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Search Results: 1 - 9 of 9 matches for " immortalization "
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A novel cell culture model for studying differentiation and apoptosis in the mouse mammary gland
Katrina E Gordon, Bert Binas, Rachel S Chapman, Kathreena M Kurian, Richard W E Clarkson, A John Clark, E Birgitte Lane, Christine J Watson
Breast Cancer Research , 2000, DOI: 10.1186/bcr57
Abstract: KIM-2 cells have a characteristic luminal epithelial cell morphology and a stable, nontransformed phenotype at the semipermissive temperature of 37°C. In contrast, at the permissive temperature of 33°C the cells have an elongated spindle-like morphology and become transformed after prolonged culture. Differentiation of KIM-2 cells at 37°C, in response to lactogenic hormones, results in the formation of polarized dome-like structures with tight junctions. This is accompanied by expression of the milk protein genes that encode β-casein and whey acidic protein (WAP), and activation of the prolactin signalling molecule, signal transducer and activator of transcription (STAT)5. Fully differentiated KIM-2 cultures at 37°C become dependent on lactogenic hormones for survival and undergo extensive apoptosis upon hormone withdrawal, as indicated by nuclear morphology and flow cytometric analysis. KIM-2 cells can be genetically modified by stable transfection and clonal lines isolated that retain the characteristics of untransfected cells.KIM-2 cells are a valuable addition, therefore, to currently available lines of mammary epithelial cells. Their capacity for extensive differentiation in the absence of exogenously added basement membrane, and ability to undergo apoptosis in response to physiological signals will provide an invaluable model system for the study of signal transduction pathways and transcriptional regulatory mechanisms that control differentiation and involution in the mammary gland.With each successive pregnancy, the mammary gland completes a cycle of growth, functional differentiation and involution. These processes are of great importance in biology in their own right, but they also provide an example of how proliferation, differentiation and apoptosis are integrated into the organization of a complex three-dimensional tissue unit, whose function changes with time. The growth and development of ductal and alveolar structures during pregnancy is dependent on
Spontaneous immortalization of human dermal microvascular endothelial cells
Ming Jiang,Yongfen Min,Laura DeBusk,Suzanne Fernandez
World Journal of Stem Cells , 2010,
Abstract: AIM: To establish and characterize a spontaneously immortalized human dermal microvascular endothelial cell line, iHDME1.METHODS: We developed a spontaneous immortalization method. This approach is based on the application of optimized culture media and culture conditions without addition of any exogenous oncogenes or carcinogens. Using this approach, we have successfully established a microvascular endothelial cell line, iHDME1, from primary human dermal microvascular endothelial cells. iHDME1 cells have been maintained in culture dishes for more than 50 passages over a period of 6 mo. Using a GFP expressing retrovirus, we generated a GFP-stable cell line (iHDME1-GFP).RESULTS: iHDME1 retain endothelial morphology and uniformly express endothelial markers such as VEGF receptor 2 and VE-cadherin but not α-smooth muscle actin (α-SM-actin) and cytokeratin 18, markers for smooth muscle cells and epithelial cells respectively. These cells retain endothelial properties, migrate in response to VEGF stimulation and form 3-D vascular structures in Matrigel, similar to the parental cells. There is no significant difference in cell cycle profile between the parental cells and iHDME1 cells. Further analysis indicates enhanced stemness in iHDME1 cells compared to parental cells. iHDME1 cells display elevated expression of CD133 and hTERT.CONCLUSION: iHDME1 cells will be a valuable source for studying angiogenesis.
Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers
Khanit Sa-ngiamsuntorn, Adisak Wongkajornsilp, Kanda Kasetsinsombat, Sunisa Duangsa-ard, Lalana Nuntakarn, Suparerk Borwornpinyo, Pravit Akarasereenont, Somchai Limsrichamrern, Suradej Hongeng
BMC Biotechnology , 2011, DOI: 10.1186/1472-6750-11-89
Abstract: The hepatocyte-like cells have been developed from hTERT plus Bmi-1-immortalized human mesenchymal stem cells to substitute the primary hepatocytes. The hepatocyte-like cells had polygonal morphology and steadily produced albumin, glycogen, urea and UGT1A1 beyond 6 months while maintaining proliferative capacity. Although these hepatocyte-like cells had low basal expression of CYP450 isotypes, their expressions could be extensively up regulated to 80 folds upon the exposure to enzyme inducers. Their inducibility outperformed the classical HepG2 cells.The hepatocyte-like cells contained the markers of hepatocytes including CYP450 isotypes. The high inducibility of CYP450 transcripts could serve as a sensitive model for profiling xenobiotic-induced expression of CYP450.Xenobiotic biotransformation has been classified into 2 phases. The majority of phase I biotransformation was implemented by cytochrome P450 (CYP450) family with 8 major isotypes in human[1]. Each isotype has overlapped spectra of substrates and catalyzes multiple reactions. Activations or suppressions of certain isotypes as a result of precipitant drugs have been associated with several clinically important drug interactions[1]. The phase II biotransformation involved several conjugation reactions (e.g., sulfonation, glucuronidation, acetylation, methylation and glutathione conjugation). These conjugations attach new functional groups to the xenobiotic that had gone through phase I metabolism[2]. The CYP450 isotypes in rodents are often different in gene regulation and enzymatic activity from those in human and thus cannot reliably predict the toxicity or metabolic profiles of xenobiotics in human[3].The idealistic cell culture model to simulate in vivo biotransformation of xenobiotics is the use of primary human hepatocytes. However, the acquisition of normal human hepatocytes is cumbersome with ethical as well as biological considerations. The cultured cells are short lived and have to be swiftly pre
Differential transforming activity of the retroviral Tax oncoproteins in human T lymphocytes
Tong Ren,Hua Cheng
Frontiers in Microbiology , 2013, DOI: 10.3389/fmicb.2013.00287
Abstract: Human T cell leukemia virus type 1 and type 2 (HTLV-1 and -2) are two closely related retroviruses. HTLV-1 causes adult T cell leukemia and lymphoma, whereas HTLV-2 infection is not etiologically linked to human disease. The viral genomes of HTLV-1 and -2 encode highly homologous transforming proteins, Tax-1 and Tax-2, respectively. Tax-1 is thought to play a central role in transforming CD4+ T lymphocytes. Expression of Tax-1 is crucial for promoting survival and proliferation of virally infected human T lymphocytes and is necessary for initiating HTLV-1-mediated oncogenesis. In transgenic mice and humanized mouse model, Tax-1 has proven to be leukemogenic. Although Tax-1 is able to efficiently transform rodent fibroblasts and to induce lymphoma in mouse model, it rarely transforms primary human CD4+ T lymphocytes. In contrast, Tax-2 efficiently immortalizes human CD4+ T cells though it exhibits a lower transforming activity in rodent cells as compared to Tax-1. We here discuss our recent observation and views on the differential transforming activity of Tax-1 and Tax-2 in human T cells.
Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor
Lima, E.M.;Rissino, J.D.;Harada, M.L.;Assump??o, P.P.;Demachki, S.;Guimar?es, A.C.;Casartelli, C.;Smith, M.A.C.;Burbano, R.R.;
Brazilian Journal of Medical and Biological Research , 2004, DOI: 10.1590/S0100-879X2004001200008
Abstract: gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. virtually all the established cell lines of gastric neoplasia were developed in asian countries, and western countries have contributed very little to this area. in the present study we describe the establishment of the cell line acp01 and characterize it cytogenetically by means of in vitro immortalization. cells were transformed from an intestinal-type gastric adenocarcinoma (t4n2m0) originating from a 48-year-old male patient. this is the first gastric adenocarcinoma cell line established in brazil. the most powerful application of the cell line acp01 is in the assessment of cytotoxicity. solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. the acp01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. these data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.
Porcine hepatocyte isolation and reversible immortalization mediated by retroviral transfer and site-specific recombination
Fan-Ying Meng, Zhi-Shui Chen, Meng Han, Xin-Peng Hu, Xing-Xing He, Yong Liu, Wen-Tao He, Wei Huang, Hui Guo, Ping Zhou
World Journal of Gastroenterology , 2010,
Abstract: AIM: To develop a hepatocyte cell line, we immortalized primary porcine hepatocytes with a retroviral vector SSR#69 containing the Simian Virus 40 T antigen (SV40Tag).METHODS: We first established a method of porcine hepatocyte isolation with a modified four-step retrograde perfusion technique. Then the porcine hepatocytes were immortalized with retroviral vector SSR#69 expressing SV40T and hygromycin-resistance genes flanked by paired loxP recombination targets. SV40T cDNA in the expanded cells was subsequently excised by Cre/LoxP site-specific recombination.RESULTS: The resultant hepatocytes with high viability (97%) were successfully immortalized with retroviral vector SSR#69. One of the immortalized clones showed the typical morphological appearance, TJPH-1, and was selected by clone rings and expanded in culture. After excision of the SV40T gene with Cre-recombinase, cells stopped growing. The population of reverted cells exhibited the characteristics of differentiated hepatocytes.CONCLUSION: In conclusion, we herein describe a modified method of hepatocyte isolation and subsequently established a porcine hepatocyte cell line mediated by retroviral transfer and site-specific recombination.
Ectopic osteogenesis with immortalized human bone marrow stromal stem cells and heterologous bone
Yong Teng,Yunyu Hu,Xusheng Li,Yucheng Guan
Journal of Applied Biomedicine , 2011,
Abstract: To resolve the problem of the insufficient availability of seed cells and to provide seed cells for tissue engineering research, an immortalized human bone marrow stromal stem cell line (MSCxj cells) was established in our department to investigate the ectopic osteogenesis of MSCxj cells.MSCxjs were grown with a heterogeneous bone scaffold for 48 h. Three groups were included: A: MSCxjs of 35 PDs were maintained with heterogeneous bone; B: MSCxjs of 128 PDs were maintained with heterogeneous bone; and C: heterogeneous bone alone. Tetracycline fluorescence staining, H&E staining, and ponceau staining, immunohistochemistry and bone histomorphometry were performed. At the same time, scanning electron microscopy was conducted to detect the growth of MSCxjs and heterogeneous bone.Scanning electron microscopy showed favorable adherence of MSCxjs to heterogeneous bone. A large number of newly generated filamentous extracellular matrix and fine granular materials were found to cover the cells. The results from staining showed that the osteogenesis was not obvious in group A/B 4 weeks after transplantation. Eight weeks after implantation, osteoid matrix deposition was noted in and around the heterogeneous bone in group A/B. Twelve weeks after implantation, osteogenesis was increased in group A/B. There were no significant differences in the time course for bone formation and the amount of newly generated bone between group A/B.Like primary hBMSCs, MSCxj cells have favourable ectopic osteogenesis and can be applied as seeded cells in bone tissue engineering.
Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor
Lima E.M.,Rissino J.D.,Harada M.L.,Assump??o P.P.
Brazilian Journal of Medical and Biological Research , 2004,
Abstract: Gastric cancer is the second most frequent type of neoplasia and also the second most important cause of death in the world. Virtually all the established cell lines of gastric neoplasia were developed in Asian countries, and western countries have contributed very little to this area. In the present study we describe the establishment of the cell line ACP01 and characterize it cytogenetically by means of in vitro immortalization. Cells were transformed from an intestinal-type gastric adenocarcinoma (T4N2M0) originating from a 48-year-old male patient. This is the first gastric adenocarcinoma cell line established in Brazil. The most powerful application of the cell line ACP01 is in the assessment of cytotoxicity. Solid tumor cell lines from different origins have been treated with several conventional and investigational anticancer drugs. The ACP01 cell line is triploid, grows as a single, non-organized layer, similar to fibroblasts, with focus formation, heterogeneous division, and a cell cycle of approximately 40 h. Chromosome 8 trisomy, present in 60% of the cells, was the most frequent cytogenetic alteration. These data lead us to propose a multifactorial triggering of gastric cancer which evolves over multiple stages involving progressive genetic changes and clonal expansion.
Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies
Min Gong, Yang Bi, Wei Jiang, Yun Zhang, Li Chen, Nali Hou, Youxue Liu, Xiaoping Wei, Jie Chen, Tingyu Li
Journal of Biomedical Science , 2011, DOI: 10.1186/1423-0127-18-87
Abstract: The retroviral vector SSR#69 expressing simian virus 40 large T (SV40T) antigen was used to construct IMSCs. IMSCs were identified by flow cytometry to detect cell surface makers. To investigate proliferation and differentiation potential of IMSCs, cell growth curve determination and mesodermal trilineage differentiation tests were performed. Neuronal differentiation characteristics of IMSCs were detected in vitro. Before IMSCs transplantation, we excluded its tumorigenicity in nude mice firstly. The Morris water maze tests and shuttle box tests were performed five weeks after HIBD models received cells transplantation therapy.In this study, reversible IMSCs were constructed successfully and had the similar morphology and cell surface makers as primary MSCs. IMSCs possessed better ability of proliferation and anti-senescence compared with primary MSCs, while maintained multilineage differentiation capacity. Neural-like cells derived from IMSCs had similar expressions of neural-specific genes, protein expression patterns and resting membrane potential (RMP) compared with their counterparts derived from primary MSCs. There was no bump formation in nude mice subcutaneously injected with IMSCs. IMSCs played same role as primary MSCs to improve learning ability and spatial memory of HIBD rats.IMSCs not only retain their features of primary MSCs but also possess the ability of high proliferation and anti-senescence. IMSCs can definitely be induced to differentiate into neuronal cells in vitro and take the place of primary MSCs for cell transplantation therapy without tumorigenesis in vivo. The stable cell line is particularly useful and valuable as an alternative to MSCs in neuronal differentiation and neuroregeneration associated studies.Mesenchymal stem cells (MSCs) are adult stem cells present in many tissues, such as bone marrow, adipose tissue and peripheral blood. They are able to differentiate into multiple mesodermal lineage cells, such as osteocytes, chondrocytes
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