondria. The supernate was then centrifuged at 225,000 g to obtain the cytosol fraction for estrogen and progestin receptor (PgR) assays. Partially purified membranes for the RDDP assays were prepared from the high-speed pellet by discontinuous sucrose density gradient centrifugation. The RDDP assay involved measuring primer-dependent poly(dT) synthesis in the presence of poly(A) as template and oligo-(dT)12-18 as primer. To date, we have studied biopsy specimens from 46 patients with breast cancer. 27 (59%) had ERP and 23 (50%) were RDDP-positive. There was no significant correlation between ERP concentration and RDDP activity. PgR data were available on 36 of the patients; 17 (47%) were positive. No correlation between RDDP and PgR was apparent. Similarly, there was no correlation between RDDP and clinical stage of the disease.
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PMID:RNA-directed DNA polymerase activity in human breast cancer biopsy specimens. Relation to estrogen receptor protein. 620 38

We describe a human (h) PRL-producing cell line, SKUT-1B-20, which we isolated as a subclone of a uterine sarcoma cell line. Although this cell line is of uterine origin, it does not use the decidual-specific upstream promoter of the hPRL gene, but transcribes the hPRL gene from the downstream pituitary-type transcription start site, as determined by Northern blot, reverse transcriptase-polymerase chain reaction and primer extension analyses. This is particularly intriguing because SKUT-1B-20 cells lack the transcription factor Pit-1. No Pit-1 messenger RNA was detectable by reverse transcriptase-polymerase chain reaction, and endogenous Pit-1 target genes (GH, PRL, and Pit-1) were refractory to transfected Pit-1 expression vector, whereas in cotransfection experiments, Pit-1 efficiently activated reporter gene fusion constructs carrying 5'-flanking sequences of the human and rat PRL or the mouse Pit-1 genes. By transfecting reporter genes containing 8.7 kilobases of DNA flanking the hPRL pituitary-specific start site (hPRL-8700/Luc) and deletions thereof, we located a Pit-1-independent cis-active region more than 7 kilobases upstream of the start site. The most distal 1650 or 880 base pairs of the hPRL genomic fragment (which extends to -8784 base pairs), when placed directly upstream of the homologous hPRL or the heterologous thymidine kinase promoters, conferred transcriptional activation to those promoters. SKUT-1B-20 cell-specific activation of hPRL-8700/Luc could not be suppressed by the introduction of an inhibitor of protein kinase A (PKA), PKI. This is the first demonstration of pituitary-type PRL gene transcription independent of Pit-1 and activation of the PKA pathway. The SKUT-1B-20 cell line was then used in reconstitution experiments to delineate the role of Pit-1 in modulating the transcriptional effects of phorbol ester, PKA, and estrogen receptor (ER) on the hPRL gene. The low response of hPRL/luciferase fusion genes to phorbol ester was greatly enhanced by cotransfected Pit-1 and was mediated by the proximal region between -250 and -38. The catalytic subunit of PKA, C beta, was able to elicit a moderate induction of hPRL-8700/Luc even in the absence of Pit-1. A potential estrogen response element has been located in the hPRL gene sequence at a position similar to that of the estrogen response element of the rat PRL gene immediately adjacent to the distal enhancer.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Pituitary-type transcription of the human prolactin gene in the absence of Pit-1. 747 71

The calbindin D9k (CaBP9k) gene is under strict estrogen control in the rat uterus. This tissue contains two CaBP9k messenger RNA (mRNA) species. We have used primer extension analysis, reverse transcriptase associated with polymerase chain reaction, and RNase H digestion to show that these two mRNA species have the same structural features, including 5'- and 3'-ends, and poly(A) tail length. Our results suggest that the difference in electrophoretic mobilities of the two mRNA species might be due to interaction with another factor. We also analyzed the imperfect estrogen-responsive element (ERE) present on the first 5'-splice site of the rat CaBP9k gene. The oligonucleotide corresponding to the CaBP9k ERE was cloned in the plasmid pBLCAT2 (where the thymidine kinase promoter governs the expression of the chloramphenicol acetyl transferase gene) and transfected into MCF7 cells. This CaBP9k ERE was found to be a hormone-inducible enhancer that worked in an orientation-independent manner on a heterologous promoter and was functional at physiological hormone concentrations. One CaBP9k ERE conferred only weak (about 2-fold) estrogen induction, but two EREs cloned in tandem were strongly synergistic (14- to 16-fold). The CaBP9k ERE also bound to the partially purified estrogen receptor (ER) and to ER expressed in COS cells by gel shift assay. Methylation interference showed that all the guanine residues in both half-sites of the CaBP9k ERE were protected by ER binding. Thus, ER binds to the CaBP9k ERE in a way similar to other EREs. The gel shift assay results indicate that the strong synergistic effect of two EREs cloned in tandem is not due to cooperative binding between the two elements. As the CaBP9k gene is under strong estrogenic control in the uterus in vivo, the imperfect CaBP9k ERE may cooperate with another trans-acting factor to become fully efficient.
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PMID:Calbindin-D9k gene expression in the uterus: study of the two messenger ribonucleic acid species and analysis of an imperfect estrogen-responsive element. 750 2

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

The estrogen receptor (ER) mediates the effects of its cognate ligand on important cellular processes such as development of female secondary sexual characteristics, establishment and maintenance of pregnancy, progression of breast cancer, and maintenance of bone mass. We have previously demonstrated that the human ER (hER) gene is transcribed from two promoters, suggesting that tissue- and cell-specific expression patterns of this gene may, at least in part, be regulated by differential promoter usage. Here we show, by using a reverse transcriptase coupled polymerase chain reaction assay, that transcripts initiated from both hER gene promoters were expressed in breast and uterus. In contrast, only transcripts originating from the distal promoter could be detected in human primary osteoblasts. Furthermore, determination of the expression levels of the two hER transcripts by quantitative polymerase chain reaction demonstrated an almost 30-fold increase of the transcripts originating from the proximal promoter in breast cancer cell lines over that detected in normal breast tissue. Taken together, our results demonstrate a previously unrecognized mechanism for regulation of hER gene expression by tissue-specific differential promoter utilization. In addition, our results suggest that estrogen-dependent cell transformation may be accompanied by a change in the relative expression levels of the two hER transcripts.
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PMID:Estrogen target tissue determines alternative promoter utilization of the human estrogen receptor gene in osteoblasts and tumor cell lines. 772 Jun 71

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


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