UNIPROT:Q9NRP7 - FACTA Search

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Query: UNIPROT:Q9NRP7 (fused)
58,367 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human selenium-dependent glutathione peroxidase (hGPx1) (EC 1.11.1.9) is thought to be involved in many critical cellular functions as a result of its role in glutathione-mediated reduction of toxic peroxides, and it is implicated as a mechanism of resistance against oxygen free radicals. Previous studies have demonstrated that the gene encoding hGPx1 (hgpx1) is more highly expressed in multidrug-resistant AdrR MCF-7 human breast cancer cells than in the parental WT MCF-7 cell line. In order to further study the transcriptional regulation of hgpx1, we have cloned the genomic hgpx1 gene and determined its nucleotide sequence. The 2550-base pair (bp) 5'-flanking sequence of hgpx1 contained the terminal 511 bp of the 3' end of a previously reported rhoH12 cDNA (Yeramian, P., Chardin, P., Madaule, P., and Tavitian, A. (1987) Nucleic Acids Res. 15, 1989), a ras-related oncogene. Further downstream from rhoH12, but before the start of transcription of hgpx1, RNase protection analysis revealed a transcribed sequence of at least 270 bp which we have called mid. RNA transcripts homologous to both rhoH12 (1.8 and 1.5 kilobase pairs (kb)) and mid (1.8 kb) are also more highly expressed in AdrR MCF-7 cells than in WT MCF-7 cells. We screened an AdrR MCF-7 cDNA library with the mid sequence and isolated a partial cDNA clone which contains both mid and rhoH12 sequences and is colinear with the genomic sequence which extends from 10 bp 3' to the rhoH12 stop codon to 810 bp 5' to the start of transcription of hgpx1. The start of transcription of hgpx1 in AdrR MCF-7 cells was determined by primer extension analysis. The promoter and 2 kb of the 5'-flanking sequence of hgpx1 was fused to the bacterial chloramphenicol acetyltransferase gene (hGPx1-CAT1). Analysis of deletion constructs of hGPx1-CAT1 revealed three possible cis-acting regulatory regions. The transcriptional regulation of hgpx1 was examined using the hGPx1-CAT hybrid genes and nuclear run-on studies. We found no evidence that increased mRNA transcript formation could account for different levels of hgpx1 RNA either in different breast cancer cell lines or in response to selenium.
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PMID:Structure and function of the 5'-flanking sequence of the human cytosolic selenium-dependent glutathione peroxidase gene (hgpx1). 155 8

A new combination of fluorescent dyes (rhodamine 123 and hydroethidine) was used to internally label hybridoma fusion partners. Murine hybridoma 520C9 (recognizing human c-erbB-2) was labeled with hydroethidine. Murine hybridoma 3G8 (recognizing human Fc gamma receptor III) was labeled with rhodamine 123, and verapamil was used to block rhodamine efflux via P-glycoprotein. Viability assays showed little cytotoxicity from these dyes at the concentrations used. The labeled cells were fused with polyethylene glycol, sorted for dual fluorescence on an Epics V cell sorter, and cloned. Hybrid hybridomas producing bispecific antibodies were selected for ability to promote lysis of SK-Br-3 breast cancer cells by human mononuclear cells. Several positive clones were obtained and shown to have a double content of DNA. Bispecific antibody produced by subclone 2B1 was purified by anion exchange chromatography and shown to bind both tumor cells and Fc gamma R III bearing cells. Using two parameter flow cytometric analysis, we were able to measure a 'bridging' effect of this bispecific antibody, which caused formation of complexes between PMNs and SK-Br-3 cells. Either parental antibody could compete with bispecific antibody to block such complexing. This fusion method provides several advantages over other techniques presently used (speed, convenience, low toxicity and automatic exclusion of dead cells) and can be applied to produce other hybrid hybridomas.
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PMID:Selection of hybrid hybridomas by flow cytometry using a new combination of fluorescent vital stains. 171 68

Loss of cell-cell adhesion in carcinoma cells may be an important step in the acquisition of an invasive, metastatic phenotype. We have examined the expression of the epithelial-specific cell adhesion molecule uvomorulin (E-cadherin, cell-CAM 120/80, L-CAM) in human breast cancer cell lines. We find that fibroblastoid, highly invasive, vimentin-expressing breast cancer cell lines do not express uvomorulin. Of the more epithelial-appearing, less invasive, keratin-expressing breast cancer cell lines, some express uvomorulin, and some do not. We examined the morphologies of the cell lines in the reconstituted basement membrane matrix Matrigel and measured the ability of the cells to traverse a Matrigel-coated filter as in vitro models for detachment of carcinoma cells from neighboring cells and invasion through basement membrane into surrounding tissue. Colonies of uvomorulin-positive cells have a characteristic fused appearance in Matrigel, whereas uvomorulin-negative cells appear detached. Cells which are uvomorulin negative and vimentin positive have a stellate morphology in Matrigel. We show that uvomorulin is responsible for the fused colony morphology in Matrigel since treatment of uvomorulin-positive MCF-7 cells with an antibody to uvomorulin caused the cells to detach from one another but did not induce invasiveness in these cells, as measured by their ability to cross a Matrigel-coated polycarbonate filter in a modified Boyden chamber assay. Two uvomorulin-negative, vimentin-negative cell lines are also not highly invasive as measured by this assay. We suggest that loss of uvomorulin-mediated cell-cell adhesion may be one of many changes involved in the progression of a carcinoma cell to an invasive phenotype.
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PMID:Cell adhesion molecule uvomorulin expression in human breast cancer cell lines: relationship to morphology and invasive capacities. 179 31

Monoclonal antibodies which bind selectively to cancer cells are currently used for tumor localization and for targeting cytotoxic reagents. The success of these approaches depends on the specificity of the antibody and its reactivity to a majority of the tumor samples. Frequently, monoclonal antibodies are generated by immunizing mice with antigenic preparations from a single tumor cell line. Antibodies generated under these conditions often react to a narrow range of tumors. In the present study, mice were immunized with multiple ovarian cancer cell lines in a sequential manner to amplify the immune response against common antigenic determinants expressed in these cell lines. Spleen cells from the immunized mice were then fused with NS-1 myeloma cells to establish hybridomas. Two cell lines were selected on the basis of their selective reactivity to ovarian cancer cells after extensive screening. Monoclonal antibodies OVX1 and OVX2 bound to all 5 ovarian carcinoma cell lines tested and did not bind to normal fibroblast cells. These antibodies recognized a unique antigenic determinant present in ovarian and breast cancer cells. Cross-blocking studies showed that the binding of OVX1 and OVX2 is not displaceable by 10 other previously described anti-ovarian antibodies including OC125. In immunocytochemical studies, OVX1 reacted to a majority of ovarian cancer tissues (17 of 20) and did not bind to normal ovarian tissues. Preliminary results indicate that OVX1 and OVX2 antibodies are directed to a high molecular weight antigen. These antibodies could be used in the preparation of cytotoxic conjugates.
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PMID:Development of two new monoclonal antibodies reactive to a surface antigen present on human ovarian epithelial cancer cells. 185 17

Murine breast cancer cell lines were developed to selectively invade the peritoneum while they proliferated in ascites form in the abdominal cavity. In a dominant form of invasion, tumor cells showed special affinity for elastin fibers and squeezed through narrow gaps in the elastic fiber meshwork of the stroma. Even in fixed tissue, such cells could be recognized as being in the process of invasive migration because of their dumbbell shape. This appearance was similar to that of diapedetic blood cells traversing bone marrow sinus endothelium. Three-dimensional STERECON graphics reconstruction from serial thick sections of 44 such cells was carried out. The reconstructions showed that, in mid-penetration, the cells spread extensively over the exterior surface of the elastic fiber meshwork. The cell surface contact of these forward projections was mainly with the elastic fiber outer coat of microfibrils, but small areas of the cell surface also fused directly to inner-core elastin. The morphological rearrangement of the cytoskeleton was minimal in both types of attachment areas. The location of these forward facing attachments is consistent with mechanisms for pulling the invasive cell through the gap. Lamellopodia formation and clustering of cytoplasmic organelles occurred more commonly at the forward-facing part of the cell. Morphometry of the reconstructions showed that a contraction of the whole cell occurred during the squeezing/migration process suggestive of an additional pushing process. However, our invasive cell lines showed marked differences in the degree of cell shrinkage. The process of adhesion and squeezing of tumor cells through elastin meshworks in vivo is clearly a complex phenomenon. Changes in cell surface activity appear to play a significant role in establishing the necessary 'foothold' component of invasion and, possibly, in the generation of tractive force as well.
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PMID:Organelle rearrangement and cell volume changes during squeezing invasion of peritoneal elastic lamina by targeted murine breast carcinoma cells. 188 32

Spleen cells from inbred Biozzi mice, immunized against the human breast cancer cell line T47D, were fused with murine myeloma SP2O cells to generate monoclonal antibodies. One of these, 1BE12, of IgM isotype, reacted with five of six human breast tumor cell lines, while no binding was detectable with normal lymphocytes, RBC, or fibroblasts. The antigen recognized by monoclonal antibody 1BE12 was localized on the surface of T47D and MCF7 cells and was detected in cell-free supernatants of cultures. The antigen was found also on the surface of milk secretory cells. Immunohistochemical staining of frozen and paraffin-embedded sections of human tissues showed apical polarized reactivity in normal breast glands, while in all breast cancers staining was either cytoplasmic or membranous and heterogeneously distributed. Immunostaining was also observed in some other normal epithelia, including salivary gland, gastroduodenal mucosa, exocrine pancreas, and cervix. The antigen was not detectable in secretory endometrium, whereas proliferative endometrium was strongly stained. Colon carcinoma, and cancers of the bladder and endometrium were strongly reactive. No staining was detected in melanoma, lymphoma, mesothelioma, non-small cell lung carcinoma, and thyroid, renal, and ovarian carcinomas. Lectin absorption of MCF7 membrane extracts reduced 1BE12 binding. A large reduction in 1BE12 reactivity was observed after digestion of T47D and MCF7 membrane extracts with proteases. Treatment with sodium periodate resulted in complete loss of antigenicity, while neuraminidase treatment did not affect 1BE12 binding. These findings suggest that the 1BE12 epitope is expressed on the carbohydrate moiety of a glycoprotein and does not contain sialic acid. Immunoblotting of the perchloric acid-soluble fraction of MCF7 membrane extracts after electrophoresis in 1% agarose detected the antigen as a high molecular weight species (Mr greater than 900,000). The antigen was purified by perchloric acid extraction of MCF7 membrane preparations followed by affinity chromatography on 1BE12 antibody coupled to Sepharose-4B and gel exclusion fast protein liquid chromatography. No reactivity of the purified material was found with monoclonal antibodies directed against human milk fat globule membrane-associated mucins HMFG1 and DF3.
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PMID:Characterization and distribution in human tissues of a glycoproteic antigen defined by monoclonal antibody 1BE12 raised against the human breast cancer cell line T47D. 222 61

Several hydroxylated derivatives of tamoxifen were tested for their effects on the growth of T47D human breast cancer cells in vitro. Compounds containing a fused seven-membered ring were used to prevent isomerization of the triphenyl-ethylenes at the double bond. This stable structure permitted the determination of the activity of the cis and trans forms of tamoxifen and the true activity of two of its metabolites, 4-hydroxytamoxifen and metabolite E. Estradiol stimulates the growth of T47D cells 3-4-fold over control after 6 days of treatment (EC50 = congruent to 3 x 10(-12) to 3 x 10(-11) M, depending upon the particular experiment). The fixed ring form of the trans isomer of tamoxifen is an antiestrogen, whereas the cis isomer is estrogenic. Fixed ring-trans-4-hydroxytamoxifen is a potent antiestrogen, and its cis isomer is a weak antiestrogen (IC50 congruent to 4 x 10(-8) to 2 x 10(-7) M). The fixed ring form of trans-metabolite E (tamoxifen without the dimethylaminoethane side chain) is only a weak partial estrogen agonist, whereas the fixed ring derivative of its cis isomer is a potent estrogen agonist (EC50 congruent to 4 x 10(-12) to 1 x 10(-11) M). These studies have determined the true biological activities of the hydroxylated derivatives of tamoxifen. This information will be valuable for the development of drug receptor models and will be particularly useful when the three-dimensional structure of the receptor complex is determined.
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PMID:Structure-function relationships of hydroxylated metabolites of tamoxifen that control the proliferation of estrogen-responsive T47D breast cancer cells in vitro. 223 1

Spleen cells from Balb/c mice immunized with human breast cancer cells (MCF-7) were fused with murine myeloma SP2/0 cells. Screening of the monoclonal antibodies produced was carried out on glutaraldehyde fixed cells coated on microtiterplates. An initial evaluation of the specificity was obtained by comparing the binding of the monoclonal antibodies to MCF-7 cells with the binding to human peripheral blood lymphocytes. Eight monoclonal antibodies reacting with different epitopes on the MCF-7 cells were obtained. On the basis of their clonal origin, isotype and reaction pattern towards the MCF-7 cells these monoclonal antibodies were subdivided into two classes. Both groups of antibodies reacted with fixed and unfixed MCF-7 cells. The cellular distribution of the antigens recognized by the monoclonal antibodies was determined. To check for specificity a panel of different cells (of human and animal origin) was evaluated by immunocytochemical techniques.
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PMID:Specific monoclonal antibodies reacting with human breast cancer cells. 243 33

Lymph node lymphocytes from patients with primary lung cancer were immortalized with Epstein-Barr virus, and culture supernatants were screened for cell-surface reactivity against allogeneic cancer cell lines. The percentage of wells containing detectable antibodies in initial screening ranged from 1 to 17%, but the vast majority of the cultures lost antibody activity on subsequent expansion. Two antibody-secreting clones, J309 and D579, derived from separate individuals and reactive with anaplastic lung cancer cell lines, were successfully expanded and fused with the NS-1 mouse myeloma cell line. The antibodies produced by these clones exhibited identical restricted serologic reactivity against cultured cell lines and detected a carbohydrate antigen present in the neutral glycolipid fraction of MCF-7 breast cancer cells. Serologic, immunochemical, and chemical analyses revealed that the antigen recognized by antibodies J309 and D579 is galactosylgloboside [Gal(beta 1----3)GalNAc(beta 1----3)Gal(alpha 1----4)Gal(beta 1----4)- GlcCer]. Conclusions regarding the significance of these findings with respect to the biology of lung cancer await further information concerning the distribution of galactosylgloboside in normal and malignant tissues and the frequency of antibodies to this structure in normal and tumor-bearing individuals.
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PMID:Recognition of galactosylgloboside by monoclonal antibodies derived from patients with primary lung cancer. 283 67

Altered steroid responsiveness leads to various pathological conditions and is a particular problem for the treatment of cancers arising in steroid-sensitive cells. To develop cellular model systems for the analysis of the molecular mechanisms mediating altered steroid responses, we have analyzed the inducibility of a steroid-responsive promoter in different cell lines. In vitro constructs containing the mouse mammary tumor virus promoter fused to the herpes simplex virus thymidine kinase gene or the bacterial neo gene were transfected into four different cell lines [Rat-2, CHO chinese hamster ovary cells, F9, and T47D). Thymidine kinase+ clones and neo-resistant clones were selected in the presence of dexamethasone (dex) and/or other steroid hormones. We find that the mouse mammary tumor virus promoter activity is completely dependent on the presence of dex in Rat-2 cells but is constitutively active in CHO cells and is inactive in F9 teratocarcinoma cells in the presence and absence of dex. In the human breast cancer cell line T47D, we observe no response to dex but do observe an inducibility by progesterone. Examination of glucocorticoid receptors in these cell lines showed that Rat-2, CHO, and F9 cells contain sufficient receptors to allow a hormonal response, whereas in T47D cells several glucocorticoid binding activities appear to be present. Our results indicate that the presence of receptor in cells is not always sufficient to allow hormonal activation and that, in some cell lines, like CHO, other factors are present that can substitute for an activated steroid hormone receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Variable responsiveness of hormone-inducible hybrid genes in different cell lines. 285 Nov

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