The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Thomas C. Spelsberg - One of the best experts on this subject based on the ideXlab platform.
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Development and characterization of a rapidly proliferating, well-differentiated Cell Line derived from normal adult human osteoblast-like Cells transfected with SV40 large T antigen.
Journal of Bone and Mineral Research, 2009Co-Authors: Philip E. Keeting, Robert E. Scott, D. S. Colvard, Marlys Anderson, Merry Jo Oursler, Thomas C. Spelsberg, Lawrence B. RiggsAbstract:A new bone Cell Line was established by transfecting normal adult human osteoblast-like (hOB) Cells, derived from a 68-year-old woman, with the plasmid pSV3 neo. The plasmid included coding sequences and promotors for the large and small T antigens of the SV40 virus as well as resistance to the antibiotics neomycin and G418. A single antibiotic-resistant colony was located and cloned. Large tumor antigen production in the Clonal Cell Line was confirmed by indirect immunofluorescence study. Treatment with 1,25-dihydroxy-vitamin D3 increased steady-state concentrations of protein and mRNA for osteocalcin and for alkaLine phosphatase. Northern blot analyses also demonstrated the presence of mRNAs for alpha(I)-procollagen, osteopontin 1a, transforming growth factor beta, and interleukin-1 beta. The plasma membrane calcium pump and osteonectin were identified by immunocytochemical analysis. These Cells produced a matrix that mineralized when beta-glycerophosphate was added to their cultures. As assessed by functional receptor assays, both estrogen and androgen receptors were present and functional, although at low concentrations. Treatment with parathyroid hormone did not stimulate adenylate cyclase activity. Thus, these Cells are a well-differentiated, steroid-responsive Clonal Cell Line that closely approximates the phenotype of the mature osteoblast. They should serve as an exCellent model for the study of osteoblast biology.
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development and characterization of a conditionally immortalized human fetal osteoblastic Cell Line
Journal of Bone and Mineral Research, 2009Co-Authors: Steven A Harris, R J Enger, B L Riggs, Thomas C. SpelsbergAbstract:We report the establishment of a human fetal osteoblast Cell Line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaLine phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB Cells at the permissive temperature (33.5 degrees C) resulted in rapid Cell division, whereas little or no Cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB Cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB Cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB Cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the Clonal Cell Line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.
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estrogen response in the hfob 1 19 human fetal osteoblastic Cell Line stably transfected with the human estrogen receptor gene
Journal of Cellular Biochemistry, 1995Co-Authors: Steven A Harris, R J Enger, B L Riggs, K R Tau, David O Toft, Thomas C. SpelsbergAbstract:The gene coding for the human wild-type estrogen receptor (ER) was stably transfected into the human fetal osteoblastic Cell Line hFOB 1.19, a Clonal Cell Line which is conditionally immortilized with a temperature sensitive mutant of SV40 large T antigen (tsA58). Five subclones were obtained which express various levels of ER mRNA and protein. The subclone with the highest level of functional (nuclear bound) ER, hFOB/ER9, contained 3,931 (+/- 1,341) 17beta-estradiol molecules bound/nucleus as determined by the nuclear binding (NB) assay. Using the dextran coated charcoal (DCC) method, the level of total cytosolic ER measured was 204 (+/- 2) fmol/mg protein. This subclone was examined further for estradiol (E2) responsiveness. The ER expressed in hFOB/ER9 Cells was shown to be functional using a transiently transfected ERE-TK-luciferase construct. Expression of luciferase from this construct increased approximately 25-fold in hFOB/ER9 Cells following 10(-9)M E2 treatment. This effect on ERE-TK-luciferase expression was both dose and steroid dependant. Further, treatment of hFOB/ER9 Cells with 10(-9)M E2 resulted in a 2.5-4.0-fold increase in endogenous progesterone receptor (PR) levels detected by steroid binding assays, and a noticeable increase in both the A and B forms of PR by western blot assay. The establishment of this estrogen responsive human osteoblastic Cell Line should provide an exCellent model system for the study of estrogen action on osteoblast function.
Marketa Brozova - One of the best experts on this subject based on the ideXlab platform.
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establishment growth and in vivo differentiation of a new Clonal human Cell Line em g3 derived from breast cancer progenitors
Breast Cancer Research and Treatment, 2007Co-Authors: Marketa Brozova, Irena Netikova, Eva Scholzova, Jana Brezinova, Jan Ševčik, A Chaloupkova, Z Kleibl, Pavel Dundr, Pavel Veselý, M ZadinovaAbstract:A new Clonal Cell Line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The Line consisted of cuboidal Cells with occasional appearance of more differentiated branched Cells apparently involved in Cell-to-Cell communication. The EM-G3 Cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins α-6, β-1, and protein CD44 by cDNA array also pointed to the character of basal/stem Cells. In contrast, dominant Cells in the human original tumor showed the luminal character (K18+, K19+, K5−, K14−, and p63−). However, Cells with the immunocytochemical profile similar to that of cultured EM-G3 Cells were found in minor clusters in the patient’s tumor sections. The EM-G3 Cells formed limited tumors in nu/nu mice. The Cells in mouse tumors were organized in primitive ductal-like structures consisting of 1–3 large central luminal-like Cells (EMA+) surrounded by peripheral myoepithelial-like Cells (p63+/EMA−). The large central Cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 Cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 Cells were derived from a premalignant population of common progenitors of luminal and myoepithelial Cells that were immortalized in an early stage of tumorigenesis.
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2-DE analysis of a new human Cell Line EM-G3 derived from breast cancer progenitor Cells and comparison with normal mammary epithelial Cells.
Proteomics, 2007Co-Authors: Irena Selicharová, Marketa Brozova, Karel Ubik, Jan Vydra, Kateřina Smutná, Miloslav Sanda, Eva Matoušková, Eva Buršíková, Jiří JiráčekAbstract:We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized Clonal Cell Line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 Cells may represent progenitors of the mammary epithelial Cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 Line with proteins from populations of normal mammary epithelial Cells (NME), and determined the phenotype of both types of Cells. NME Cells are a mixture of both main Cell types in breast epithelia, myoepithelial and luminal Cells. The EM-G3 breast cancer Cell Line has a unique basal-like phenotype. We identified proteins that are differently expressed in these Cells. Cytokeratin 16, cytokeratin 19, squamous Cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME Cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intraCellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 Cells. The proteins up-regulated in EM-G3 Cells may represent potential protein markers of mammary epithelial Cells progenitors and may be important in early phase of carcinogenesis.
Jiří Jiráček - One of the best experts on this subject based on the ideXlab platform.
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2-DE analysis of a new human Cell Line EM-G3 derived from breast cancer progenitor Cells and comparison with normal mammary epithelial Cells.
Proteomics, 2007Co-Authors: Irena Selicharová, Marketa Brozova, Karel Ubik, Jan Vydra, Kateřina Smutná, Miloslav Sanda, Eva Matoušková, Eva Buršíková, Jiří JiráčekAbstract:We performed a 2-DE analysis of proteins of the newly established spontaneously immortalized Clonal Cell Line EM-G3 derived from a primary lesion of infiltrating ductal breast carcinoma. EM-G3 Cells may represent progenitors of the mammary epithelial Cells spontaneously immortalized in early phase of cancerogenesis. We compared the protein profile of EM-G3 Line with proteins from populations of normal mammary epithelial Cells (NME), and determined the phenotype of both types of Cells. NME Cells are a mixture of both main Cell types in breast epithelia, myoepithelial and luminal Cells. The EM-G3 breast cancer Cell Line has a unique basal-like phenotype. We identified proteins that are differently expressed in these Cells. Cytokeratin 16, cytokeratin 19, squamous Cell carcinoma antigen 1, caphepsin B and caspase 14 were predominantly expressed by NME Cells. Cytokeratin 13, isoelectric variant of annexin 5, isoelectric variant of chloride intraCellular channel protein 1, glyoxalase 1 and glutamine synthetase were predominantly expressed by EM-G3 Cells. The proteins up-regulated in EM-G3 Cells may represent potential protein markers of mammary epithelial Cells progenitors and may be important in early phase of carcinogenesis.
M Zadinova - One of the best experts on this subject based on the ideXlab platform.
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establishment growth and in vivo differentiation of a new Clonal human Cell Line em g3 derived from breast cancer progenitors
Breast Cancer Research and Treatment, 2007Co-Authors: Marketa Brozova, Irena Netikova, Eva Scholzova, Jana Brezinova, Jan Ševčik, A Chaloupkova, Z Kleibl, Pavel Dundr, Pavel Veselý, M ZadinovaAbstract:A new Clonal Cell Line, EM-G3, was derived from a primary lesion of human infiltrating ductal breast carcinoma. The Line consisted of cuboidal Cells with occasional appearance of more differentiated branched Cells apparently involved in Cell-to-Cell communication. The EM-G3 Cells, population doubling time 34 h, are dependent on the epidermal growth factor. Multicolor fluorescence in situ hybridization (mFISH) analysis demonstrated a stable diploid genome with several genetic changes. Immunocytochemical analysis of EM-G3 in vitro revealed positivity for keratins (K) K5, K14, K18, nuclear protein p63, epithelial membrane antigen (EMA) and other proteins indicative of a pattern of mammary epithelium bipotent progenitors. Detection of integrins α-6, β-1, and protein CD44 by cDNA array also pointed to the character of basal/stem Cells. In contrast, dominant Cells in the human original tumor showed the luminal character (K18+, K19+, K5−, K14−, and p63−). However, Cells with the immunocytochemical profile similar to that of cultured EM-G3 Cells were found in minor clusters in the patient’s tumor sections. The EM-G3 Cells formed limited tumors in nu/nu mice. The Cells in mouse tumors were organized in primitive ductal-like structures consisting of 1–3 large central luminal-like Cells (EMA+) surrounded by peripheral myoepithelial-like Cells (p63+/EMA−). The large central Cells gradually disintegrated, forming a pseudolumen. Apparently, EM-G3 Cells are able to partially differentiate in vivo as well as in vitro. Our results indicate that EM-G3 Cells were derived from a premalignant population of common progenitors of luminal and myoepithelial Cells that were immortalized in an early stage of tumorigenesis.
C Belloli - One of the best experts on this subject based on the ideXlab platform.
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A Clonal Cell Line (BME-UV1) as a possible model to study bovine mammary epithelial metabolism: metabolism and cytotoxicity of aflatoxin B1.
Toxicon, 2009Co-Authors: A. Altafini, P. Ormas, Anna Zaghini, C Belloli, M Caruso, Andrea Mariotti, Claudia ZizzadoroAbstract:Abstract Despite the toxicological risks to which humans and animals are exposed due to the transfer of toxic xenobiotic metabolites into milk of domestic animals, studies on the metabolizing mechanisms occurring in ruminant mammary gland are totally lacking. To investigate the possible biotransformation capabilities of a bovine mammary epithelial Cell Line (BME-UV1), monolayers were exposed to aflatoxin B1 (AFB1 – 1.0–8.0 μM). Starting from 4 h of exposure, the hydroxylate metabolite aflatoxin M1 (AFM1) was detected in media by high performance liquid chromatography. AFM1 concentration increased Linearly with time for 36–48 h and the percent biotransformation of AFB1 (2–4 μM) at 48 h was about 12–14%. Parallel cytotoxicity assays (neutral red uptake-NRU and MTT assays) were performed to investigate the possible interference of AFB1 cytotoxicity with Cellular metabolism. MTT assay (from 24 h of Cell exposure) and NRU assay (from 16 h of Cell exposure) showed time-dependent and time/concentration-dependent decrease of Cell viability, respectively, and the former assay being more successful at revealing cytotoxic effects (NRU: CC 50 at 48 h = 12.00 ± 2.66 μM; MTT: CC 50 at 72 h = 20.42 ± 7.30 μM). The results suggest that BME-UV1 Cells express metabolizing enzymes having catalytic activity, thus representing a potential in vitro model for studying biotransformation in bovine mammary gland.