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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As a model for tissue-specific gene expression, our laboratory has been studying the expression of vitellogenin and FOSP-1 (frog oviduct-specific protein-1) genes in Xenopus laevis which are expressed exclusively in the liver and oviduct, respectively, both strictly regulated by estrogen. Whereas the structure and function of Xenopus vitellogenin mRNAs and the upstream regulatory sequences (URS) of their genes are well established, little or no similar information is available for FOSP-1 genes. In this study, using a combination of 5' rapid amplification of cDNA ends (RACE) and reverse-transcriptase PCR, we have identified two gene copies of FOSP-1, termed FOSP-1A and FOSP-1B. Comparison of the sequences of full-length FOSP-1A and partial FOSP-1B cDNAs revealed a high degree of similarity at the 5' end. We next isolated FOSP-1A and FOSP-1B genomic clones. Dot-plot comparison of their URS showed both similarities and differences. Two estrogen-responsive elements (EREs), termed proximal (pERE) and distal (dERE), were identified at -1070/-1082 and -1167/-1179, respectively, in FOSP-1B, but not FOSP-1A, URS. Quantitative electrophoretic mobility shift assay (EMSA) and DNA footprinting with recombinant Xenopus estrogen receptor (xER) expressed in insect Sf9 cells, showed that xER interacted with a higher affinity with dERE than pERE in a hormone-independent manner, and that the two EREs do not act cooperatively. Functional studies involving transient transfection of human MCF-7 cells with a FOSP-1B URS-tkCAT construct confirmed that both EREs act as hormone-inducible cis-acting elements. These studies now pave the way for analysis of tissue specificity of estrogen-inducible gene expression in Xenopus liver and oviduct.
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PMID:Structural and functional characterization and cloning of Xenopus FOSP-1 (frog oviduct-specific protein-1) genes. 774 34

The recent demonstration that bone sialoprotein (BSP) can be detected in human breast cancer tissue by immunoperoxidase suggests that this phosphoprotein is ectopically expressed by malignant mammary epithelial cells. Its detection in human breast cancer cells raises questions about its potential role(s) during breast cancer progression. Because BSP is secreted and is present in the serum, the positivity of breast cancer cells for BSP could have been the result of an uptake of the circulating phosphoprotein by the cells rather than of an intrinsic expression. We examined the expression of BSP at both the protein and mRNA levels in nine human breast cancer samples as well as in three human breast cancer cell lines (MCF-7, T47-D, and MDA-MB-231) using immunohistochemistry, flow cytometric analysis, immunoblot, and reverse-transcriptase PCR. BSP was detected at both protein and mRNA levels in human breast cancer tissue and in the three human breast cancer cell lines. Using a specific polyclonal anti-BSP antibody, we showed by both fluorescence-activated cell sorter analysis and immunohistochemistry experiments that all of the human breast cancer cell lines studied express BSP. This was localized at the cell surface and in the cytosol of the estrogen receptor-positive MCF-7 and T47-D cell lines, whereas it was detected only in the cytosol of the estrogen receptor-negative MDA-MB-231 cells. Using the same polyclonal anti-BSP antibody, we were able to identify an approximately 97-kd band on total protein extracts from the three cell lines by immunoblotting. Reverse-transcriptase PCR reactions using specific oligonucleotides performed on total RNA of nine human breast cancer biopsy samples and the three cell lines demonstrated the presence of BSP mRNA in all of the samples examined. This study is the first demonstration that human malignant breast epithelial cell lines express BSP at the protein and mRNA levels. Our study identified MCF-7, T47-D, and MDA-MB-231 cells as useful models for the examination of the molecular mechanisms involved in the ectopic expression of BSP in breast malignant lesions.
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PMID:Detection of bone sialoprotein in human breast cancer tissue and cell lines at both protein and messenger ribonucleic acid levels. 876 20

Sex hormone binding globulin (SHBG) is a high affinity binding protein for estrogens and androgens. SHBG has been found in breast tissue and cell lines through immunostaining. The goal of this series of experiments was to determine whether mRNA for SHBG is expressed in breast cancer cell lines and tumor tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect SHBG and beta-2 microglobulin (control for tissue extractions). Three breast cancer cell lines, ZR-75-1, MCF-7, and MDA-MB-231 and 56 breast tissue samples were collected and analysed for SHBG mRNA expression. mRNA was successfully extracted from 30 of these breast tissue samples. SHBG mRNA was detected in ZR-75-1, MCF-7 and MDA-MB-231 cells, and in 11 of the breast tissue samples. Two PCR products were routinely amplified from the breast cancer cell line RNA, one at approximately 500 bp and another at approximately 300 bp. The DNA sequence of the 300 bp PCR produce was consistent with alternate splicing of the SHBG mRNA, where exon 7 is deleted, and is accompanied by a point deletion at the beginning of exon 8. SHBG protein production from the three breast cancer cell lines was detected by immunoprecipitation using an affinity purified SHBG antibody. SHBG mRNA was found in 11 of 30 samples of breast tissue. Some samples expressed only the 500 bp or the 300 bp PCR product, whereas others expressed both PCR products. The presence of SHBG mRNA in these samples was not associated with either the presence or absence of steroid receptors. SHBG mRNA is thus expressed in breast cancer cell lines, and in some breast tissue samples.
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PMID:Sex hormone binding globulin mRNA in human breast cancer: detection in cell lines and tumor samples. 901 Mar 21

GnRH receptors belong to the family of G protein-coupled receptor proteins and have been localized to the anterior pituitary, brain and reproductive organs as well as many steroid-dependent tumor tissues. Recently, cDNAs for the GnRH receptors of several species including the human have been cloned. To determine the structure of the gene encoding the human GnRH receptor, we isolated the receptor gene clones from the human genomic libraries. Comparison of the genomic and cDNA sequences revealed that the human GnRH receptor gene is composed of three exons and two introns and spans over 20 kb in size. Exon 1 encodes the 5' untranslated sequence and nucleotide +1 to +522 in the open reading frame, exon 2 encodes nucleotide +523 to +742 and exon 3 encodes nucleotide +743 to +987 in the open reading frame as well as the 3' untranslated sequence. Southern blot analysis of genomic DNA and localization of the GnRH receptor gene to a single site on human chromosome 4 (4q13) indicate the presence of a single copy of the gene in the human genome. Several regulatory sequences for various hormones and other regulatory factors were identified, including PEA-3, AP-1, AP-2, and Pit-1 sites. In addition, glucocorticoid/progesterone response element thyroid hormone response element, and cAMP response element sequences were identified. Reverse transcriptase-primer extension and 5' RACE analysis of the human pituitary RNA demonstrated the presence of multiple transcriptional start sites upstream of the translational start site. Analysis of the 5' flanking region of the gene also revealed the presence of multiple TATA and CAAT sequences. The finding of multiple transcriptional start sites raises the possibility of tissue-specific regulation and the existence of variable size transcripts. Chimeras containing 1.26 kb (-534 to 728) of the 5' flanking region of the receptor gene and the luciferase (Luc) gene expressed a significant luciferase activity when transfected into a human endometrial tumor cell line (HEC-1A) and a breast tumor cell line (MCF-7) but not in a mouse pituitary gonadotrope cell line (alpha T3-1), suggesting the existence of multiple promoter elements in the gene. These findings indicate a multiplicity of regulation of expression of the GnRH receptor and provide the substrate for detailed investigation in the reproductive system.
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PMID:Molecular structure of the human gonadotropin-releasing hormone receptor gene. 1102 3

Basic fibroblast growth factor (bFGF) is a classical mitogen in fibroblasts and endothelial cells. Our previous studies have demonstrated that bFGF inhibits the growth of MCF-7 human breast cancer cells. The aim of the present study was to examine the effect of bFGF on cis-diamminedichloroplatinum(cisplatin)-induced cytotoxicity in MCF-7 breast cancer cells as compared to normal endothelial cells. MCF-7/NCF cells transduced with a vector expressing the bFGF gene and overexpressing its product, and MCF-7/N2 cells transduced with the backbone vector were incubated with a combination of bFGF and cisplatin for 5 days; results were compared with those obtained with bovine aortic endothelial cells. Cell proliferation was assessed with the sulforhodamine B colorimetric cytotoxicity assay. Apoptosis was quantitatively determined by flow-cytometric analysis for DNA damage and the apoptotic death assay for DNA fragmentation, and qualitatively by electron microscopy. Reverse transcriptase/polymerase chain reaction analysis and an enzyme immunoassay were used to determine the mRNA and protein level, respectively, of the anti-apoptotic bcl-2 gene product. We found that bFGF enhanced cisplatin-induced cytotoxicity in MCF-7 breast cancer sublines. bFGF enhanced proliferation of normal endothelial cells and did not increase cisplatin-induced cytotoxicity. This effect was accompanied by down-regulation of the anti-apoptotic protooncogene bcl-2 and the enhancement of cisplatin-induced apoptosis. We suggest that the improved understanding of the role of bFGF in the differential modulation of the response of breast cancer and normal endothelial cells to chemotherapy may enable active intervention to alter the therapeutic ratio favorably in breast cancer patients.
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PMID:Basic fibroblast growth factor potentiates cisplatinum-induced cytotoxicity in MCF-7 human breast cancer cells. 1047 68

We have investigated the mechanisms by which radiation inhibits proliferation of human breast cancer cells in culture. Radiation, within the dose range used for treatment of humans, decreased the rate of proliferation of estrogen-independent MDA-MB-231 cells more effectively than it did that of estrogen-dependent MCF-7 cells. The rate of proliferation of MDA-MB-231 cells was also inhibited to a greater extent than that of MCF-7 cells by purified TGFB1. Using an ELISA specific for activated TGFB1, we found that conditioned medium from irradiated MDA-MB-231 or MCF-7 cells contained twofold more TGFB1 than that from nonirradiated cells. Conditioned medium from irradiated breast cancer cells, but not from nonirradiated cells, inhibited the growth of untreated MDA-MB-231 cells. The inhibitory activity was blocked by an anti-TGFB1 neutralizing antibody. An approximately twofold increase in the TGFB1 mRNA in irradiated cells compared to controls was found using semiquantitative reverse-transcriptase PCR. Last, the mRNA for insulin-like growth factor binding protein 3, a reported target of the cell inhibitory activity of TGFB1, was increased threefold upon irradiation. Our results demonstrate that the TGFB1 is increased after irradiation and that the activation of the TGFB1 signaling pathway may sensitize cells to the effects of radiation.
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PMID:Regulation of transforming growth factor beta1 by radiation in cells of two human breast cancer cell lines. 1052 25

Indole-3-carbinol (I3C), a compound naturally occurring in Brassica vegetables, can induce a G(1) cell cycle arrest of human MCF-7 breast cancer cells that is accompanied by the selective inhibition of cyclin-dependent kinase 6 (CDK6) expression. Reverse transcriptase-polymerase chain reaction analysis of CDK6 mRNA decay rates revealed that I3C had no effect on CDK6 transcript stability. We report the first identification and functional characterization of the CDK6 promoter in order to determine whether I3C inhibits CDK6 transcription. In MCF-7 cells stably transfected with CDK6 promoter-linked luciferase reporter plasmids, I3C inhibited CDK6 promoter activity in an I3C-specific response that was not a consequence of the growth-arrested state of the cells. Deletion analysis revealed a 167-base pair I3C-responsive region of the CDK6 promoter between -805 and -638. Site-specific mutations within this region revealed that both Sp1 and Ets-like sites, which are spaced 5 base pairs apart, were necessary for I3C responsiveness in the context of the CDK6 promoter. Electrophoretic mobility shift analysis of protein-DNA complexes formed with nuclear proteins isolated from I3C-treated and -untreated cells, in combination with supershift assays using Sp1 antibodies, demonstrated that the Sp1-binding site in the CDK6 promoter forms a specific I3C-responsive DNA-protein complex that contains the Sp1 transcription factor. Taken together, our results suggest that I3C down-regulates CDK6 transcription by targeting Sp1 at a composite DNA site in the CDK6 promoter.
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PMID:Indole-3-carbinol inhibits CDK6 expression in human MCF-7 breast cancer cells by disrupting Sp1 transcription factor interactions with a composite element in the CDK6 gene promoter. 1129 39

We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro.
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PMID:Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes. 1167 67

Breast cancer is a serious illness affecting approximately one in nine women in the United States. Although an actual cause for breast cancer is unknown, genetic and environmental factors have been associated with its onset. Elevated levels of estrogen and heightened expression of the WNT10B proto-oncogene have been implicated in the development of human malignant breast tumors because they enhance the proliferation of mammary tissue. Two pyrethroid insecticides, sumithrin and fenvalerate, have been shown to mimic estrogenic activity in MCF-7 human breast carcinoma cells by inducing pS2 expression whereas two other pyrethroids, permethrin and d-trans allethrin do not have the same capability. To investigate if estrogen and these four pyrethroid insecticides could affect the expression of a gene related to mammary gland development, WNT10B expression in pyrethroid-treated MCF-7 cells was examined. MCF-7 cells under normal growth conditions do not express WNT10B. Reverse-transcriptase polymerase chain reaction (RT-PCR), nested PCR and Southern hybridization were employed to detect WNT10B expression. As controls, cells were treated with either ethanol, corn oil, or Vista LPA solvent. When compared to the solvent-treated controls, sumithrin, fenvalerate and estrogen treated MCF-7 cells all had increased levels of WNT10B expression. The non-estrogenic pyrethroids, d-trans allethrin and permethrin, demonstrated a similar elevation of WNT10B expression at a lower concentration, but not at the higher concentration. The results suggest that pyrethroid insecticides and estrogen can enhance the expression of the WNT10B proto-oncogene. However, since both the estrogenic and non-estrogenic substances amplified Wnt10B expression, the mechanism likely involves multiple distinct pathways.
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PMID:Effects of pyrethroid insecticides and estrogen on WNT10B proto-oncogene expression. 1243 93

Nuclear clusterin (nCLU) is an ionizing radiation (IR)-inducible protein that binds Ku70, and triggers apoptosis when overexpressed in MCF-7 cells. We demonstrate that endogenous nCLU synthesis is a product of alternative splicing. Reverse transcriptase-PCR analyses revealed that exon II, containing the first AUG and encoding the endoplasmic reticulum-targeting peptide, was omitted. Exons I and III are spliced together placing a downstream AUG in exon III as the first available translation start site. This shorter mRNA produces the 49-kDa precursor nCLU protein. Ku70 binding activity was localized to the C-terminal coiled-coil domain of nCLU. Leucine residues 357, 358, and 361 of nCLU were necessary for Ku70-nCLU interaction. The N- and C-terminal coiled-coil domains of nCLU interacted with each other, suggesting that the protein could dimerize or fold. Mutation analyses indicate that the C-terminal NLS was functional in nCLU with the same contribution from N-terminal NLS. The C-terminal coiled-coil domain of nCLU was the minimal region required for Ku binding and apoptosis. MCF-7 cells show nuclear as well as cytoplasmic expression of GFP-nCLU in apoptotic cells. Cytosolic aggregation of GFP-nCLU was found in viable cells. These results indicate that an inactive precursor of nCLU exists in the cytoplasm of non-irradiated MCF-7 cells, translocates into the nucleus following IR, and induces apoptosis.
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PMID:Synthesis and functional analyses of nuclear clusterin, a cell death protein. 1255 33


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