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Sample records for human immortalized cholangiocytes

  1. Altered chromosome 6 in immortal human fibroblasts.

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    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  2. Altered chromosome 6 in immortal human fibroblasts

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    Hubbard-Smith, K.; Pardinas, J.R.; Jha, K.K.; Ozer, H.L. (New Jersey Medical School, Newark, NJ (United States)); Patsalis, P.; Henderson, A.S. (City Univ. of New York, NY (United States))

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. 66 refs., 6 figs., 2 tabs.

  3. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

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    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  4. Cholangiocytes and blood supply

    Institute of Scientific and Technical Information of China (English)

    Eugenio Gaudio; Antonio Franchitto; Luigi Pannarale; Guido Carpino; Gianfranco Alpini; Heather Francis; Shannon Glaser; Domenico Alvaro; Paolo Onori

    2006-01-01

    The microvascular supply of the biliary tree, the peribiliary plexus (PBP), stems from the hepatic artery branches and flows into the hepatic sinusoids. A detailed three-dimensional study of the PBP has been performed by using the Scanning Electron Microscopy vascular corrosion casts (SEMvcc) technique. Considering that the PBP plays a fundamental role in supporting the secretory and absorptive functions of the biliary epithelium, their organization in either normalcy and pathology is explored. The normal liver shows the PBP arranged around extra- and intrahepatic biliary tree.In the small portal tract PBP was characterized by a single layer of capillaries which progressively continued with the extrahepatic PBP where it showed a more complex vascular network. After common duct ligation (BDL), progressive modifications of bile duct and PBP proliferation are observed. The PBP presents a threedimensional network arranged around many bile ducts and appears as bundles of vessels, composed by capillaries of homogeneous diameter with a typical round mesh structure. The PBP network is easily distinguishable from the sinusoidal network which appears normal. Considering the enormous extension of the PBP during BDL, the possible role played by the Vascular Endothelial Growth Factor (VEGF) is evaluated. VEGF-A,VEGF-C and their related receptors appeared highly immunopositive in proliferating cholangiocytes of BDL rats. The administration of anti-VEGF-A or anti-VEGF-C antibodies to BDL rats as well as hepatic artery ligation induced a reduced bile duct mass. The administration of rVEGF-A to BDL hepatic artery ligated rats prevented the decrease of cholangiocyte proliferation and VEGF-A expression as compared to BDL control rats. These data suggest the role of arterial blood supply of the biliary tree in conditions of cholangiocyte proliferation, such as it occurs during chronic cholestasis. On the other hand,the role played by VEGF as a tool of cross-talk between cholangiocytes

  5. SV40-mediated immortalization of human fibroblasts.

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    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  6. Genes involved in immortalization of human mammary cells

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    Stampfer, Martha R.; Yaswen, Paul

    2001-09-27

    Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions--a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). Inappropriate expression of specific oncogenes in a key cellular signaling pathway (Ras, Raf) can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence-like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to overexpressed oncogenic Raf changes from being tumor-suppressive to tumor enhancing, and what are the molecular underpinnings of this response. Our data indicate: (1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of immortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited. (2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor-suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response. Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor-suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means of enforcing a Raf

  7. Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines.

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    Bao G Vu

    Full Text Available BACKGROUND: Human adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin. METHODOLOGY/PRINCIPAL FINDINGS: Primary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8. CONCLUSIONS/SIGNIFICANCE: Endotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to

  8. Immortalization of human alveolar epithelial cells to investigate nanoparticle uptake.

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    Kemp, Sarah J; Thorley, Andrew J; Gorelik, Julia; Seckl, Michael J; O'Hare, Michael J; Arcaro, Alexandre; Korchev, Yuri; Goldstraw, Peter; Tetley, Teresa D

    2008-11-01

    Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 microm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 microm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-microm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cell-associated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

  9. Immortalization of human articular chondrocytes and induction of their phenotype

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    何清义; 李起鸿; 杨柳; 许建中

    2003-01-01

    Objective To immortalize human articular chondrocytes (HACs) using gene transfection and to maintain stable phenotype of transformed HACs after induction.Methods HACs were transfected with the retroviral vector pLXSN encoding human papillomavirus 16E7 (HPV16E7), and the transformed clones were sorted and proliferated. Karyotype analysis, clone forming tests and nude mice tumor forming tests were applied to check the characteristics of the transformation. Type Ⅱ collagen of transformed chondrocytes was inducted with free serum medium (FSM) supplemented with nutridoma-sp and ascorbate. Results Immortalized HACs were isolated with fifty passages achieved. The HPV16E7 transformed cells were confirmed to be benign. Induction of FSM with nutridoma-sp and ascorbate promoted type Ⅱ collagen of transformed chondrocytes to the high levels of normal chondrocytes. Conclusion HACs transformed with HPV16E7 survive for long periods in vitro, and type Ⅱ collagen can maintain stability after induction.

  10. Immortalization of human normal and NF1 neurofibroma Schwann cells.

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    Li, Hua; Chang, Lung-Ji; Neubauer, Debbie R; Muir, David F; Wallace, Margaret R

    2016-10-01

    Neurofibromas, which are benign Schwann cell tumors, are the hallmark feature in the autosomal dominant condition neurofibromatosis 1 (NF1) and are associated with biallelic loss of NF1 gene function. There is a need for effective therapies for neurofibromas, particularly the larger, plexiform neurofibromas. Tissue culture is an important tool for research. However, it is difficult to derive enriched human Schwann cell cultures, and most enter replicative senescence after 6-10 passages, impeding cell-based research in NF1. Through exogenous expression of human telomerase reverse transcriptase and murine cyclin-dependent kinase (mCdk4), normal (NF1 wild-type), neurofibroma-derived Schwann cells heterozygous for NF1 mutation, and neurofibroma-derived Schwann cells homozygous for NF1 mutation were immortalized, including some matched samples from the same NF1 patient. Initial experiments employed retroviral vectors, while subsequent work utilized lentiviral vectors carrying these genes because of improved efficiency. Expression of both transgenes was required for immortalization. Molecular and immunohistochemical analysis indicated that these cell lines are of Schwann cell lineage and have a range of phenotypes, many of which are consistent with their primary cultures. This is the first report of immortalization and detailed characterization of multiple human NF1 normal nerve and neurofibroma-derived Schwann cell lines, which will be highly useful research tools to study NF1 and other Schwann tumor biology and conditions.

  11. Excretory/secretory products of the carcinogenic liver fluke are endocytosed by human cholangiocytes and drive cell proliferation and IL6 production.

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    Chaiyadet, Sujittra; Smout, Michael; Johnson, Michael; Whitchurch, Cynthia; Turnbull, Lynne; Kaewkes, Sasithorn; Sotillo, Javier; Loukas, Alex; Sripa, Banchob

    2015-10-01

    Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south

  12. Differentiation and apoptosis in human immortalized sebocytes.

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    Wróbel, Anna; Seltmann, Holger; Fimmel, Sabine; Müller-Decker, Karin; Tsukada, Miki; Bogdanoff, Birgit; Mandt, Nathalie; Blume-Peytavi, Ulrike; Orfanos, Constantin E; Zouboulis, Christos C

    2003-02-01

    Increased cell volume, accumulation of lipid droplets in the cytoplasm, and nuclear degeneration are phenomena indicating terminal differentiation of human sebocytes followed by holocrine secretion and cell death. The molecular pathways of natural and induced sebocyte elimination are still unknown, however. In this study, SZ95 sebocytes were found to exhibit DNA fragmentation after a 6 h culture followed by increased lactate dehydrogenase release after 24 h, indicating cell damage. With the help of morphologic studies and using Oil Red detection of cellular lipids, cell enlargement, accumulation of lipid droplets in the cytoplasm, and nuclear fragmentation could be observed under treatment with arachidonic acid. Staurosporine, a potent inhibitor of phospholipid Ca2+-dependent protein kinase, increased externalized phosphatidylserine levels on SZ95 sebocytes, detected by annexin V/propidium iodide flow cytometry, as early as after 1 h, whereas dose-dependent reduction of bcl-2 mRNA and protein expression, enhanced DNA fragmentation, and increased caspase 3 levels, detected by caspase 3 inhibitor/propidium iodide flow cytometry, were found after 6 h of treatment. SZ95 sebocyte death was detected as early as after 6 h of SZ95 sebocyte treatment with high staurosporine concentrations (10(-6)-10(-5) M). 5Alpha-dihydrotestosterone (10(-8)-10(-5) M) did not affect externalized phosphatidylserine levels and DNA fragmentation in SZ95 sebocytes but slightly decreased lactate dehydrogenase cell release. Neither acitretin nor 13-cis retinoic acid (10(-8)-10(-5) M) affected externalized phosphatidylserine levels, DNA fragmentation, and lactate dehydrogenase cell release, despite the increased caspase 3 levels under treatment with 13-cis retinoic acid. The combined staurosporine and 13-cis retinoic acid treatment enhanced DNA fragmentation in SZ95 sebocytes to the same magnitude as in cells only treated with staurosporine. In conclusion, SZ95 sebocytes in vitro undergo apoptosis

  13. Network signatures of cellular immortalization in human lymphoblastoid cell lines

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    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: jaepiljeon@hanmail.net [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  14. Bile acid interactions with cholangiocytes

    Institute of Scientific and Technical Information of China (English)

    Xuefeng Xia; Heather Francis; Shannon Glaser; Gianfranco Alpini; Gene LeSage

    2006-01-01

    Cholangiocytes are exposed to high concentrations of bile acids at their apical membrane. A selective transporter for bile acids, the Apical Sodium Bile Acid Cotransporter (ASBT) (also referred to as Ibat; gene name Slc10a2)is localized on the cholangiocyte apical membrane. On the basolateral membrane, four transport systems have been identified (t-ASBT, multidrug resistance (MDR)3,an unidentified anion exchanger system and organic solute transporter (Ost) heteromeric transporter, OstαOstβ. Together, these transporters unidirectionally move bile acids from ductal bile to the circulation. Bile acids absorbed by cholangiocytes recycle via the peribiliaryplexus back to hepatocytes for re-secretion into bile.This recycling of bile acids between hepatocytes and cholangiocytes is referred to as the cholehepatic shunt pathway. Recent studies suggest that the cholehepatic shunt pathway may contribute in overall hepatobiliary transport of bile acids and to the adaptation to chronic cholestasis due to extrahepatic obstruction. ASBT is acutely regulated by an adenosine 3', 5'-monophosphate (cAMP)-dependent translocation to the apical membrane and by phosphorylation-dependent ubiquitination and proteasome degradation. ASBT is chronically regulated by changes in gene expression in response to biliary bile acid concentration and inflammatory cytokines.Another potential function of cholangiocyte ASBT is to allow cholangiocytes to sample biliary bile acids in order to activate intracellular signaling pathways. Bile acids trigger changes in intracellular calcium, protein kinase C (PKC), phosphoinositide 3-kinase (PI3K), mitogenactivated protein (MAP) kinase and extracellular signalregulated protein kinase (ERK) intracellular signals.Bile acids significantly alter cholangiocyte secretion,proliferation and survival. Different bile acids have differential effects on cholangiocyte intracellular signals,and in some instances trigger opposing effects on cholangiocyte secretion

  15. Differential gene expression in SV40-mediated immortalization of human fibroblasts.

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    Pardinas, J; Pang, Z; Houghton, J; Palejwala, V; Donnelly, R J; Hubbard, K; Small, M B; Ozer, H L

    1997-06-01

    Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of

  16. Motoneuron differentiation of immortalized human spinal cord cell lines.

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    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  17. Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

    NARCIS (Netherlands)

    Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

    Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

  18. Inducible immortality in hTERT-human mesenchymal stem cells.

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    Piper, Samantha L; Wang, Miqi; Yamamoto, Akira; Malek, Farbod; Luu, Andrew; Kuo, Alfred C; Kim, Hubert T

    2012-12-01

    Human mesenchymal stem cells (hMSCs) are attractive candidates for tissue engineering and cell-based therapy because of their multipotentiality and availability in adult donors. However, in vitro expansion and differentiation of these cells is limited by replicative senescence. The proliferative capacity of hMSCs can be enhanced by ectopic expression of telomerase, allowing for long-term culture. However, hMSCs with constitutive telomerase expression demonstrate unregulated growth and even tumor formation. To address this problem, we used an inducible Tet-On gene expression system to create hMSCs in which ectopic telomerase expression can be induced selectively by the addition of doxycycline (i-hTERT hMSCs). i-hTERT hMSCs have inducible hTERT expression and telomerase activity, and are able to proliferate significantly longer than wild type hMSCs when hTERT expression is induced. They stop proliferating when hTERT expression is turned off and can be rescued when expression is re-induced. They retain multipotentiality in vitro even at an advanced age. We also used a selective inhibitor of telomere elongation to show that the mechanism driving immortalization of hMSCs by hTERT is dependent upon maintenance of telomere length. Thanks to their extended lifespan, preserved multipotentiality and controlled growth, i-hTERT hMSCs may prove to be a useful tool for the development and testing of novel stem cell therapies. Copyright © 2012 Orthopaedic Research Society.

  19. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

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    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  20. Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

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    Masao Takeuchi

    Full Text Available Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN; these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5, which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1, the cell surface receptor for hedgehog (Hh signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional

  1. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha

    2008-01-01

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged...

  2. Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality

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    Solinas-Toldo, Sabina; Dürst, Matthias; Lichter, Peter

    1997-01-01

    High risk human papillomaviruses (HPVs) known to be closely associated with cervical cancer, such as HPV16 and HPV18, have the potential to immortalize human epithelial cells in culture. Four lines of HPV-transfected keratinocytes were analyzed by comparative genomic hybridization at different time points after transfection. A number of chromosomal imbalances was found to be highly characteristic for the cultures progressing toward immortality. Whereas several of these were new and previously not found as recurrent aberrations in cervical tumors, some were identical to chromosomal changes observed during cervical carcinogenesis. The data put new emphasis on the studied cell system as a relevant model for HPV-induced pathogenesis. PMID:9108068

  3. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Woodworth, C.D.; Doniger, J.; DiPaolo, J.A.

    1989-01-01

    Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

  4. Molecular hydrogen attenuates hypoxia/reoxygenation injury of intrahepatic cholangiocytes by activating Nrf2 expression.

    Science.gov (United States)

    Yu, Jianhua; Zhang, Weiguang; Zhang, Rongguo; Jiang, Guixing; Tang, Haijun; Ruan, Xinxian; Ren, Peitu; Lu, Baochun

    2015-11-01

    Hypoxia/reoxygenation (H/R) injury of cholangiocytes causes serious biliary complications during hepatobiliary surgeries. Molecular hydrogen (H2) has been shown to be effective in protecting various cells and organs against oxidative stress injury. Human liver cholangiocytes were used to determine the potential protective effects of hydrogen against cholangiocyte H/R injury and explore the underlying mechanisms. We found that H2 ameliorated H/R-induced cholangiocytes apoptosis. Our study revealed that H2 activated NF-E2-related factor 2 (Nrf2) and downstream cytoprotective protein expression. However, the protective function of H2 was abolished when Nrf2 was silenced. Apoptosis in cholangiocytes isolated from a rat model of liver ischemia/reperfusion injury indicated that H2 significantly attenuates ischemia/reperfusion cholangiocyte injury in vivo. In conclusion, our study shows that H2 protects intrahepatic cholangiocytes from hypoxia/reoxygenation-induced apoptosis in vitro or in vivo, and this phenomenon may depend on activating Nrf2 expression.

  5. Development of a full-thickness human gingiva equivalent constructed from immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    J.K. Buskermolen; C.M.A. Reijnders; S.W. Spiekstra; T. Steinberg; C.J. Kleverlaan; A.J. Feilzer; A.D. Bakker; S. Gibbs

    2016-01-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines

  6. Tissue distribution and engraftment of human mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene

    DEFF Research Database (Denmark)

    Bentzon, J F; Stenderup, K; Hansen, F D

    2005-01-01

    Engraftment of mesenchymal stem cells (MSC) in peripheral tissues for replenishing of local stem cell function has been proposed as a therapeutic approach to degenerative diseases. We have previously reported the development of an immortalized human telomerase reverse transcriptase transduced MSC...... that infused cells were efficiently arrested in microvasculature during first-pass, but only for a fraction of the infused cells was arrest followed by vascular emigration and tissue engraftment. Few engrafted cells in lungs, heart, and kidney glomeruli remained after 4 weeks. These observations are consistent...

  7. The People Paradox: Self-Esteem Striving, Immortality Ideologies, and Human Response to Climate Change

    Directory of Open Access Journals (Sweden)

    Janis L. Dickinson

    2009-06-01

    Full Text Available In 1973, Ernest Becker, a cultural anthropologist cross-trained in philosophy, sociology, and psychiatry, invoked consciousness of self and the inevitability of death as the primary sources of human anxiety and repression. He proposed that the psychological basis of cooperation, competition, and emotional and mental health is a tendency to hold tightly to anxiety-buffering cultural world views or "immortality projects" that serve as the basis for self-esteem and meaning. Although he focused mainly on social and political outcomes like war, torture, and genocide, he was increasingly aware that materialism, denial of nature, and immortality-striving efforts to control, rather than sanctify, the natural world were problems whose severity was increasing. In this paper I review Becker's ideas and suggest ways in which they illuminate human response to global climate change. Because immortality projects range from belief in technology and materialism to reverence for nature or belief in a celestial god, they act both as barriers to and facilitators of sustainable practices. I propose that Becker's cross-disciplinary "science of man," and the predictions it generates for proximate-level determinants of social behavior, add significantly to our understanding of and potential for managing the people paradox, i.e., that the very things that bring us symbolic immortality often conflict with our prospects for survival. Analysis of immortality projects as one of the proximate barriers to addressing climate change is both cautionary and hopeful, providing insights that should be included in the cross-disciplinary quest to uncover new pathways toward rational, social change.

  8. Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

    Science.gov (United States)

    Raymon, H K; Thode, S; Zhou, J; Friedman, G C; Pardinas, J R; Barrere, C; Johnson, R M; Sah, D W

    1999-07-01

    A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

  9. Protein composition of catalytically active human telomerase from immortal cells

    DEFF Research Database (Denmark)

    Cohen, Scott B; Graham, Mark E; Lovrecz, George O

    2007-01-01

    Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on th...

  10. Cholangiocyte anion exchange and biliary bicarbonate excretion

    Institute of Scientific and Technical Information of China (English)

    Jesús M Banales; Jesús Prieto; Juan F Medina

    2006-01-01

    Primary canalicular bile undergoes a process of fluidization and alkalinization along the biliary tract that is influenced by several factors including hormones, innervation/neuropeptides, and biliary constituents. Theexcretion of bicarbonate at both the canaliculi and the bile ducts is an important contributor to the generation of the so-called bile-salt independent flow. Bicarbonate is secreted from hepatocytes and cholangiocytes through parallel mechanisms which involve chloride efflux through activation of Cl- channels, and further bicarbonate secretion via AE2/SLC4A2-mediated Cl-/HCO3-exchange. Glucagon and secretin are two relevant hormones which seem to act very similarly in their target cells (hepatocytes for the former and cholangiocytes for the latter). These hormones interact with their specific G protein-coupled receptors, causing increases in intracellular levels of cAMP and activation of cAMP-dependent Cl- and HCO3- secretory mechanisms. Both hepatocytes and cholangiocytes appear to have cAMP-responsive intracellular vesicles in which AE2/SLC4A2 colocalizes with cell specific Cl- channels (CFTR in cholangiocytes and not yet determined in hepatocytes) and aquaporins (AQP8 in hepatocytes and AQP1 in cholangiocytes). cAMP-induced coordinated trafficking of these vesicles to either canalicular or cholangiocyte lumenal membranes and further exocytosis results in increased osmotic forces and passive movement of water with net bicarbonate-rich hydrocholeresis.

  11. A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

    Directory of Open Access Journals (Sweden)

    Xiangshan Zhao

    2011-01-01

    Full Text Available Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs immortalized with human telomerase reverse transcriptase (hTERT possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with g-radiation, share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

  12. Establishment and transformation of telomerase-immortalized human small airway epithelial cells by heavy ions

    Science.gov (United States)

    Zhao, Y. L.; Piao, C. Q.; Hei, T. K.

    Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of 1 GeV/nucleon of 56Fe ions accelerated at the Brookhaven National Laboratory. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

  13. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Buskermolen, Jeroen K; Reijnders, Christianne M A; Spiekstra, Sander W; Steinberg, Thorsten; Kleverlaan, Cornelis J; Feilzer, Albert J; Bakker, Astrid D; Gibbs, Susan

    2016-08-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  14. In Search of Rationality in Human Longevity and Immortality.

    Science.gov (United States)

    Bhar, Gopal C

    2016-01-01

    The human body is machine-like, but self-moving, self-regulating, and self-adjusting, governed by willpower and intelligence. Aging of the body is basically a maintenance problem and so it could perhaps be postponed by thorough and frequent maintenance. Aging brings on a cascade of ills and health problems leading to deterioration of physical, mental, emotional, and social dimensions of life. This paper deals with solution of the problem philosophically in the light of Indian scriptures without entering into traditional bioethical issues. With a meaningful reason for existence, life can be extended. Examining the scientific perspectives on aging, some common manipulations for its extension are discussed. These are calorie restriction, vitamin and antioxidant treatment, exercise and hormonal interventions, etc. Finally, the question of longevity is explored through pursuance of eternal value-based activity and spirituality in the tradition of Indian heritage.

  15. Bradykinin promotes migration and invasion of human immortalized trophoblasts

    Directory of Open Access Journals (Sweden)

    Lisboa Francisco

    2011-07-01

    Full Text Available Abstract Having demonstrated that the bradykinin B2 receptor (B2R is expressed in cells that participate in trophoblast invasion in humans and guinea-pigs, we investigated the role of bradykinin (BK on cell migration and invasion in the HTR-8/SVneo trophoblast cell line using wound healing and invasion assays. First, we documented that HTR-8/SVneo cells expressed kallikrein, B2R, B1R, MMP-2 and MMP-9 using immunocytochemistry. Incubation with BK (10.0 microMol/L for 18 hours increased the migration index 3-fold in comparison to controls or to cells preincubated with the B2R antagonist HOE-140. BK (10.0 microMol/L incubation yielded a similar number of proliferating and viable cells as controls, therefore the enhanced closure of the wound cannot be attributed to proliferating cells. Incubation with BK (10.0 microMol/L for 18 hours increased the invasion index 2-fold in comparison to controls or to cells preincubated with the antagonist of the B2R. Neither the B1R ligand Lys-des-Arg9 BK, nor its antagonist Lys-(des-Arg9-Leu8, modified migration and invasion. Further support for the stimulatory effect of B2R activation on migration and invasion is provided by the 3-fold increase in the number of filopodia per cell versus controls or cells preincubated with the B2R antagonist. Bradykinin had no effect on the cellular protein content of the B2R, nor the MMP-9 and MMP-2 gelatinase activity in the culture media varied after incubation with BK. This study adds bradykinin-acting on the B2R-to the stimuli of trophoblast migration and invasion, an effect that should be integrated to other modifications of the kallikrein-kinin system in normal and pathological pregnancies.

  16. Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

    Directory of Open Access Journals (Sweden)

    Mamchaoui Kamel

    2011-11-01

    Full Text Available Abstract Background Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

  17. Benzo (a) pyrene induced tumorigenesity of human immortalized oral epithelial cells: transcription profiling

    Institute of Scientific and Technical Information of China (English)

    LI Jin-zhong; PAN Hong-ya; ZHENG Jia-wei; ZHOU Xiao-jian; ZHANG Ping; CHEN Wan-tao; ZHANG Zhi-yuan

    2008-01-01

    Background The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo (a) pyrene.Methods The human immortalized oral epithelial cells, which have been established through transfection of E6/E7 genasof human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo (a) pyrene.Tumorigenesity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance (ANOVA). The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction.Results There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction,possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, MET were validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens.Concluslona This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo (a) pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to

  18. Immortalization of Human Precartilaginous Stem Cells by Transfecting SV40Tag

    Institute of Scientific and Technical Information of China (English)

    Junfang WANG; Huang FANG; Renyun XIA; Anming CHEN; Hao CHENG

    2009-01-01

    Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs.Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection.Colonies were isolated by puromycin selection and expanded by multiple passages,lmmunohistochemistry,RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines.The positive colonies were isolated and subcultured,designated immortalized precartilaginous stem cells (IPSCs),which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR.SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting,and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR.These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

  19. Immortality of cancers

    Science.gov (United States)

    Duesberg, Peter; McCormack, Amanda

    2013-01-01

    Immortality is a common characteristic of cancers, but its origin and purpose are still unclear. Here we advance a karyotypic theory of immortality based on the theory that carcinogenesis is a form of speciation. Accordingly, cancers are generated from normal cells by random karyotypic rearrangements and selection for cancer-specific reproductive autonomy. Since such rearrangements unbalance long-established mitosis genes, cancer karyotypes vary spontaneously but are stabilized perpetually by clonal selections for autonomy. To test this theory we have analyzed neoplastic clones, presumably immortalized by transfection with overexpressed telomerase or with SV40 tumor virus, for the predicted clonal yet flexible karyotypes. The following results were obtained: (1) All immortal tumorigenic lines from cells transfected with overexpressed telomerase had clonal and flexible karyotypes; (2) Searching for the origin of such karyotypes, we found spontaneously increasing, random aneuploidy in human fibroblasts early after transfection with overexpressed telomerase; (3) Late after transfection, new immortal tumorigenic clones with new clonal and flexible karyotypes were found; (4) Testing immortality of one clone during 848 unselected generations showed the chromosome number was stable, but the copy numbers of 36% of chromosomes drifted ± 1; (5) Independent immortal tumorigenic clones with individual, flexible karyotypes arose after individual latencies; (6) Immortal tumorigenic clones with new flexible karyotypes also arose late from cells of a telomerase-deficient mouse rendered aneuploid by SV40 virus. Because immortality and tumorigenicity: (1) correlated exactly with individual clonal but flexible karyotypes; (2) originated simultaneously with such karyotypes; and (3) arose in the absence of telomerase, we conclude that clonal and flexible karyotypes generate the immortality of cancers. PMID:23388461

  20. Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells.

    Science.gov (United States)

    Yang, X; Nakao, Y; Pater, M M; Tang, S C; Pater, A

    1997-09-01

    We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis.

  1. A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice

    NARCIS (Netherlands)

    Parashar, B; Moshage, H; Tanaka, KE; Engelhardt, D; Rabbani, E; Roy-Chowdhury, N; Roy-Chowdhury, J

    2000-01-01

    Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transfe

  2. Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

    Science.gov (United States)

    Pantic, Boris; Borgia, Doriana; Giunco, Silvia; Malena, Adriana; Kiyono, Tohru; Salvatori, Sergio; De Rossi, Anita; Giardina, Emiliano; Sangiuolo, Federica; Pegoraro, Elena; Vergani, Lodovica; Botta, Annalisa

    2016-03-01

    Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.

  3. Bioinformatic approach for understanding the heterogeneity of cholangiocytes

    Institute of Scientific and Technical Information of China (English)

    Koji Fukushima; Yoshiyuki Ueno

    2006-01-01

    It is remarkable that microarray technologies have nearly reached a pinnacle. Establishment of further analysis and management of enormous data derived from microarray technology is currently the highest priority. The heterogeneous functions of cholangiocytes regulate the pathophysiology of the biliary epithelium in relation to secretory, proliferative and apoptotic activities. Distinct expression profiles of two murine cholangiocyte lines, termed small and large have been revealed by microarray analysis. The features of the two cholangiocyte cell lines, categorized partly according to gene ontology, indicate the specific physiological role of each cell line. The large cholangiocytes are characterized as "transport" and "immune/ inflammatory responses".In contrast, small cholangiocytes are associated with properties of limited physiological functional ability and proliferating/migrating potential with specific molecules like Eph receptors, comparable to mesenchymal cells.'Omic study will be of great help in understanding the heterogeniety of cholangiocytes.

  4. Immortalization protocols used in cell culture models of human breast morphogenesis

    DEFF Research Database (Denmark)

    Gudjonsson, T; Villadsen, R; Rønnov-Jessen, L

    2004-01-01

    of the tissue of origin. In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern. This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application...

  5. Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

    Science.gov (United States)

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

    Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis. Copyright © 2012 John Wiley & Sons, Ltd.

  6. Exome-wide Mutation Profile in Benzo[a]pyrene-derived Post-stasis and Immortal Human Mammary Epithelial Cells

    Science.gov (United States)

    Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; Futscher, Bernard W.

    2014-01-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and towards immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. PMID:25435355

  7. Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

    Science.gov (United States)

    Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

    1998-11-01

    Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

  8. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells.

    Science.gov (United States)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

    2008-02-15

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor gamma (PPARgamma) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPARgamma agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPARgamma-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.

  9. Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

    Science.gov (United States)

    Yildiz, Gokhan; Arslan-Ergul, Ayca; Bagislar, Sevgi; Konu, Ozlen; Yuzugullu, Haluk; Gursoy-Yuzugullu, Ozge; Ozturk, Nuri; Ozen, Cigdem; Ozdag, Hilal; Erdal, Esra; Karademir, Sedat; Sagol, Ozgul; Mizrak, Dilsa; Bozkaya, Hakan; Ilk, Hakki Gokhan; Ilk, Ozlem; Bilen, Biter; Cetin-Atalay, Rengul; Akar, Nejat; Ozturk, Mehmet

    2013-01-01

    Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene

  10. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression.

    Science.gov (United States)

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X; Kemp, Sadie E; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2014-04-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells.

  11. Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

    Institute of Scientific and Technical Information of China (English)

    BIAN Chang; ZHAO Kui; TONG Guo-xin; ZHU Yong-liang; CHEN Peng

    2005-01-01

    Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods:Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor,hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

  12. HPV16 E7 protein and hTERT proteins defective for telomere maintenance cooperate to immortalize human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Jonathan Miller

    Full Text Available Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT protein can functionally replace the human papillomavirus type 16 (HPV-16 E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A, elongation-defective hTERT (hTERT-HA, and telomere recruitment-defective hTERT (hTERT N+T also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.

  13. Zeno's Paradox of Immortality.

    Science.gov (United States)

    Olshansky, S Jay; Carnes, Bruce A

    2013-01-01

    Scientists who speculate on the future of human longevity have a broad range of views ranging from the promise of immortality, to radical life extension, to declines in life expectancy. Among those who contend that radical life extension is already here, or on the horizon, or immortality is forthcoming, elements of their reasoning appear surprisingly close, if not identical, to a famous mathematical paradox posed by the ancient Greek mathematician known as Zeno. Here we examine the underlying assumptions behind the views that much longer life expectancies are forthcoming or have already arrived, and place their line of reasoning within the context of a new Zeno paradox described here as The Paradox of Immortality. Copyright © 2012 S. Karger AG, Basel.

  14. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

    Science.gov (United States)

    Lehr, Elizabeth E; Qadadri, Brahim; Brown, Calla R; Brown, Darron R

    2003-09-30

    Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

  15. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available Arsenic is a lung toxicant that can lead to respiratory illness through inhalation and ingestion, although the most common exposure is through contaminated drinking water. Lung effects reported from arsenic exposure include lung cancer and obstructive lung disease, as well as reductions in lung function and immune response. As part of their role in innate immune function, airway epithelial cells provide a barrier that protects underlying tissue from inhaled particulates, pathogens, and toxicants frequently found in inspired air. We evaluated the effects of a five-day exposure to environmentally relevant levels of arsenic {<4μM [~300 μg/L (ppb] as NaAsO2} on airway epithelial barrier function and structure. In a primary mouse tracheal epithelial (MTE cell model we found that both micromolar (3.9 μM and submicromolar (0.8 μM arsenic concentrations reduced transepithelial resistance, a measure of barrier function. Immunofluorescent staining of arsenic-treated MTE cells showed altered patterns of localization of the transmembrane tight junction proteins claudin (Cl Cl-1, Cl-4, Cl-7 and occludin at cell-cell contacts when compared with untreated controls. To better quantify arsenic-induced changes in tight junction transmembrane proteins we conducted arsenic exposure experiments with an immortalized human bronchial epithelial cell line (16HBE14o-. We found that arsenic exposure significantly increased the protein expression of Cl-4 and occludin as well as the mRNA levels of Cl-4 and Cl-7 in these cells. Additionally, arsenic exposure resulted in altered phosphorylation of occludin. In summary, exposure to environmentally relevant levels of arsenic can alter both the function and structure of airway epithelial barrier constituents. These changes likely contribute to the observed arsenic-induced loss in basic innate immune defense and increased infection in the airway.

  16. Development and characterization of a hydrogen peroxide-resistant cholangiocyte cell line: A novel model of oxidative stress-related cholangiocarcinoma genesis

    Energy Technology Data Exchange (ETDEWEB)

    Thanan, Raynoo [Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Techasen, Anchalee [Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Faculty of Associated Medical Science, Khon Kaen University, Khon Kaen 40002 (Thailand); Hou, Bo [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507 (Japan); Jamnongkan, Wassana; Armartmuntree, Napat [Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Yongvanit, Puangrat, E-mail: puangrat@kku.ac.th [Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002 (Thailand); Murata, Mariko, E-mail: mmurata@doc.medic.mie-u.ac.jp [Department of Environmental and Molecular Medicine, Mie University Graduate School of Medicine, Tsu, Mie 514-8507 (Japan)

    2015-08-14

    Oxidative stress is a cause of inflammation–related diseases, including cancers. Cholangiocarcinoma is a liver cancer with bile duct epithelial cell phenotypes. Our previous studies in animal and human models indicated that oxidative stress is a major cause of cholangiocarcinoma development. Hydrogen peroxide (H{sub 2}O{sub 2}) can generate hydroxyl radicals, which damage lipids, proteins, and nucleic acids, leading to cell death. However, some cells can survive by adapting to oxidative stress conditions, and selective clonal expansion of these resistant cells would be involved in oxidative stress-related carcinogenesis. The present study aimed to establish H{sub 2}O{sub 2}-resistant cell line from an immortal cholangiocyte cell line (MMNK1) by chronic treatment with low-concentration H{sub 2}O{sub 2} (25 μM). After 72 days of induction, H{sub 2}O{sub 2}-resistant cell lines (ox-MMNK1-L) were obtained. The ox-MMNK1-L cell line showed H{sub 2}O{sub 2}-resistant properties, increasing the expression of the anti-oxidant genes catalase (CAT), superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), and superoxide dismutase-3 (SOD3) and the enzyme activities of CAT and intracellular SODs. Furthermore, the resistant cells showed increased expression levels of an epigenetics-related gene, DNA methyltransferase-1 (DNMT1), when compared to the parental cells. Interestingly, the ox-MMNK1-L cell line had a significantly higher cell proliferation rate than the MMNK1 normal cell line. Moreover, ox-MMNK1-L cells showed pseudopodia formation and the loss of cell-to-cell adhesion (multi-layers) under additional oxidative stress (100 μM H{sub 2}O{sub 2}). These findings suggest that H{sub 2}O{sub 2}-resistant cells can be used as a model of oxidative stress-related cholangiocarcinoma genesis through molecular changes such as alteration of gene expression and epigenetic changes. - Highlights: • An H{sub 2}O{sub 2}-resistant ox-MMNK1-L cells was established from

  17. Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

    DEFF Research Database (Denmark)

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal...

  18. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B

    1999-01-01

    were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A...

  19. Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.

    Science.gov (United States)

    Martinho, F C; Camargo, S E A; Fernandes, A M M; Campos, M S; Prado, R F; Camargo, C H R; Valera, M C

    2017-04-25

    To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers. Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level. The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P sealers tested (P epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05). A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against immortalized pulp cells compared to an epoxy resin-based sealer (AH Plus), a methacrylate-based sealer (EndoRez) and a calcium hydroxide-based sealer (Apexit). © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  20. Artichoke compound cynarin differentially affects the survival, growth and stress response of normal, immortalized and cancerous human cells

    DEFF Research Database (Denmark)

    Gezer, Ceren; Yücecan, Sevinç; Rattan, Suresh Inder Singh

    2015-01-01

    Cynarin (CYN) is the main derivative of caffeoylquinic acid, found in leaves and heads of artichoke. Potential health-beneficial effects of CYN include as being choloretic-cholesterol lowering, hepatoprotective, anti-atherosclerotic, and antioxidative. We have tested the effects of various doses...... of CYN on the proliferative potential, survival, morphology, and stress response (SR) markers haemoxygenase-1 (HO-1) and heat shock protein-70 (HSP70) in normal human skin fibroblasts (FSF-1), telomerase-immortalized mesenchymal stem cells (hTERT-MSC) and cervical cancer cells, HeLa. Effects of CYN...

  1. Artichoke compound cynarin differentially affects the survival, growth and stress response of normal, immortalized and cancerous human cells

    DEFF Research Database (Denmark)

    Gezer, Ceren; Yücecan, Sevinç; Rattan, Suresh Inder Singh

    2015-01-01

    Cynarin (CYN) is the main derivative of caffeoylquinic acid, found in leaves and heads of artichoke. Potential health-beneficial effects of CYN include as being choloretic-cholesterol lowering, hepatoprotective, anti-atherosclerotic, and antioxidative. We have tested the effects of various doses...... of CYN on the proliferative potential, survival, morphology, and stress response (SR) markers haemoxygenase-1 (HO-1) and heat shock protein-70 (HSP70) in normal human skin fibroblasts (FSF-1), telomerase-immortalized mesenchymal stem cells (hTERT-MSC) and cervical cancer cells, HeLa. Effects of CYN...

  2. Fluorescent immortalized human adipose derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microvesicles.

    Science.gov (United States)

    Coccè, Valentina; Balducci, Luigi; Falchetti, Maria Laura; Pascucci, Luisa; Ciusani, Emilio; Brini, Anna Teresa; Sisto, Francesca; Piovani, Giovanna; Alessandri, Giulio; Parati, Eugenio; Cabeza, Laura; Pessina, Augusto

    2017-03-27

    A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASC) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigate for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would be less resource and time consuming and it could possibly contribute to simplification of GMP procedures. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Immortality in Empedocles

    OpenAIRE

    Long, Alex

    2017-01-01

    Most of the work for the paper was undertaken in Toronto during a Leverhulme Research Fellowship. The paper examines Empedocles’ attributions of immortality. I argue that Empedocles does not withhold immortality from the gods but rather has an unorthodox conception of what immortality is. Immortality does not mean, or imply, endless duration. A god’s immortality is its continuity, as one and the same organism, over a long but finite period. This conception of divine immortality then influe...

  4. Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I

    Energy Technology Data Exchange (ETDEWEB)

    Rosendahl, Ann H., E-mail: ann.rosendahl@med.lu.se [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden); Lund University and Skåne University Hospital, Department of Clinical Sciences Lund, Division of Oncology and Pathology, Lund (Sweden); Gundewar, Chinmay; Said Hilmersson, Katarzyna [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden); Ni, Lan; Saleem, Moin A. [University of Bristol, School of Clinical Sciences, Children' s Renal Unit and Academic Renal Unit, Bristol (United Kingdom); Andersson, Roland [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden)

    2015-01-15

    Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer. - Highlights: • Generation of human conditionally immortalized human pancreatic stellate cell lines. • Temperature-sensitive SV40LT allows switch to primary PSC phenotype characteristics. • Proteome profiling revealed distinct expression patterns between TPSC and NPSC. • Enhanced IGF-I-stimulated proliferation and motility by TPSC compared to NPSC.

  5. Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James; Takenaka, Yasuhiro; Stampfer, Martha R.; Gilley, David; Yaswen, Paul

    2004-12-23

    We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.

  6. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

    Directory of Open Access Journals (Sweden)

    Kee Hang Lee

    Full Text Available Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs immortalized by the human telomerase reverse transcriptase (hTERT gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM cells were injected into adult (4-6-week-old Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL, they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases.

  7. Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate.

    Science.gov (United States)

    Zhao, Xiangshan; Malhotra, Gautam K; Lele, Subodh M; Lele, Manjiri S; West, William W; Eudy, James D; Band, Hamid; Band, Vimla

    2010-08-10

    There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

  8. Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

    Science.gov (United States)

    Merkley, Mark A; Hildebrandt, Ellen; Podolsky, Robert H; Arnouk, Hilal; Ferris, Daron G; Dynan, William S; Stöppler, Hubert

    2009-08-23

    Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

  9. Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

    Directory of Open Access Journals (Sweden)

    Arnouk Hilal

    2009-08-01

    Full Text Available Abstract Background Infection with high-risk type human papilloma viruses (HPVs is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Results Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE. The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23% of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2% were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1; and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27. Conclusion This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

  10. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer.

    Science.gov (United States)

    Chen, Yi; Pirisi, Lucia; Creek, Kim E

    2013-09-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.

  11. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yi; Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC (United States)

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  12. Expression of human cell cycle regulators in the primary cell line of the African savannah elephant (loxodonta africana) increases proliferation until senescence, but does not induce immortalization.

    Science.gov (United States)

    Fukuda, Tomokazu; Iino, Yuuka; Onuma, Manabu; Gen, Bando; Inoue-Murayama, Miho; Kiyono, Tohru

    2016-01-01

    The African savannah elephant (Loxodonta africana) is one of the critically endangered animals. Conservation of genetic and cellular resources is important for the promotion of wild life-related research. Although primary cultured cells are a useful model for the physiology and genomics of the wild-type animals, their distribution is restricted due to the limited number of cell divisions allowed in them. Here, we tried to immortalize a primary cell line of L. africana with by overexpressing human mutant form of cyclin-dependent kinase 4 (CDK4R24C), cyclin D, and telomerase (TERT). It has been shown before that the combination of human CDK4R24C, cyclin D, and TERT induces the efficient cellular immortalization of cells derived from humans, bovine, swine, and monkeys. Interestingly, although the combination of these three genes extended the cellular proliferation of the L. africana-derived cells, they did not induce cellular immortalization. This study suggest that control of cellular senescence in L. africana-derived cells would be different molecular mechanisms compared to those governing human, bovine, swine, and monkey cells.

  13. Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b

    Directory of Open Access Journals (Sweden)

    Brosens Jan J

    2009-07-01

    Full Text Available Abstract Background Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. Methods Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. Results St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82. Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. Conclusion St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural

  14. Foetal hepatic progenitor cells assume a cholangiocytic cell phenotype during two-dimensional pre-culture.

    Science.gov (United States)

    Anzai, Kazuya; Chikada, Hiromi; Tsuruya, Kota; Ida, Kinuyo; Kagawa, Tatehiro; Inagaki, Yutaka; Mine, Tesuya; Kamiya, Akihide

    2016-06-23

    Liver consists of parenchymal hepatocytes and other cells. Liver progenitor cell (LPC) is the origin of both hepatocytes and cholangiocytic cells. The analyses of mechanism regulating differentiation of LPCs into these functional cells are important for liver regenerative therapy using progenitor cells. LPCs in adult livers were found to form cysts with cholangiocytic characteristics in 3D culture. In contrast, foetal LPCs cannot form these cholangiocytic cysts in the same culture. Thus, the transition of foetal LPCs into cholangiocytic progenitor cells might occur during liver development. Primary CD45(-)Ter119(-)Dlk1(+) LPCs derived from murine foetal livers formed ALBUMIN (ALB)(+)CYTOKERATIN (CK)19(-) non-cholangiocytic cysts within 3D culture. In contrast, when foetal LPCs were pre-cultured on gelatine-coated dishes, they formed ALB(-)CK19(+) cholangiocytic cysts. When hepatocyte growth factor or oncostatin M, which are inducers of hepatocytic differentiation, was added to pre-culture, LPCs did not form cholangiocytic cysts. These results suggest that the pre-culture on gelatine-coated dishes changed the characteristics of foetal LPCs into cholangiocytic cells. Furthermore, neonatal liver progenitor cells were able to form cholangiocytic cysts in 3D culture without pre-culture. It is therefore possible that the pre-culture of mid-foetal LPCs in vitro functioned as a substitute for the late-foetal maturation step in vivo.

  15. Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast.

    Science.gov (United States)

    Sirchia, Rosalia; Longo, Alessandra; Luparello, Claudio

    2008-10-01

    Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96 h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl(2) at 5 microM concentration, corresponding to the IC(50) for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.

  16. Separation and identification of differentially expressed nuclear matrix proteins between human esophageal immortalized and carcinomatous cell lines

    Institute of Scientific and Technical Information of China (English)

    Xing-Dong Xiong; En-Min Li; Li-Yan Xu; Hai-Bin Chen; Ling Chen; Wei-Jia Cai; Ya-Li Han; Zhong-Ying Shen; Yi Zeng

    2003-01-01

    AIM: To separate and identify differentially expressed nuclear matrix proteins (NMPs) between the immortalized human esophageal epithelial cell line (SHEE) and the malignantly transformed esophageal carcinoma cell line (SHEEC), and to provide new ways for finding specific markers and the pathogenesis of esophageal carcinoma.METHODS: SHEE and SHEEC cell lines were used to extract NMPs. The quality of NMPs was monitored by Western blot analysis including DNA topoisomerase Ⅱα, proliferation cell nuclear antigen (PCNA) and histone. NMPs of SHEE and SHEEC were analyzed by two-dimensional electrophoresis (2-DE), silver staining and PDQuest6.2 image analysis software. Three spots in which the differentially expressed NMlPs were more obvious, were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI- TOF-MS) and database search.RESULTS: Western blot analysis revealed that DNA topoisomerase Ⅱα and PCNA were detected, and the majority of histones were deleted in NMPs of SHEE and SHEEC. After 2-DE image analysis by PDQuest6.2 software, the 2-DE maps were detected with an average of 106±7.1 spots in SHEE and 132±5.0 spots in SHEEC. Most of them were matched one another (r=0.72), only 16 protein spots were found differing in intensity. Three NMPs including cytoskeletal tropomyosin,FK506bindingprotein6,similartoretinoblastoma binding protein 8 were preliminarily identified by MALDI- TOF-MS.CONCLUSION: These differentially expressed NMPs may play an important role during malignant transformation from SHEE to SHEEC. Their separation and identification will contribute to searching for specific markers and probing into the pathogenesis of esophageal carcinoma.

  17. Persistent Exposure to Porphyromonas gingivalis Promotes Proliferative and Invasion Capabilities, and Tumorigenic Properties of Human Immortalized Oral Epithelial Cells

    Science.gov (United States)

    Geng, Fengxue; Liu, Junchao; Guo, Yan; Li, Chen; Wang, Hongyang; Wang, Hongyan; Zhao, Haijiao; Pan, Yaping

    2017-01-01

    Recent epidemiological studies revealed a significant association between oral squamous cell carcinoma (OSCC) and Porphyromonas gingivalis, a major pathogen of periodontal disease. As a keystone pathogen of periodontitis, P. gingivalis is known not only to damage local periodontal tissues, but also to evade the host immune system and eventually affect systemic health. However, its role in OSCC has yet to be defined. To explore the underlying effect of chronic P. gingivalis infection on OSCC and to identify relevant biomarkers as promising targets for therapy and prevention, we established a novel model by exposing human immortalized oral epithelial cells (HIOECs) to P. gingivalis at a low multiplicity of infection (MOI) for 5–23 weeks. The P. gingivalis infected HIOECs were monitored for tumor biological alteration by proliferation, wound healing, transwell invasion, and gelatin zymography assays. Microarray and proteomic analyses were performed on HIOECs infected with P. gingivalis for 15 weeks, and some selected data were validated by quantitative real-time PCR and (or) western blot on cells infected for 15 and 23 weeks. Persistent exposure to P. gingivalis caused cell morphological changes, increased proliferation ability with higher S phase fraction in the cell cycle, and promoted cell migratory and invasive properties. In combining results of bioinformatics analyses and validation assays, tumor-related genes such as NNMT, FLI1, GAS6, lncRNA CCAT1, PDCD1LG2, and CD274 may be considered as the key regulators in tumor-like transformation in response to long-time exposure of P. gingivalis. In addition, some useful clinical biomarkers and novel proteins were also presented. In conclusion, P. gingivalis could promote tumorigenic properties of HIOECs, indicating that chronic P. gingivalis infection may be considered as a potential risk factor for oral cancer. The key regulators detected from the present model might be used in monitoring the development of OSCC with

  18. Immortalization of Werner syndrome and progeria fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Saito, H.; Moses, R.E. (Baylor College of Medicine, Houston, TX (USA))

    1991-02-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

  19. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B

    1999-01-01

    To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized....... The promoter sequence revealed an internal 379 bp fragment not reported in previous publications. This novel fragment contains consensus sequences for several transcription factors including SpI and GATA. DNA from 48 unrelated individuals was PCR amplified, in this region, to test for allelic variations. All...

  20. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M

    2017-01-01

    3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during...

  1. Generation and evaluation of a human corneal model cell system for ophthalmologic issues using the HPV16 E6/E7 oncogenes as uniform immortalization platform.

    Science.gov (United States)

    Schulz, Simon; Steinberg, Thorsten; Beck, David; Tomakidi, Pascal; Accardi, Rosita; Tommasino, Massimo; Reinhard, Thomas; Eberwein, Philipp

    2013-01-01

    The present study aimed at employing the human papillomavirus type 16 (HPV16) E6/E7 gene platform, to create a uniform authentic in vitro model cell system of the human cornea for ophthalmologic issues and here especially for prospective biomaterial evaluations for therapeutic regenerative approaches. Therefore, HPV16 E6/E7 genes were employed as uniform platform to immortalize primary human corneal keratinocytes (IHCK), fibroblasts (IHCF), and endothelial (IHCE) cells. qPCR revealed that E6/E7 mRNA transcription persisted at rising passages and FISH detection of the chromosome portfolio 1, 8, 10 and 18 showed fairly the disomic cytogenetic status. Hot spot passages proved oscillation of aneuploidies in the entire passage spectrum under study, while hot spot aneuploidies annotated prevalence for distinct chromosomes. Though IIF revealed general endurance, tissue-innate corneal biomarkers were modulated, i.e. expressed in a temporal-confluence, temporal-spatial or passage-dependent manner. In summary, by the fairly normal chromosomal status, and expression of tissue-innate biomarkers, we created for the first time a uniform authentic in vitro model cell system of the human cornea, by application of the HPV16 E6/E7 immortalization platform only. This system renders a precious tool for prospective iterative in vitro studies on issues such as corneal tissue homeostasis, pharmaceutical generics, and/or evaluation of new biomaterials for clinical corneal applications. Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  2. Adult Mouse Liver Contains Two Distinct Populations of Cholangiocytes

    Directory of Open Access Journals (Sweden)

    Bin Li

    2017-08-01

    Full Text Available The biliary system plays an important role in several acquired and genetic disorders of the liver. We have previously shown that biliary duct epithelium contains cells giving rise to proliferative Lgr5+ organoids in vitro. However, it remained unknown whether all biliary cells or only a specific subset had this clonogenic activity. The cell surface protease ST14 was identified as a positive marker for the clonogenic subset of cholangiocytes and was used to separate clonogenic and non-clonogenic duct cells by fluorescence-activated cell sorting. Only ST14hi duct cells had the ability to generate organoids that could be serially passaged. The gene expression profiles of clonogenic and non-clonogenic duct cells were similar, but several hundred genes were differentially expressed. RNA fluorescence in situ hybridization showed that clonogenic duct cells are interspersed among regular biliary epithelium at a ∼1:3 ratio. We conclude that adult murine cholangiocytes can be subdivided into two populations differing in their proliferative capacity.

  3. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

    Energy Technology Data Exchange (ETDEWEB)

    Senthilkumar, P.K. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Robertson, L.W. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States); Ludewig, G., E-mail: Gabriele-ludewig@uiowa.edu [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States)

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  4. Characterization of vitamin C-induced cell sheets formed from primary and immortalized human corneal stromal cells for tissue engineering applications.

    Science.gov (United States)

    Grobe, Gesa Maria; Reichl, Stephan

    2013-01-01

    The purpose of this study was to compare the ability of primary human corneal stromal cells (HuFib cells) and SV40-immortalized human corneal keratocytes (HCK cells) to synthesize their own extracellular matrix induced by vitamin C supplementation. Therefore, the amount of collagen secreted and resulting biomechanical properties based on the culture duration were assessed. Cells were cultivated for several weeks with or without vitamin C. The amount of collagen secreted by the cells was quantified based on the culture duration. Cell viability was simultaneously determined via the MTT assay. Collagen secretion was increased as a result of vitamin C supplementation. The effect was stronger in primary cells. In addition, vitamin C supplementation had a positive effect on HuFib cell viability. Vitamin C supplementation induced the formation of detachable cell sheets in both primary and immortalized cells. The biomechanical properties of the sheets were evaluated using a static material testing machine, and the ultrastructure of the cell sheets was examined using scanning electron microscopy. The cell sheets formed from HuFib cells had a higher percentage of light transmission between 400 and 800 nm and were superior in terms of E-modulus and ultimate strength testing. Indirect immunofluorescence and Western blot confirmed the presence of collagen type I in the HuFib and HCK cell cultures. Stimulating secretion of the extracellular matrix in corneal stromal cells is a promising approach for corneal stroma reconstruction for tissue engineering applications. Copyright © 2013 S. Karger AG, Basel.

  5. Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation

    Directory of Open Access Journals (Sweden)

    Nadella Kiran S

    2008-06-01

    Full Text Available Abstract Background Adult T-cell leukemia/lymphoma (ATLL is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1; however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated. Results We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1α (MIP-1α, a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was

  6. La aportación de Étienne Gilson al problema de la inmortalidad del alma humana en Cayetano / The Contribution of Étienne Gilson to the Problem of the Immortality of the Human Soul in Cajetan

    Directory of Open Access Journals (Sweden)

    Ceferino P. D. Muñoz

    2013-12-01

    Full Text Available In his article the author reviews Cajetan’s different positions on the prob-lem of the immortality of the human soul, and investigates possible reasons which led the Cardinal to dissent with the position of Thomas Aquinas. For this purpose, he invokes selected interpretations of distinguished scholars, with special reference to the approach of Étienne Gilson. Against the back-ground of his analyses the authors attempts to give a synthesis which, as he hopes, can become a modest contribution to contemporary comments on the thought of this great French medievalist about the problem of the immortality of the human soul in Cajetan.

  7. Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

    Energy Technology Data Exchange (ETDEWEB)

    Tatrai, Peter, E-mail: peter.tatrai@biomembrane.hu [Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest (Hungary); Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Szepesi, Aron, E-mail: aron.szepesi@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Matula, Zsolt, E-mail: matula.zsolt@gmail.com [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Szigeti, Anna, E-mail: anna.szigeti@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Buchan, Gyoengyi, E-mail: buchan@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Madi, Andras, E-mail: madi@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Stem Cell, Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Uher, Ferenc, E-mail: uher@biomembrane.hu [Stem Cell Laboratory, Hungarian National Blood Transfusion Service, Dioszegi ut 64, H-1113 Budapest (Hungary); and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. Black-Right-Pointing-Pointer hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. Black-Right-Pointing-Pointer SV40T introduced along with hTERT abrogates proliferation control and multipotency. Black-Right-Pointing-Pointer hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC{sup hTERT}, ASC{sup Bmi-1}, ASC{sup Bmi-1+hTERT} and ASC{sup SV40T+hTERT} were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC{sup Bmi-1} had limited replicative potential, while the rapidly proliferating ASC{sup SV40T+hTERT} acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC{sup hTERT} and ASC{sup hTERT+Bmi-1}, on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC{sup hTERT} also acquired aberrant karyotype and showed signs of transformation after long-term culture

  8. A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

    Directory of Open Access Journals (Sweden)

    Brittney-Shea Herbert

    Full Text Available Autosomal dominant polycystic kidney disease (ADPKD is associated with a variety of cellular phenotypes in renal epithelial cells. Cystic epithelia are secretory as opposed to absorptive, have higher proliferation rates in cell culture and have some characteristics of epithelial to mesenchymal transitions. In this communication we describe a telomerase immortalized cell line that expresses proximal tubule markers and is derived from renal cysts of an ADPKD kidney. These cells have a single detectable truncating mutation (Q4004X in polycystin-1. These cells make normal appearing but shorter cilia and fail to assemble polycystin-1 in the cilia, and less uncleaved polycystin-1 in membrane fractions. This cell line has been maintained in continuous passage for over 35 passages without going into senescence. Nephron segment specific markers suggest a proximal tubule origin for these cells and the cell line will be useful to study mechanistic details of cyst formation in proximal tubule cells.

  9. Suppression of the HPA axis during extrahepatic biliary obstruction induces cholangiocyte proliferation in the rat.

    Science.gov (United States)

    Quinn, Matthew; Ueno, Yoshiyuki; Pae, Hae Yong; Huang, Li; Frampton, Gabriel; Galindo, Cheryl; Francis, Heather; Horvat, Darijana; McMillin, Matthew; Demorrow, Sharon

    2012-01-01

    Cholestatic patients often present with clinical features suggestive of adrenal insufficiency. In the bile duct-ligated (BDL) model of cholestasis, the hypothalamic-pituitary-adrenal (HPA) axis is suppressed. The consequences of this suppression on cholangiocyte proliferation are unknown. We evaluated 1) HPA axis activity in various rat models of cholestasis and 2) effects of HPA axis modulation on cholangiocyte proliferation. Expression of regulatory molecules of the HPA axis was determined after BDL, partial BDL, and α-naphthylisothiocyanate (ANIT) intoxication. The HPA axis was suppressed by inhibition of hypothalamic corticotropin-releasing hormone (CRH) expression by central administration of CRH-specific Vivo-morpholinos or by adrenalectomy. After BDL, the HPA axis was reactivated by 1) central administration of CRH, 2) systemic ACTH treatment, or 3) treatment with cortisol or corticosterone for 7 days postsurgery. There was decreased expression of 1) hypothalamic CRH, 2) pituitary ACTH, and 3) key glucocorticoid synthesis enzymes in the adrenal glands. Serum corticosterone and cortisol remained low after BDL (but not partial BDL) compared with sham surgery and after 2 wk of ANIT feeding. Experimental suppression of the HPA axis increased cholangiocyte proliferation, shown by increased cytokeratin-19- and proliferating cell nuclear antigen-positive cholangiocytes. Conversely, restoration of HPA axis activity inhibited BDL-induced cholangiocyte proliferation. Suppression of the HPA axis is an early event following BDL and induces cholangiocyte proliferation. Knowledge of the role of the HPA axis during cholestasis may lead to development of innovative treatment paradigms for chronic liver disease.

  10. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    Science.gov (United States)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  11. Science, self, and immortality.

    Science.gov (United States)

    Haught, John F

    2011-10-01

    The following considers the concept of scientific naturalism in relation to life after death and contrasts three alternative perspectives on immortality of the soul, including naturalistic fatalism, otherworldly optimism, and long-suffering hope. © 2011 New York Academy of Sciences.

  12. Functional pharmacological evidence for EP2 and EP4 prostanoid receptors in immortalized human trabecular meshwork and non-pigmented ciliary epithelial cells.

    Science.gov (United States)

    Crider, J Y; Sharif, N A

    2001-02-01

    The aim of these studies was to characterize the molecular pharmacology of the prostanoid receptors positively coupled to stimulation of adenylyl cyclase activity in immortalized human trabecular meshwork (TM-3) cells and to compare these results with that of the receptors in immortalized human nonpigmented epithelial (NPE) cells. In general, the TM-3 and NPE cells showed a similar profile with respect to their responses to various prostaglandin (PG) receptor agonists. The rank order of potency (EC50; means +/- SEM) for these compounds in the TM-3 cells was: PGE2 (124 +/- 21 nM) > 13,14-dihydro-PGE1 (430 +/- 110 nM) = PGE1 (522 +/- 345 nM) > 11-deoxy-PGE1 (1063 +/- 118 nM) = 16,16-dimethyl-PGE2 (1776 +/- 460 nM) = butaprost (1920 +/- 527 nM) > PGD2 = PGI2 = PGF2alpha (n = 3 - 12). While the agonist profile indicated the presence of EP2 receptors, the effects of the EP4 receptor antagonists suggested the additional expression of EP4 receptors in both of these cells. Thus, the EP4 receptor antagonist, AH23848B, at a concentration of 30 microM, caused a dextral shift in the PGE2 concentration-response curves in both TM-3 and NPE cells coupled with a 20-28% decrease in the maximal response of PGE2, indicating apparent noncompetitive antagonism profiles. The antagonist potency of AH23848B in these cells was: Kb = 38.4 +/- 14.8 microM and 23.5 +/- 4.5 microM; -log Kb = 4.7. The other EP4 receptor antagonist, AH22921 (-log Kb = 4.1 - 4.7), was weaker than AH23848B. Taken together, these pharmacological studies have shown than TM-3 and NPE cells apparently contain functional EP2 and EP4 prostanoid receptors positively coupled to adenylyl cyclase.

  13. Conditionally immortalized human proximal tubular epithelial cells isolated from the urine of a healthy subject express functional calcium-sensing receptor.

    Science.gov (United States)

    Di Mise, Annarita; Tamma, Grazia; Ranieri, Marianna; Svelto, Maria; Heuvel, Bert van den; Levtchenko, Elena N; Valenti, Giovanna

    2015-06-01

    The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, which plays an essential role in regulating Ca(2+) homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Immunolocalization studies of polarized ciPTEC revealed the apical localization of the receptor. By Western blotting of ciPTEC lysates, both monomeric and dimeric forms of CaSR at 130 and ∼250 kDa, respectively, were detected. Functional studies indicated that both external calcium and the positive CaSR allosteric modulator, NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to its agonists. Calcium depletion from the endoplasmic reticulum using cyclopiazonic acid abolished the increase in cytosolic calcium elicited by NPS-R568, confirming calcium exit from intracellular stores. Activation of CaSR by NPS-R significantly reduced the increase in cAMP elicited by forskolin (FK), a direct activator of adenylate cyclase, further confirming the functional expression of the receptor in this cell line. CaSR expressed in ciPTEC was found to interact with Gq as a downstream effector, which in turn can cause release of calcium from intracellular stores via phospholipase C activation. We conclude that human proximal tubular ciPTEC express functional CaSR and respond to its activation with a release of calcium from intracellular stores. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients. Copyright © 2015 the American Physiological Society.

  14. Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M. [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States); Mondal, Sumona [Department of Mathematics, Clarkson University, Potsdam, NY 13699-5805 (United States); Woodworth, Craig D., E-mail: woodworth@clarkson.edu [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States)

    2012-03-30

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

  15. Universal mortality law and immortality

    Science.gov (United States)

    Azbel', Mark Ya.

    2004-10-01

    Well-protected human and laboratory animal populations with abundant resources are evolutionarily unprecedented. Physical approach, which takes advantage of their extensively quantified mortality, establishes that its dominant fraction yields the exact law, which is universal for all animals from yeast to humans. Singularities of the law demonstrate new kinds of stepwise adaptation. The law proves that universal mortality is an evolutionary by-product, which at any given age is reversible, independent of previous life history, and disposable. Life expectancy may be extended, arguably to immortality, by minor biological amendments in the animals. Indeed, in nematodes with a small number of perturbed genes and tissues it increased 6-fold (to 430 years in human terms), with no apparent loss in health and vitality. The law relates universal mortality to specific processes in cells and their genetic regulation.

  16. Differential expression of cholangiocyte and ileal bile acid transporters following bile acid supplementation and depletion

    Institute of Scientific and Technical Information of China (English)

    N. Sertac Kip; Konstantinos N. Lazaridis; Anatoliy I. Masyuk; Patrick L. Splinter; Robert C. Huebert; Nicholas F. LaRusso

    2004-01-01

    AIM: We have previously demonstrated that cholangiocytes,the epithelial cells lining intrahepatic bile ducts, encode two functional bile acid transporters via alternative splicing of a single gene to facilitate bile acid vectorial transport.Cholangiocytes possess ASBT, an apical sodium-dependent bile acid transporter to take up bile acids, and t-ASBT, a basolateral alternatively spliced and truncated form of ASBT to efflux bile acids. Though hepatocyte and ileal bile acid transporters are in part regulated by the flux of bile acids,the effect of alterations in bile acid flux on the expression of t-ASBT in terminal ileocytes remains unclear. Thus, we tested the hypothesis that expression of ASBT and t-ASBT in cholangiocytes and ileocytes was regulated by bile acid flux. METHODS: Expression of ASBT and t-ASBT message and protein in cholangiocytes and ileocytes isolated from pairfed rats given control (C) and 1% taurocholate (TCA) or 5% cholestyramine (CY) enriched diets, were assessed by both quantitative RNase protection assays and quantitative immunoblotting. The data obtained from each of the control groups were pooled to reflect the changes observed following TCA and CY treatments with respect to the control diets.Cholangiocyte taurocholate uptake was determined using a novel microperfusion technique on intrahepatic bile duct units (IBDUs) derived from C, TCA and CY fed rats.RESULTS: In cholangiocytes, both ASBT and t-ASBT message RNA and protein were significantly decreased in response to TCA feeding compared to C diet. In contrast,message and protein of both bile acid transporters significantly increased following CY feeding compared to C diet. In the ileum, TCA feeding significantly up-regulated both ASBT and t-ASBT message and protein compared to C diet, while CY feeding significantly down-regulated message and protein of both bile acid transporters compared to C diet. As anticipated from alterations in cholangiocyte ASBT expression, the uptake of

  17. Symbiotic Plant Peptides Eliminate Candida albicans Both In Vitro and in an Epithelial Infection Model and Inhibit the Proliferation of Immortalized Human Cells

    Directory of Open Access Journals (Sweden)

    Lilla Ördögh

    2014-01-01

    Full Text Available The increasing number of multidrug-resistant microbes now emerging necessitates the identification of novel antimicrobial agents. Plants produce a great variety of antimicrobial peptides including hundreds of small, nodule-specific cysteine-rich NCR peptides that, in the legume Medicago truncatula, govern the differentiation of endosymbiotic nitrogen fixing bacteria and, in vitro, can display potent antibacterial activities. In this study, the potential candidacidal activity of 19 NCR peptides was investigated. Cationic NCR peptides having an isoelectric point above 9 were efficient in killing Candida albicans, one of the most common fungal pathogens of humans. None of the tested NCR peptides were toxic for immortalized human epithelial cells at concentrations that effectively killed the fungus; however, at higher concentrations, some of them inhibited the division of the cells. Furthermore, the cationic peptides successfully inhibited C. albicans induced human epithelial cell death in an in vitro coculture model. These results highlight the therapeutic potential of cationic NCR peptides in the treatment of candidiasis.

  18. Quantitative assessment of the relative antineoplastic potential of the n-butanolic leaf extract of Annona muricata Linn. in normal and immortalized human cell lines.

    Science.gov (United States)

    George, V Cijo; Kumar, D R Naveen; Rajkumar, V; Suresh, P K; Kumar, R Ashok

    2012-01-01

    Natural products have been the target for cancer therapy for several years but there is still a dearth of information on potent compounds that may protect normal cells and selectively destroy cancerous cells. The present study was aimed to evaluate the cytotoxic potential of n-butanolic leaf extract of Annona muricata L. on WRL-68 (normal human hepatic cells), MDA-MB-435S (human breast carcinoma cells) and HaCaT (human immortalized keratinocyte cells) lines by XTT assay. Prior to cytotoxicity testing, the extract was subjected to phytochemical screening for detecting the presence of compounds with therapeutic potential. Their relative antioxidant properties were evaluated using the reducing power and DPPH* radical scavenging assay. Since most of the observed chemo-preventive potential invariably correlated with the amount of total phenolics present in the extract, their levels were quantified and identified by HPLC analysis. Correlation studies indicated a strong and significant (PAnnona muricata. Isolation of the active metabolites from the extract is in prospect.

  19. Role of gp91phox Homolog Nox1 in Induction of Premalignant Spindle Phenotypes of HPV 16 E6/E7—Immortalized Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Walee Chamulitrat

    2010-01-01

    Full Text Available The NADPH oxidase (Nox family of superoxide- and hydrogen peroxide—producing proteins has been recognized as important for signal transduction that regulates receptor-mediated functions, including cytoskeleton remodeling, cell proliferation, migration, differentiation, and cell death. Nox1 was the first of the Nox catalytic subunits to be cloned and shown to induce tumorigenic conversion of mouse fibroblasts. While Nox1 has been shown to be expressed in human colon and prostate cancers, however, limited studies have demonstrated the involvement of Nox1 in an early step of cell transformation. The aim of this review is to provide an overview on the role of Nox1 in cancer, as well as the contribution of our studies to demonstrate the involvement of Nox1 on neoplastic progression of human keratinocytes beyond the immortalization step. The generation of spindle phenotypes concomitant with anchorage-independent growth and invasiveness will be highlighted and discussed in relation to the possible role of Nox1 in epithelial-mesenchymal transition. Understanding these mechanisms may provide insight into Nox1 and redox signaling components as potential therapeutic targets to inhibit tumor progression.

  20. Hedgehog Signaling Overcomes an EZH2-Dependent Epigenetic Barrier to Promote Cholangiocyte Expansion

    Science.gov (United States)

    Lu, Jie; Almada, Luciana L.; Lomberk, Gwen; Fernandez-Zapico, Martin E.; Urrutia, Raul; Huebert, Robert C.

    2016-01-01

    Background & Aims Developmental morphogens play an important role in coordinating the ductular reaction and portal fibrosis occurring in the setting of cholangiopathies. However, little is known about how membrane signaling events in ductular reactive cells (DRCs) are transduced into nuclear transcriptional changes to drive cholangiocyte maturation and matrix deposition. Therefore, the aim of this study was to investigate potential mechanistic links between cell signaling events and epigenetic regulators in DRCs. Methods Using directed differentiation of induced pluripotent stem cells (iPSC), isolated DRCs, and in vivo models, we examine the mechanisms whereby sonic hedgehog (Shh) overcomes an epigenetic barrier in biliary precursors and promotes both cholangiocyte maturation and deposition of fibronectin (FN). Results We demonstrate, for the first time, that Gli1 influences the differentiation state and fibrogenic capacity of iPSC-derived hepatic progenitors and isolated DRCs. We outline a novel pathway wherein Shh-mediated Gli1 binding in key cholangiocyte gene promoters overcomes an epigenetic barrier conferred by the polycomb protein, enhancer of zeste homolog 2 (EZH2) and initiates the transcriptional program of cholangiocyte maturation. We also define previously unknown functional Gli1 binding sites in the promoters of cytokeratin (CK)7, CK19, and FN. Our in vivo results show that EZH2 KO mice fed the choline-deficient, ethanolamine supplemented (CDE) diet have an exaggerated cholangiocyte expansion associated with more robust ductular reaction and increased peri-portal fibrosis. Conclusion We conclude that Shh/Gli1 signaling plays an integral role in cholangiocyte maturation in vitro by overcoming an EZH2-dependent epigenetic barrier and this mechanism also promotes biliary expansion in vivo. PMID:27936185

  1. Borges, immortality and the circular ruins.

    Science.gov (United States)

    Bronstein, Catalina

    2002-06-01

    The author explores ideas surrounding immortality and death focusing on the interplay between their development in two stories by Borges ('The circular ruins' and 'The immortal') and their manifestation in a patient. With the help of Borges's stories, the author addresses the desperate necessity experienced by some individuals to search for immortality. This is not just an expression of the universal wish to live forever but, at a deeper level, arises from the impossibility of bearing the mental pain of experiencing ordinary human vulnerability and loss - death being the ultimate expression of such vulnerability. It is suggested that the relentless pursuit of immortality in such individuals expresses an omnipotent phantasy of ridding the self of the emotional pain and fear that arises through being alive. It leads to a denial of the emotional significance of passage of time, of separation and sexual differences. In actuality, the individual's state of not feeling approximates to a complete loss of human identity and emotional death, with no place for any meaningful others. The individual him/herself becomes a 'mere image', living in a delusional world peopled by him/herself and his/ her projections, and ending up trapped inside the circular ruins he/she has generated. The horror experienced at the stark awareness of the individual's emotional death and the wish to re-establish contact with the good internal objects that have been attacked sets in motion the long process of searching for the recovery of a sense of temporality (that would still include the wish for immortality) and, with it, a sense of identity.

  2. Ageing and immortality.

    Science.gov (United States)

    Rose, M R; Mueller, L D

    2000-01-01

    The concept of the force of natural selection was developed to explain the evolution of ageing. After ageing, however, comes a period in which mortality rates plateau and some individual organisms could, in theory, live forever. This late-life immortality has no presently agreed upon explanation. Two main theories have been offered. The first is heterogeneity within ageing cohorts, such that only extremely robust individuals survive ageing. This theory can be tested by comparisons of more and less robust cohorts. It can also be tested by fitting survival data to its models. The second theory is that late-life plateaus in mortality reflect the inevitable late-life plateau in the force of natural selection. This theory can be tested by changing the force of natural selection in evolving laboratory populations, particularly the age at which the force plateaus. This area of research has great potential for elucidating the overall structure of life-history evolution, particularly the interrelationship between the three life-history phases of development, ageing and immortality. PMID:11127912

  3. Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    C.M.A. Reijnders; A. van Lier; S. Roffel; D. Kramer; R.J. Scheper; S. Gibbs

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  4. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

    NARCIS (Netherlands)

    Reijnders, Christianne M. A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.; Gibbs, Susan

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  5. Stages based molecular mechanisms for generating cholangiocytes from liver stem/progenitor cells.

    Science.gov (United States)

    Liu, Wei-Hui; Ren, Li-Na; Chen, Tao; Liu, Li-Ye; Tang, Li-Jun

    2013-11-07

    Except for the most organized mature hepatocytes, liver stem/progenitor cells (LSPCs) can differentiate into many other types of cells in the liver including cholangiocytes. In addition, LSPCs are demonstrated to be able to give birth to other kinds of extra-hepatic cell types such as insulin-producing cells. Even more, under some bad conditions, these LSPCs could generate liver cancer stem like cells (LCSCs) through malignant transformation. In this review, we mainly concentrate on the molecular mechanisms for controlling cell fates of LSPCs, especially differentiation of cholangiocytes, insulin-producing cells and LCSCs. First of all, to certificate the cell fates of LSPCs, the following three features need to be taken into account to perform accurate phenotyping: (1) morphological properties; (2) specific markers; and (3) functional assessment including in vivo transplantation. Secondly, to promote LSPCs differentiation, systematical attention should be paid to inductive materials (such as growth factors and chemical stimulators), progressive materials including intracellular and extracellular signaling pathways, and implementary materials (such as liver enriched transcriptive factors). Accordingly, some recommendations were proposed to standardize, optimize, and enrich the effective production of cholangiocyte-like cells out of LSPCs. At the end, the potential regulating mechanisms for generation of cholangiocytes by LSPCs were carefully analyzed. The differentiation of LSPCs is a gradually progressing process, which consists of three main steps: initiation, progression and accomplishment. It's the unbalanced distribution of affecting materials in each step decides the cell fates of LSPCs.

  6. SOX17 Regulates Cholangiocyte Differentiation and Acts as a Tumor Suppressor in Cholangiocarcinoma

    DEFF Research Database (Denmark)

    Merino-Azpitarte, M; Lozano, E; Perugorria, M J

    2017-01-01

    BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a biliary malignancy linked to genetic and epigenetic abnormalities, such as hypermethylation of SOX17 promoter. Here, the role of SOX17 in cholangiocyte differentiation and cholangiocarcinogenesis was studied. METHODS: SOX17 expression/function was ...

  7. Long-term exposure to cigarette smoke extract induces hypomethylation at the RUNX3 and IGF2-H19 loci in immortalized human urothelial cells.

    Directory of Open Access Journals (Sweden)

    Li-Mei Chen

    Full Text Available Cigarette smoking is the single most important epidemiological risk factor for bladder cancer but it is not known whether exposure of urothelial cells to the systemic soluble contents of cigarette smoke is directly causative to bladder cancer and the associated epigenetic changes such as tumor suppressor gene hypermethylation. We undertook this study to investigate if long-term treatment of human urothelial cells with cigarette smoke extract (CSE results in tumor suppressor gene hypermethylation, a phenotype that was previously associated with long-term constant CSE treatment of airway epithelial cells. We chronically treated an immortalized human urothelial cell line UROtsa with CSE using a cyclic daily regimen but the cells were cultured in CSE-free medium between daily treatments. Bisulfite sequencing and real-time PCR array-based methylation profiling were employed to evaluate methylation changes at tumor suppressor gene loci in the chronically CSE-treated cells versus the passage-matched untreated control cells. The RUNX3 tumor suppressor gene promoter was hypomethylated with a significant increase in proportion of the completely unmethylated haplotype after the long-term CSE treatment; whereas RUNX3 promoter hypermethylation was previously reported for bladder cancers of smokers. Hypomethylation induced by the long-term CSE treatment was also observed for the IGF2-H19 locus. The methylation status at the PRSS8/prostasin and 16 additional loci however, was unaffected by the chronic CSE treatment. Transient CSE treatment over 1 daily regimen resulted in transcriptional down-regulation of RUNX3 and H19, but only the H19 transcription was down-regulated in the chronically CSE-treated urothelial cells. Transcription of a key enzyme in one-carbon metabolism, dihydrofolate reductase (DHFR was greatly reduced by the long-term CSE treatment, potentially serving as a mechanism for the hypomethylation phenotype via a reduced supply of methyl donor

  8. The Immortal Fire Within

    Science.gov (United States)

    Sheehan, William

    2007-12-01

    Preface; Key to abbreviations in notes; 1. Through rugged ways; 2. Ardent and faithful work with a telescope; 3. Mars; his moons and his heavens; 4. A seeker of comets; 5. Vanderbilt astronomer; 6. In the realm of the nebulae; 7. Go west, young man!; 8. Hanging fire; 9. On Mt. Hamilton; 10. A year of wonders; 11. The young rebel; 12. 'I am tired here'; 13. Immortality; 14. Travels and travails; 15. Barnard and Mars; 16. Nature's true artisan; 17. A tide in his affairs; 18. Yerkes observatory; 19. Disappointments and triumphs; 20. The comet and Milky Way photographs; 21. Comet tales; 22. Observer of all that shines - or obscures; 23. Eclipse and decline; 24. Ad astra; Index.

  9. Low-Dose Methylmercury-Induced Genes Regulate Mitochondrial Biogenesis via miR-25 in Immortalized Human Embryonic Neural Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Xinjin Wang

    2016-12-01

    Full Text Available Mitochondria are essential organelles and important targets for environmental pollutants. The detection of mitochondrial biogenesis and generation of reactive oxygen species (ROS and p53 levels following low-dose methylmercury (MeHg exposure could expand our understanding of underlying mechanisms. Here, the sensitivity of immortalized human neural progenitor cells (ihNPCs upon exposure to MeHg was investigated. We found that MeHg altered cell viability and the number of 5-ethynyl-2′-deoxyuridine (EdU-positive cells. We also observed that low-dose MeHg exposure increased the mRNA expression of cell cycle regulators. We observed that MeHg induced ROS production in a dose-dependent manner. In addition, mRNA levels of peroxisome-proliferator-activated receptor gammacoactivator-1α (PGC-1α, mitochondrial transcription factor A (TFAM and p53-controlled ribonucleotide reductase (p53R2 were significantly elevated, which were correlated with the increase of mitochondrial DNA (mtDNA copy number at a concentration as low as 10 nM. Moreover, we examined the expression of microRNAs (miRNAs known as regulatory miRNAs of p53 (i.e., miR-30d, miR-1285, miR-25. We found that the expression of these miRNAs was significantly downregulated upon MeHg treatment. Furthermore, the overexpression of miR-25 resulted in significantly reducted p53 protein levels and decreased mRNA expression of genes involved in mitochondrial biogenesis regulation. Taken together, these results demonstrated that MeHg could induce developmental neurotoxicity in ihNPCs through altering mitochondrial functions and the expression of miRNA.

  10. A novel method for banking stem cells from human exfoliated deciduous teeth: lentiviral TERT immortalization and phenotypical analysis

    OpenAIRE

    Yin, Zhanhai; Wang, Qi; Li, Ye; Wei, Hong; Shi, Jianfeng; Li, Ang

    2016-01-01

    Background Stem cells from human exfoliated deciduous teeth (SHED) have recently attracted attention as novel multipotential stem cell sources. However, their application is limited due to in vitro replicative senescence. Ectopic expression of telomerase reverse transcriptase (TERT) is a promising strategy for overcoming this replicative senescence. Nevertheless, its potential application and the phenotype as well as tumorigenicity have never been assessed in SHED. Methods TERT expression was...

  11. Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen.

    Science.gov (United States)

    van Leeuwen, E B; Veenstra, R; van Wijk, R; Molema, G; Hoekstra, A; Ruiters, M H; van der Meer, J

    2000-01-01

    Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but aSMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.

  12. Immortality versus resurrection in the Christian tradition.

    Science.gov (United States)

    Murphy, Nancey

    2011-10-01

    For those in contemporary society who believe in an afterlife, there are a number of views available. The most common may be based on belief in an immortal soul. However, the early Christian account was, instead, bodily resurrection. As Christianity moved throughout the Mediterranean world, apologists and theologians adapted their teaching on human nature and the afterlife to Greek and Roman philosophies. By the time of Augustine (d. 430), the doctrines of body-soul dualism and immortality of the soul were firmly entrenched in Christian teaching. The incorporation of the concept of an immortal soul into Christian accounts of life after death produced a hybrid account. The body dies, the soul (at least of those who were to be saved) travels to heaven. At the end of history, there would be a general resurrection, and the souls would be reunited with their bodies, although the bodies would be in a transformed, indestructible state. This hybrid account of life after death went largely uncontested until the twentieth century. In this essay, I describe this history and argue for a return to the early Christian view of humans as a unity, not a duality, and for belief in resurrection of the body as the appropriate expectation for eternal life. This would not only be truer to Christian sources, but, valuable, I believe, in focusing Christian attention on the need to care for the environment. © 2011 New York Academy of Sciences.

  13. EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

    Science.gov (United States)

    da Rosa, Marina Rolo Pinheiro; Falcão, Aline Semblano Carreira; Fuzii, Hellen Thais; da Silva Kataoka, Maria Sueli; Ribeiro, André L R; Boccardo, Enrique; de Siqueira, Adriane Sousa; Jaeger, Ruy G; de Jesus Viana Pinheiro, João; de Melo Alves Júnior, Sérgio

    2014-11-01

    Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion

  14. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE.

    Science.gov (United States)

    Graur, Dan; Zheng, Yichen; Price, Nicholas; Azevedo, Ricardo B R; Zufall, Rebecca A; Elhaik, Eran

    2013-01-01

    A recent slew of ENCyclopedia Of DNA Elements (ENCODE) Consortium publications, specifically the article signed by all Consortium members, put forward the idea that more than 80% of the human genome is functional. This claim flies in the face of current estimates according to which the fraction of the genome that is evolutionarily conserved through purifying selection is less than 10%. Thus, according to the ENCODE Consortium, a biological function can be maintained indefinitely without selection, which implies that at least 80 - 10 = 70% of the genome is perfectly invulnerable to deleterious mutations, either because no mutation can ever occur in these "functional" regions or because no mutation in these regions can ever be deleterious. This absurd conclusion was reached through various means, chiefly by employing the seldom used "causal role" definition of biological function and then applying it inconsistently to different biochemical properties, by committing a logical fallacy known as "affirming the consequent," by failing to appreciate the crucial difference between "junk DNA" and "garbage DNA," by using analytical methods that yield biased errors and inflate estimates of functionality, by favoring statistical sensitivity over specificity, and by emphasizing statistical significance rather than the magnitude of the effect. Here, we detail the many logical and methodological transgressions involved in assigning functionality to almost every nucleotide in the human genome. The ENCODE results were predicted by one of its authors to necessitate the rewriting of textbooks. We agree, many textbooks dealing with marketing, mass-media hype, and public relations may well have to be rewritten.

  15. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S; Abdallah, Basem M; Guldberg, Per

    2005-01-01

    Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation...... or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had...... tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer....

  16. Investigation of the in vitro therapeutic efficacy of nilotinib in immortalized human NF2-null vestibular schwannoma cells.

    Directory of Open Access Journals (Sweden)

    Nesrin Sabha

    Full Text Available Vestibular schwannomas (VS are a common posterior fossa brain tumor, and though benign can cause significant morbidity, particularly loss of hearing, tinnitus, vertigo and facial paralysis. The current treatment options for VS include microsurgical resection, stereotactic radiosurgery or close surveillance monitoring, with each treatment option carrying associated complications and morbidities. Most importantly, none of these options can definitively reverse hearing loss or tinnitus. Identification of a novel medical therapy, through the use of targeted molecular inhibition, is therefore a highly desirable treatment strategy that may minimize complications arising from both tumor and treatment and more importantly be suitable for patients whose options are limited with respect to surgical or radiosurgical interventions. In this study we chose to examine the effect of Nilotinib on VS. Nilotinib (Tasigna® is a second-generation receptor tyrosine kinase (RTK inhibitor with a target profile similar to that of imatinib (Gleevec®, but increased potency, decreased toxicity and greater cellular and tissue penetration. Nilotinib targets not only the BCR-ABL oncoprotein, but also platelet-derived growth factor (PDGF receptor signalling. In this preclinical study, the human NF2-null schwannoma cell line HEI-193 subjected to nilotinib inhibition demonstrated decreased viability, proliferation and anchorage-independent growth, and increased apoptosis. A daily dose of nilotinib for 5 days inhibited HEI-I93 proliferation at a clinically-relevant concentration in a dose-dependent manner (IC(50 3-5 µmol/L in PDGF-stimulated cells. These anti-tumorigenic effects of nilotinib were correlated to inhibited activation of PDGFR-α and PDGFR-β and major downstream signalling pathways. These experiments support a therapeutic potential for Nilotinib in VS.

  17. Immortality in the Christian Physicalistic Theology: A Critical Survey

    Directory of Open Access Journals (Sweden)

    Hasan Ahmadizade

    2017-07-01

    Full Text Available Physicalistic Theology is a term that has no exact definition in theologian views. In the 20th century some of Christian thinkers on theology, like Nancy Murphy and Peter van Inwagen, by accepting a Physicalistic approach on human being, tried to analyze the Christian beliefs about human identity and his immortality. This approach today is called Physicalistic Theology. According to this approach, human is not but this physical body itself and so we can simply analyze the immortality problem. In this article we try to by an analytic and descriptive method, analyze the immortality of human according to the view of Physicalistic Theology. We will analyze the most important reasoning of Physicalistic Theology that is: no-interaction between the material and the immaterial, interaction between the person and the body, and the physicalism in Christian beliefs. One of the conclusions of this article is that according to Physicalistic view, the person that at some time has not been in the world, must exists any time to destroyed forever because the Christians believe to things that cannot justify rationally. The problem of immortality is one of these matters. Physicalistic Theology try to prove the immortality based on the miracles and the absolute power of God.

  18. Triggering of toll-like receptors 2 and 4 by Aspergillus fumigatus conidia in immortalized human corneal epithelial cells to induce inflammatory cytokines

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jie; WU Xin-yi

    2008-01-01

    Background Cornea epithelial cells play early and crucial roles in the initiation of ocular surface responses to pathogens.Participation of toll-like receptor(TLR)2 and TLR4,which are major forms of fungi receptors,may be involved in Aspergillus fumigatus induced immune responses.The obiective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-κB activation and production of proinflammaory cytokines,and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells (THCEs).This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi.Methods Aspergillus fumigatus conidia were used to challenge THCE cells.THCE cells were harvested after 0.5,1,2or 4 hours incubation.Real-time quantitative PCR was performed to determine the expression of TLR2,TLR4,TNF-α and IL-8.Western blotting was performed to determine the expression of NF-κB.Enzyme-linked immunosorbent assay (EUSA)was performed to determine the expression of TNF-α and IL-8.And the release of TNF-α and IL-8 in the cell supematant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies.Results Aspergillus fumigatus conidia elicited the expression of TLR2,TLR4,TNF-α and IL-8 mRNA in THCEs.Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-κB activation,which increased at 30 minutes (increased from 11.35±2.74 in the controls to 19.12±3.48,P<0.05)and thereafter increased steadily up to 4 hours after challenge(P<0.01).Concomitant with NF-κB acfivation,secretion of TNF-α and IL-8 in conidia-challenged cells was increased in a time-dependent manner.Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in jnhibifion of conidia-induced TNF-α and IL-8 secretion(P<0.05),TLR2 antibody and TLR4 antibody together significantly increased

  19. A Polyphenol-Enriched Fraction of Rose Oil Distillation Wastewater Inhibits Cell Proliferation, Migration and TNF-α-Induced VEGF Secretion in Human Immortalized Keratinocytes.

    Science.gov (United States)

    Wedler, Jonas; Rusanov, Krasimir; Atanassov, Ivan; Butterweck, Veronika

    2016-07-01

    Water steam distillation of rose flowers separates the essential oil from the polyphenol-containing rose oil distillation wastewater. Recently, a strategy was developed to separate rose oil distillation wastewater into a polyphenol depleted water fraction and a polyphenol-enriched fraction [RF20-(SP-207)]. The objective of the present study was to investigate RF20-(SP-207) and fraction F(IV), augmented in quercetin and ellagic acid, for possible antiproliferative effects in immortalized human keratinocytes (HaCaT) since rose petals are known to contain compounds with potential antiproliferative activity.RF20-(SP-207) revealed dose-dependent antiproliferative activity (IC50 of 9.78 µg/mL). In a nontoxic concentration of 10 µg/mL, this effect was stronger than that of the two positive controls LY294002 (10 µM, PI3 K-inhibitor, 30 % inhibition) and NVP-BEZ235 (100 nM, dual PI3 K/mTOR inhibitor, 30 % inhibition) and clearly exceeded the antiproliferative action of quercetin (50 µM, 25 % inhibition) and ellagic acid (1 µM, 15 % inhibition). Time-lapse microscopy detected a significant impairment of cell migration of RF20-(SP-207) and F(IV). At concentrations of 10 µg/mL of both, extract and fraction, cell migration was strongly suppressed (51 % and 28 % gap closure, respectively, compared to 95 % gap closure 24 hours after control treatment). The suppression of cell migration was comparable to the positive controls LY294002, NVP-BEZ235, and quercetin. Furthermore, basal and TNF-α-stimulated VEGF-secretion was significantly reduced by RF20-(SP-207) and F(IV) at 10 µg/mL (44 % vs. untreated control).In conclusion, RF20-(SP-207) showed promising antiproliferative and antimigratory effects and could be developed as a supportive, therapy against hyperproliferation-involved skin diseases.

  20. Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype

    OpenAIRE

    2015-01-01

    This is the final version. It was first published by OUP at http://dx.doi.org/10.1093/infdis/jiv291 Background.  Throughout Asia there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes including chronic inflammation and secretion of parasite proteins into the biliary epithelium drive infection towards cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown. ...

  1. Establishment of Immortalized Human Lymphocytes Cell Lines with Multiple SNP Genotyping in Chinese Han Population%中国汉族人群代谢酶基因分型永生化淋巴细胞库的建立

    Institute of Scientific and Technical Information of China (English)

    张娟; 熊梦祯; 尹立红; 浦跃朴

    2011-01-01

    目的 建立汉族人群代谢酶基因分型的永生化淋巴细胞库,为单核苷酸多态(SNP)结构对毒物诱导的基因功能表达的影响建立研究平台.方法 用EB病毒(Epstein-Barr Virus,EBV)转化中国汉族健康成人外周血B淋巴细胞,通过对中国汉族人群CYP2E1和NQ01的单核苷酸多态(SNP)位点信息分析,选择中国人群特异性CYP2EI、NQ01基因SNP位点CYP2E1rs2070673,CYP2E1rs2031921,CYP2E13813866,NQO1rs 1800566,NQO1rs10517,应用适配器连接介导的等位基因特异性扩增法(ALM-SAS)对上述位点进行基因分型.将供体信息和细胞株基因分型的信息录入,构建中国汉族基因分型的永生化淋巴细胞库.结果 通过对细胞株进行上述5个SNP位点的基因分型,获得每株细胞的基因分型信息,获得CYP2E1rs2070673,CYP2E1rs2031921,CYP2E13813866,NQO1rs 1800566,NQO1rs10517不同基因分型的永生化淋巴细胞株.结论 通过永生化细胞库的建立、基因分型,初步建立中国的汉族人群代谢酶基因分型永生化淋巴细胞库,该细胞库的进一步建设,可以为研究中国人群环境毒物和药物代谢酶基因结构和功能的关系提供技术平台.%Objective To establish immortalized human lymphocytes cell lines with multiple SNP genotyping of Chinese Han population which could afford a platform to study gene function expression influenced by SNPs structure induced by chemical Methods EBV (Epstein-Bart Virus) were used to immortalize peripheral B lymphocytes derived from Chinese Hah population. After analyzing CYP2EI、NQOI SNP database of Chinese population, the five Chinese specific SNP sites: CYP2EIrs2070673,CYP2Elrs2031921, CYP2E13813866,NQOlrs 1800566, NQOl rs10517 were genotyped by an adapter-ligation mediated allele specific amplification (ALM-ASA) method. The information of providers and genotypes of cell lines were input to establish the database of immortalized human lymphocytes cell lines with multiple SNP genotyping

  2. Long-term culture of cholangiocytes from liver fibro-granulomatous lesions

    Directory of Open Access Journals (Sweden)

    Borojevic Radovan

    2006-04-01

    Full Text Available Abstract Background Extensive bile duct proliferation is a key feature of the tissue reaction to clinical and experimental forms of liver injury. Experimental infection of mice by Schistosoma mansoni is a well-studied model of liver fibrosis with bile duct hyperplasia. However, the regulatory mechanisms of bile duct changes are not well understood. In this study we report the reproducible isolation of long-term cultures of cholangiocytes from mice livers with schistosomal fibrosis. Methods We have isolated a cholangiocyte cell line from Schistosoma-induced liver granulomas using a combination of methods including selective adhesion and isopyknic centrifugation in Percoll. Results The cell line was characterized by morphological criteria in optical and transmission electron microscopy, ability to form well differentiated ductular structures in collagen gels and by a positive staining for cytokeratin 18 and cytokeratin 19. To our knowledge, this is the first murine cholangiocyte cell line isolated from schistosomal fibrosis reported in the literature. Conclusion After 9 months and 16 passages this diploid cell line maintained differentiated characteristics and a high proliferative capacity. We believe the method described here may be a valuable tool to study bile duct changes during hepatic injury.

  3. The illusion of cell immortality

    Science.gov (United States)

    Hayflick, L

    2000-01-01

    Normal cultured cell populations are mortal but cells that are immortal are abnormal and most have properties of cancer cells. Nevertheless, this distinction becomes blurred because the terms ‘mortality’ and ‘immortality’ are subject to enormous variations in understanding. Forty years ago we showed that cell mortality and immortality are inextricably linked to longevity determination, ageing and cancer. We suggested that a counting mechanism existed in normal cells and that has now been identified as telomere attrition. This replicometer, in combination with the discovery of the enzyme telomerase, has gone very far in explaining why most normal somatic cells have a finite capacity to replicate both in vivo and in vitro and how immortal cancer cells circumvent this inevitability. It is suggested that telomere attrition may be better understood as a direct measure of longevity determination and to only have an indirect association with age changes. © 2000 Cancer Research Campaign PMID:10970682

  4. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

    Directory of Open Access Journals (Sweden)

    Ken Kono

    Full Text Available Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

  5. Immortalization of Fetal Bovine Colon Epithelial Cells by Expression of Human Cyclin D1, Mutant Cyclin Dependent Kinase 4, and Telomerase Reverse Transcriptase: An In Vitro Model for Bacterial Infection.

    Directory of Open Access Journals (Sweden)

    Kengo Kuroda

    Full Text Available Cattle are the economically important animals in human society. They are essential for the production of livestock products such as milk and meats. The production efficiency of livestock products is negatively impacted by infection with zoonotic pathogens. To prevent and control infectious diseases, it is important to understand the interaction between cattle tissue and pathogenic bacteria. In this study, we established an in vitro infection model of an immortalized bovine colon-derived epithelial cell line by transducing the cells with lentiviral vectors containing genes encoding cell cycle regulators cyclin D1, mutant cyclin dependent kinase 4 (CDK4, and human telomerase reverse transcriptase (TERT. The established cell line showed continuous cell proliferation, expression of epithelial markers, and an intact karyotype, indicating that the cells maintained their original nature as colon-derived epithelium. Furthermore, we exposed the established cell line to two strains of Salmonella enterica and EHEC. Interestingly, S. Typhimurium showed higher affinity for the established cell line and invaded the cytoplasm than S. Enteritidis. Quantitative RT-PCR revealed that gene expression of Toll-like receptor 1 (TLR1, TLR 2 and TLR 3, whereas TLR 4, 5 and 6 were not detectable in established cells. Our established immortalized colon-derived epithelial cell should be a useful tool for studies evaluating the molecular mechanisms underlying bacterial infection.

  6. Immortalization capacity of HPV types is inversely related to chromosomal instability

    DEFF Research Database (Denmark)

    Schütze, Denise M; Krijgsman, Oscar; Snijders, Peter J F

    2016-01-01

    High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality....... This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.......All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed...

  7. Immortality of cell lines: challenges and advantages of establishment.

    Science.gov (United States)

    Maqsood, Muhammad Irfan; Matin, Maryam M; Bahrami, Ahmad Reza; Ghasroldasht, Mohammad M

    2013-10-01

    Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.There are also some other factors and mechanisms involved in immortalisation, which need to be discovered. Immortalisation of cells derived from different sources and establishment of immortal cell lines has proven useful in understanding the molecular pathways governing cell developmental cascades in eukaryotic, especially human, cells. After the breakthrough of achieving the immortal cells and understanding their critical importance in the field of molecular biology, intense efforts have been dedicated to establish cell lines useful for elucidating the functions of telomerase, developmental lineage of progenitors, self-renewal potency, cellular transformation, differentiation patterns and some bioprocesses, like odontogenesis. Meanwhile, discovering the exact mechanisms of immortality, a major challenge for science yet, is believed to open new gateways toward understanding and treatment of cancer in the long term. This review summarises the methods involved in establishing immortality, its advantages and the challenges still being faced in this field. © 2013 International Federation for Cell Biology.

  8. Effects of hTERT immortalization on osteogenic and adipogenic differentiation of dental pulp stem cells

    Directory of Open Access Journals (Sweden)

    Ikbale El-Ayachi

    2016-03-01

    Full Text Available These data relate to the differentiation of human dental pulp stem cells (DPSC and DPSC immortalized by constitutively expressing human telomerase reverse transcriptase (hTERT through both osteogenic and adipogenic lineages (i.e. to make bone producing and fat producing cells from these dental pulp stem cells. The data augment another study to characterize immortalized DPSC for the study of neurogenetic “Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders” [1]. Two copies of one typical control cell line (technical replicates were used in this study. The data represent the differentiation of primary DPSC into osteoblast cells approximately 60% more effectively than hTERT immortalized DPSC. Conversely, both primary and immortalized DPSC are poorly differentiated into adipocytes. The mRNA expression levels for both early and late adipogenic and osteogenic gene markers are shown.

  9. The Golden Bough: Literature and the Idea of Immortality.

    Science.gov (United States)

    Klotz, Kenneth

    1979-01-01

    All works of literature reflect man's obsession with death and his creative attempts to evade or postpone it. Art cannot solve the problem, but within the limits of human capabilities, we come to art, whether as artists or audience, for our own small share in the "intimations of immortality." (Author/SJL)

  10. Immortality of Cu damascene interconnects

    Science.gov (United States)

    Hau-Riege, Stefan P.

    2002-04-01

    We have studied short-line effects in fully-integrated Cu damascene interconnects through electromigration experiments on lines of various lengths and embedded in different dielectric materials. We compare these results with results from analogous experiments on subtractively-etched Al-based interconnects. It is known that Al-based interconnects exhibit three different behaviors, depending on the magnitude of the product of current density, j, and line length, L: For small values of (jL), no void nucleation occurs, and the line is immortal. For intermediate values, voids nucleate, but the line does not fail because the current can flow through the higher-resistivity refractory-metal-based shunt layers. Here, the resistance of the line increases but eventually saturates, and the relative resistance increase is proportional to (jL/B), where B is the effective elastic modulus of the metallization system. For large values of (jL/B), voiding leads to an unacceptably high resistance increase, and the line is considered failed. By contrast, we observed only two regimes for Cu-based interconnects: Either the resistance of the line stays constant during the duration of the experiment, and the line is considered immortal, or the line fails due to an abrupt open-circuit failure. The absence of an intermediate regime in which the resistance saturates is due to the absence of a shunt layer that is able to support a large amount of current once voiding occurs. Since voids nucleate much more easily in Cu- than in Al-based interconnects, a small fraction of short Cu lines fails even at low current densities. It is therefore more appropriate to consider the probability of immortality in the case of Cu rather than assuming a sharp boundary between mortality and immortality. The probability of immortality decreases with increasing amount of material depleted from the cathode, which is proportional to (jL2/B) at steady state. By contrast, the immortality of Al-based interconnects is

  11. Disruptive chemicals, senescence and immortality.

    Science.gov (United States)

    Carnero, Amancio; Blanco-Aparicio, Carmen; Kondoh, Hiroshi; Lleonart, Matilde E; Martinez-Leal, Juan Fernando; Mondello, Chiara; Scovassi, A Ivana; Bisson, William H; Amedei, Amedeo; Roy, Rabindra; Woodrick, Jordan; Colacci, Annamaria; Vaccari, Monica; Raju, Jayadev; Al-Mulla, Fahd; Al-Temaimi, Rabeah; Salem, Hosni K; Memeo, Lorenzo; Forte, Stefano; Singh, Neetu; Hamid, Roslida A; Ryan, Elizabeth P; Brown, Dustin G; Wise, John Pierce; Wise, Sandra S; Yasaei, Hemad

    2015-06-01

    Carcinogenesis is thought to be a multistep process, with clonal evolution playing a central role in the process. Clonal evolution involves the repeated 'selection and succession' of rare variant cells that acquire a growth advantage over the remaining cell population through the acquisition of 'driver mutations' enabling a selective advantage in a particular micro-environment. Clonal selection is the driving force behind tumorigenesis and possesses three basic requirements: (i) effective competitive proliferation of the variant clone when compared with its neighboring cells, (ii) acquisition of an indefinite capacity for self-renewal, and (iii) establishment of sufficiently high levels of genetic and epigenetic variability to permit the emergence of rare variants. However, several questions regarding the process of clonal evolution remain. Which cellular processes initiate carcinogenesis in the first place? To what extent are environmental carcinogens responsible for the initiation of clonal evolution? What are the roles of genotoxic and non-genotoxic carcinogens in carcinogenesis? What are the underlying mechanisms responsible for chemical carcinogen-induced cellular immortality? Here, we explore the possible mechanisms of cellular immortalization, the contribution of immortalization to tumorigenesis and the mechanisms by which chemical carcinogens may contribute to these processes. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  12. Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation

    DEFF Research Database (Denmark)

    Abdallah, Basem M; Haack-Sørensen, Mandana; Burns, Jorge S

    2005-01-01

    Human bone marrow mesenchymal stem cells (hMSC) represent a population of stem cells that are capable of differentiation into multiple lineages. However, these cells exhibit senescence-associated growth arrest and phenotypic changes during long-term in vitro culture. We have recently demonstrated...

  13. Human T-cell leukemia virus type 1 expressing nonoverlapping tax and rex genes replicates and immortalizes primary human T lymphocytes but fails to replicate and persist in vivo.

    Science.gov (United States)

    Younis, Ihab; Yamamoto, Brenda; Phipps, Andrew; Green, Patrick L

    2005-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is an oncogenic retrovirus associated primarily with adult T-cell leukemia and neurological disease. HTLV-1 encodes the positive trans-regulatory proteins Tax and Rex, both of which are essential for viral replication. Tax activates transcription initiation from the viral long terminal repeat and modulates the transcription or activity of a number of cellular genes. Rex regulates gene expression posttranscriptionally by facilitating the cytoplasmic expression of incompletely spliced viral mRNAs. Tax and Rex mutants have been identified that have defective activities or impaired biochemical properties associated with their function. To ultimately determine the contribution of specific protein activities on viral replication and cellular transformation of primary T cells, mutants need to be characterized in the context of an infectious molecular clone. Since the tax and rex genes are in partially overlapping reading frames, mutation in one gene frequently disrupts the other, confounding interpretation of mutational analyses in the context of the virus. Here we generated and characterized a unique proviral clone (H1IT) in which the tax and rex genes were separated by expressing Tax from an internal ribosome entry site. We showed that H1IT expresses both functional Tax and Rex. In short- and long-term coculture assays, H1IT was competent to infect and immortalize primary human T cells similar to wild-type HTLV-1. In contrast, H1IT failed to efficiently replicate and persist in inoculated rabbits, thus emphasizing the importance of temporal and quantitative regulation of specific mRNA for viral survival in vivo.

  14. Does 'Immortal DNA strand' exist in 'immortal' stem cells?

    Institute of Scientific and Technical Information of China (English)

    Linheng Li

    2007-01-01

    @@ Stem cells function to generate differentiated cells,and,at the same time,are maintained as‘immortal'cells through self-renewal.Accumulated evidence indicates that adult stem cells may be the most common precursor for cancers in adult mammalian tissue[1],and this concept is gaining increasing support[2,3].Meanwhile,this also raises the question of how stem cells,which have a much longer lifespan compared to other progenitor and mature ceils,can avoid accumulating mutations from DNA replica-tion errors.

  15. Immortality of cancers: a consequence of inherent karyotypic variations and selections for autonomy.

    Science.gov (United States)

    Duesberg, Peter; McCormack, Amanda

    2013-03-01

    Immortality is a common characteristic of cancers, but its origin and purpose are still unclear. Here we advance a karyotypic theory of immortality based on the theory that carcinogenesis is a form of speciation. Accordingly, cancers are generated from normal cells by random karyotypic rearrangements and selection for cancer-specific reproductive autonomy. Since such rearrangements unbalance long-established mitosis genes, cancer karyotypes vary spontaneously but are stabilized perpetually by clonal selections for autonomy. To test this theory we have analyzed neoplastic clones, presumably immortalized by transfection with overexpressed telomerase or with SV40 tumor virus, for the predicted clonal yet flexible karyotypes. The following results were obtained: (1) All immortal tumorigenic lines from cells transfected with overexpressed telomerase had clonal and flexible karyotypes; (2) Searching for the origin of such karyotypes, we found spontaneously increasing, random aneuploidy in human fibroblasts early after transfection with overexpressed telomerase; (3) Late after transfection, new immortal tumorigenic clones with new clonal and flexible karyotypes were found; (4) Testing immortality of one clone during 848 unselected generations showed the chromosome number was stable, but the copy numbers of 36% of chromosomes drifted ± 1; (5) Independent immortal tumorigenic clones with individual, flexible karyotypes arose after individual latencies; (6) Immortal tumorigenic clones with new flexible karyotypes also arose late from cells of a telomerase-deficient mouse rendered aneuploid by SV40 virus. Because immortality and tumorigenicity: (1) correlated exactly with individual clonal but flexible karyotypes; (2) originated simultaneously with such karyotypes; and (3) arose in the absence of telomerase, we conclude that clonal and flexible karyotypes generate the immortality of cancers.

  16. Progressive transformation of immortalized esophageal epithelial cells

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Li-Yan Xu; Min-Hua Chen; Jian Shen; Wei-Jia Cai; Yi Zeng

    2002-01-01

    AIM: To investigate the progressive transformation of immortal cells of human fetal esophageal epithelium induced by human papillomavirus, and to examine biological criteria of sequential passage of cells, including cellular phenotype, proliferative rate, telomerase, chromosome and tumorigenicity.METHODS: The SHEE cell series consisted of immortalized embryonic esophageal epithelium which was in malignant transformation when cultivated over sixty passages without co-carcinogens. Cells of the 10th, 31st, 60th and 85th passages were present in progressive development after being transfected with HPV. Cells were cultivated in a culture flask and 24-hole cultural plates. Progressive changes of morphology, cell growth, contact-inhibition, and anchoragedependent growth characteristics were examined by phase contrast microscopy. The cell proliferation rate was assayed by flow cytometry. The modal number of chromosomes was analyzed. HPV18E6E7 was detected by Western blot methods and activities of telomerase were analyzed by TRAP.Tumorigenicity of cells was detected with soft agar plates cultivated and with tumor formation in SCID mice.RESULTS: In morphological examination the 10th passage cells were in good differentiation, the 60th and 85th passages cells were in relatively poor differentiation, and the 31st passage cells had two distinct differentiations. The characteristics of the 85th and 60th passage cells were weakened at contact-inhibition and anchorage-dependent growth. Karyotypes of four stages of cells belonged to hyperdiploid or hypotriploid, and bimodal distribution of chromosomes appeared in the 31st and 60th passage cells. All of these characteristics combined with a increasing trend. The activities of telomerase were expressed in the latter three passages. Four fourths of SCID mice in the 85th passage cells and one fourth of SCID mice in the 60th passage cells developed tumors, but the cells in the 10th and 31st passage displayed no tumor formation

  17. Inactivation of p16INK4a, with retention of pRB and p53/p21cip1 function, in human MRC5 fibroblasts that overcome a telomere-independent crisis during immortalization.

    Science.gov (United States)

    Taylor, Lisa M; James, Alexander; Schuller, Christine E; Brce, Jesena; Lock, Richard B; Mackenzie, Karen L

    2004-10-15

    Recent investigations, including our own, have shown that specific strains of fibroblasts expressing telomerase reverse transcriptase (hTERT) have an extended lifespan, but are not immortal. We previously demonstrated that hTERT-transduced MRC5 fetal lung fibroblasts (MRC5hTERTs) bypassed senescence but eventually succumbed to a second mortality barrier (crisis). In the present study, 67 MRC5hTERT clones were established by limiting dilution of a mass culture. Whereas 39/67 clones had an extended lifespan, all 39 extended lifespan clones underwent crisis. 11 of 39 clones escaped crisis and were immortalized. There was no apparent relationship between the fate of clones at crisis and the level of telomerase activity. Telomeres were hyperextended in the majority of the clones analyzed. There was no difference in telomere length of pre-crisis compared with post-crisis and immortal clones, indicating that hyperextended telomeres were conducive for immortalization and confirming that crisis was independent of telomere length. Immortalization of MRC5hTERT cells was associated with repression of the cyclin-dependent kinase inhibitor p16INK4a and up-regulation of pRB. However, the regulation of pRB phosphorylation and the response of the p53/p21cip1/waf1 pathway were normal in immortal cells subject to genotoxic stress. Overexpression of oncogenic ras failed to de-repress p16INK4a in immortal cells. Furthermore, expression of ras enforced senescent-like growth arrest in p16INK4a-positive, but not p16INK4a-negative MRC5hTERT cells. Immortal cells expressing ras formed small, infrequent colonies in soft agarose, but were non-tumorigenic. Overall, these results implicate the inactivation of p16INK4a as a critical event for overcoming telomere-independent crisis, immortalizing MRC5 fibroblasts and overcoming ras-induced premature senescence.

  18. Strategies for immortalization of primary hepatocytes

    Science.gov (United States)

    Eva, Ramboer; Bram, De Craene; Joery, De Kock; Tamara, Vanhaecke; Geert, Berx; Vera, Rogiers; Mathieu, Vinken

    2014-01-01

    The liver has the unique capacity to regenerate in response to a damaging event. Liver regeneration is hereby largely driven by hepatocyte proliferation, which in turn relies on cell cycling. The hepatocyte cell cycle is a complex process that is tightly regulated by several well-established mechanisms. In vitro, isolated hepatocytes do not longer retain this proliferative capacity. However, in vitro cell growth can be boosted by immortalization of hepatocytes. Well-defined immortalization genes can be artificially overexpressed in hepatocytes or the cells can be conditionally immortalized leading to controlled cell proliferation. This paper discusses the current immortalization techniques and provides a state-of-the-art overview of the actually available immortalized hepatocyte-derived cell lines and their applications. PMID:24911463

  19. Differential regulation of two forms of gonadotropin-releasing hormone messenger ribonucleic acid by gonadotropins in human immortalized ovarian surface epithelium and ovarian cancer cells.

    Science.gov (United States)

    Choi, Jung-Hye; Choi, Kyung-Chul; Auersperg, Nelly; Leung, Peter C K

    2006-06-01

    Although gonadotropin-releasing hormone (GnRH) has been shown to play a role as an autocrine/ paracrine regulator of cell growth in ovarian surface epithelium and ovarian cancer, the factors which regulate the expression of GnRH and its receptor in these cells are not well characterized. In the present study, we employed real-time PCR to determine the potential regulatory effect of gonadotropins on the expression levels of GnRH I (the mammalian GnRH), GnRH II (a second form of GnRH) and their common receptor (GnRHR) in immortalized ovarian surface epithelial (IOSE-80 and IOSE-80PC) cells and ovarian cancer cell lines (A2780, BG-1, CaOV-3, OVCAR-3 and SKOV-3). The cells were treated with increasing concentrations (100 and 1000 ng/ml) of recombinant follicle-stimulating hormone (FSH) or luteinizing hormone (LH) for 24 h. Treatment with FSH or LH reduced GnRH II mRNA levels in both IOSE cell lines and in three out of five ovarian cancer cell lines (A2780, BG-1 and OVCAR-3). A significant decrease in GnRHR mRNA levels was observed in IOSE and ovarian cancer cells, except CaOV-3 cells, following treatment with FSH or LH. In contrast, treatment with either FSH or LH had no effect on GnRH I mRNA levels in these cells, suggesting that gonadotropins regulate the two forms of GnRH and its receptor differentially. In separate experiments, the effect of gonadotropins on the anti-proliferative action of GnRH I and GnRH II agonists in IOSE-80, OVCAR-3 and SKOV-3 cells was investigated. The cells were pretreated with FSH or LH (100 ng/ml) for 24 h after which they were treated with either GnRH I or GnRH II (100 ng/ml) for 2 days, and cell growth was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Pretreatment of the cells with FSH or LH significantly reversed the growth inhibitory effect of GnRH I and GnRH II agonists in these cell types. These results provide the first demonstration of a potential interaction between gonadotropins and the

  20. Knockdown of Myo-Inositol Transporter SMIT1 Normalizes Cholinergic and Glutamatergic Function in an Immortalized Cell Line Established from the Cerebral Cortex of a Trisomy 16 Fetal Mouse, an Animal Model of Human Trisomy 21 (Down Syndrome).

    Science.gov (United States)

    Cárdenas, Ana María; Fernández-Olivares, Paola; Díaz-Franulic, Ignacio; González-Jamett, Arlek M; Shimahara, Takeshi; Segura-Aguilar, Juan; Caviedes, Raúl; Caviedes, Pablo

    2017-07-10

    The Na(+)/myo-inositol cotransporter (SMIT1) is overexpressed in human Down syndrome (DS) and in trisomy 16 fetal mice (Ts16), an animal model of the human condition. SMIT1 overexpression determines increased levels of intracellular myo-inositol, a precursor of phophoinositide synthesis. SMIT1 is overexpressed in CTb cells, an immortalized cell line established from the cerebral cortex of a Ts16 mouse fetus. CTb cells exhibit impaired cytosolic Ca(2+) signals in response to glutamatergic and cholinergic stimuli (increased amplitude and delayed time-dependent kinetics in the decay post-stimulation), compared to our CNh cell line, derived from the cerebral cortex of a euploid animal. Considering the role of myo-inositol in intracellular signaling, we normalized SMIT1 expression in CTb cells using specific mRNA antisenses. Forty-eight hours post-transfection, SMIT1 levels in CTb cells reached values comparable to those of CNh cells. At this time, decay kinetics of Ca(2+) signals induced by either glutamate, nicotine, or muscarine were accelerated in transfected CTb cells, to values similar to those of CNh cells. The amplitude of glutamate-induced cytosolic Ca(2+) signals in CTb cells was also normalized. The results suggest that SMIT1 overexpression contributes to abnormal cholinergic and glutamatergic Ca(2+) signals in the trisomic condition, and knockdown of DS-related genes in our Ts16-derived cell line could constitute a relevant tool to study DS-related neuronal dysfunction.

  1. Epidermal growth factor suppresses insulin-like growth factor binding protein 3 levels in human papillomavirus type 16-immortalized cervical epithelial cells and thereby potentiates the effects of insulin-like growth factor 1.

    Science.gov (United States)

    Hembree, J R; Agarwal, C; Eckert, R L

    1994-06-15

    Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b

  2. Senescence mechanism and immortalization of human bone marrow mesenchymal stem cells%人骨髓间充质干细胞的衰老机制及其"永生化"技术

    Institute of Scientific and Technical Information of China (English)

    黄炜; 吕安林; 刘博武; 候婧; 李垚; 燕学波

    2011-01-01

    背景:人类骨髓间充质干细胞由于其自身特性,被认为是最有潜力修复坏死心肌的干细胞,并已成为心肌组织工程研究的热点.目的:回顾分析人类骨髓间充质干细胞的衰老机制及根据这些机制建立的人类骨髓间充质干细胞永生化方法.方法:由第一作者检索1990/2010 PubMed数据库、万方数据库及Springer、Science Direct数据库有关骨髓间充质干细胞衰老机制及其永生化方法等方面的文献.结果与结论:骨髓间充质干细胞在体外培养传代过程中存在着细胞老化、干细胞样特征丢失的现象,阻碍了其在临床的广泛应用.通过对p53-p21及p16-pRb等信号通路细胞衰老分子机制的深入了解,研究者们试图建立永生化的骨髓间充质干细胞,包括将HPV16 E6E7蛋白的基因转入人骨髓间充质干细胞建立的KP细胞系,或将端粒末端转移酶反转录酶的片段 phTERT -IRES2-EGFP转染入KP细胞,建立3A6细胞系等方法,均在一定程度上延长了骨髓间充质干细胞寿命.但由于利用病毒载体进行基因转染方法带来的基因重组和插入突变等风险及细胞的固有抗病毒反应导致转染成功率较低,因而大量研究集中在了寻找非基因干涉方法上,建立标准化的永生化骨髓间充质干细胞的方法有待进一步研究.%BACKGROUND: Human bone marrow mesenchymal stem cells (hBMSCs) due to its property are considered one of the most promising stem cell for regeneration of the injured myocardiocytes, and have become the hot point for the study of myocardial tissue engineering.OBJECTIVE: To retrospectively analyze senescence mechanism of hBMSCs and to establish immortalized method of hBMSCs according to these mechanisms.METHODS: The first author retrieved PubMed Database, Wanfang Database, Springer and Science Direct Database for articles on senescence mechanism of hBMSCs and the immortalized method published from 1990 to 2010.RESULTS AND CONCLUSION

  3. Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells.

    Science.gov (United States)

    Basar, Norazah; Oridupa, Olayinka Ayotunde; Ritchie, Kenneth J; Nahar, Lutfun; Osman, Nashwa Mostafa M; Stafford, Angela; Kushiev, Habibjon; Kan, Asuman; Sarker, Satyajit D

    2015-06-01

    Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra.

  4. "Tuck Everlasting": Winnie Foster's Intimations of Immortality.

    Science.gov (United States)

    Berger, Peter N.

    1998-01-01

    Tells the plot of the novel "Tuck Everlasting" by Natalie Babbitt--the Tucks are invulnerable and immortal, but everlasting life holds trials for them. Provides seven questions for stimulating student response to the novel. (PA)

  5. "Tuck Everlasting": Winnie Foster's Intimations of Immortality.

    Science.gov (United States)

    Berger, Peter N.

    1998-01-01

    Tells the plot of the novel "Tuck Everlasting" by Natalie Babbitt--the Tucks are invulnerable and immortal, but everlasting life holds trials for them. Provides seven questions for stimulating student response to the novel. (PA)

  6. The immortality of humoral immunity.

    Science.gov (United States)

    Elgueta, Raul; de Vries, Victor C; Noelle, Randolph J

    2010-07-01

    Decades of high-titered antibody are sustained due to the persistence of memory B cells and long-lived plasma cells (PCs). The differentiation of each of these subsets is antigen- and T-cell driven and is dependent on signals acquired and integrated during the germinal center response. Inherent in the primary immune response must be the delivery of signals to B cells to create these populations, which have virtual immortality. Differences in biology and chemotactic behavior disperse memory B cells and long-lived PCs to a spectrum of anatomic sites. Each subset must rely on survival factors that can support their longevity. This review focuses on the generation of each of these subsets, their survival, and renewal, which must occur to sustain serological memory. In this context, we discuss the role of antigen, bystander inflammation, and cellular niches. The contribution of BAFF (B-cell activating factor belonging to the tumor necrosis factor family) and APRIL (a proliferation-inducing ligand) to the persistence of memory B cells and PCs are also detailed. Insights that have been provided over the past few years in the regulation of long-lived B-cell responses will have profound impact on vaccine development, the treatment of pre-sensitized patients for organ transplantation, and therapeutic interventions in both antibody- and T-cell-mediated autoimmunity.

  7. Transcription profiles of non-immortalized breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Holland James F

    2006-04-01

    Full Text Available Abstract Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs were used in addition to commercially-available normal breast epithelial cells (HMECs, established breast cancer cell lines (T-est and established normal breast cells (N-est. The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research.

  8. Therapeutic targeting of replicative immortality.

    Science.gov (United States)

    Yaswen, Paul; MacKenzie, Karen L; Keith, W Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G; Amedei, Amedeo; Amin, Amr; Helferich, Bill; Boosani, Chandra S; Guha, Gunjan; Ciriolo, Maria Rosa; Chen, Sophie; Mohammed, Sulma I; Azmi, Asfar S; Bhakta, Dipita; Halicka, Dorota; Niccolai, Elena; Aquilano, Katia; Ashraf, S Salman; Nowsheen, Somaira; Yang, Xujuan

    2015-12-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persistent cytostasis. This state, termed "senescence," can be triggered by intrinsic cellular processes such as telomere dysfunction and oncogene expression, and by exogenous factors such as DNA damaging agents or oxidative environments. Despite differences in upstream signaling, senescence often involves convergent interdependent activation of tumor suppressors p53 and p16/pRB, but can be induced, albeit with reduced sensitivity, when these suppressors are compromised. Doses of conventional genotoxic drugs required to achieve cancer cell senescence are often much lower than doses required to achieve outright cell death. Additional therapies, such as those targeting cyclin dependent kinases or components of the PI3K signaling pathway, may induce senescence specifically in cancer cells by circumventing defects in tumor suppressor pathways or exploiting cancer cells' heightened requirements for telomerase. Such treatments sufficient to induce cancer cell senescence could provide increased patient survival with fewer and less severe side effects than conventional cytotoxic regimens. This positive aspect is countered by important caveats regarding senescence reversibility, genomic instability, and paracrine effects that may increase heterogeneity and adaptive resistance of surviving cancer cells. Nevertheless, agents that effectively disrupt replicative immortality will likely be valuable components of new combinatorial approaches to cancer therapy. Copyright © 2015 The Authors

  9. Inefficiency in macromolecular transport of SCS-based microcapsules affects viability of primary human mesenchymal stem cells but not of immortalized cells

    DEFF Research Database (Denmark)

    Sanz-Nogués, Clara; Horan, Jason; Thompson, Kerry

    2015-01-01

    Microcapsules made of sodium cellulose sulphate (SCS) and poly-diallyl-dimethyl-ammonium chloride (pDADMAC) have been employed to encapsulate a wide range of established cell lines for several applications. However, little is known about the encapsulation of primary cells including human mesenchy...

  10. Immortality in view of Maimonides and Spinoza

    Directory of Open Access Journals (Sweden)

    Morteza Shajari

    2014-12-01

    Full Text Available Desire for immortality can be seen as the essential natural impulse. Therefore, different religions and thinkers have attempted to see the issue from different viewpoints. The great Jewish philosopher. Maimonides, due to deep fixation to Judaism, has tried to express their issues to be consistent with the Bible and his own community believes. He, in his discussion of resurrection, believed to three basic steps: The Messiah, the resurrection, and the world hereafter. His standpoint of eternity is dedicated to the hereafter. And we can be immortalized only by acting and teachings in accordance with the Bible and righteousness. Like Maimonides, Spinoza – the other Jewish philosopher - considered the immortality as Ultimate bliss through which the “immutable and eternal love of God" can be achieved. In his opinion, a person reaches this stage, when the lusts and emotions can reasonably be overcome, and also, when the power and anger and contempt and disregard others will respond with love and dignity. Thus, a man can be reached its proper perfection and immortality is reached. The difference between these two philosophers is that Maimonides believes through "actual intellect" -that is Emanation of the active intellect- can be immortalized but, for Spinoza, eternity can be reached through the adequate Ideas.

  11. Amoebal endosymbiont Parachlamydia acanthamoebae Bn9 can grow in immortal human epithelial HEp-2 cells at low temperature; an in vitro model system to study chlamydial evolution.

    Directory of Open Access Journals (Sweden)

    Chikayo Yamane

    Full Text Available Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37 °C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30 °C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30 °C compared to at 37 °C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.

  12. Amoebal Endosymbiont Parachlamydia acanthamoebae Bn9 Can Grow in Immortal Human Epithelial HEp-2 Cells at Low Temperature; An In Vitro Model System to Study Chlamydial Evolution

    Science.gov (United States)

    Nakamura, Shinji; Matsuo, Junji; Ishida, Kasumi; Yamazaki, Sumire; Oguri, Satoshi; Shouji, Natsumi; Hayashi, Yasuhiro; Yoshida, Mitsutaka; Yimin; Yamaguchi, Hiroyuki

    2015-01-01

    Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7–1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37°C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30°C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30°C compared to at 37°C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells. PMID:25643359

  13. Modern embalming, circulation of fluids, and the voyage through the human arterial system: Carl L. Barnes and the culture of immortality in America.

    Science.gov (United States)

    Podgorny, Irina

    2011-01-01

    By considering the work of American embalmer, lawyer, and physician Carl Lewis Barnes (1872-1927), this paper analyzes the emergence of modern embalming in America. Barnes experimented with and exhibited the techniques by which embalming fluids travelled into the most remote cavities of the human body. In this sense, modem embalmers based their skills and methods on experimental medicine, turning the anatomy of blood vessels, physiology of circulation, and composition of blood into a circuit that allowed embalming fluids to move throughout the corpse. Embalmers in the late 19th century took ownership of the laws of hydrodynamics and the physiology of blood circulation to market their fluids and equipment, thus playing the role of physiologists of death, performing and demonstrating physiological experiments with dead bodies.

  14. Human papillomavirus(HPV) oncogene-induced cell immortalization%人乳头瘤病毒癌基因诱导细胞永生化的研究进展

    Institute of Scientific and Technical Information of China (English)

    王珊珊; 王伟; 于莹莹; 李辉; 王宁

    2012-01-01

    永生化细胞是研究细胞增殖、分化、凋亡及衰老等的理想细胞模型.目前人类已建立多种细胞永生的方法,其中人乳头瘤病毒(HPV)癌基因(E6和E7)被广泛用于永生化细胞研究.E6蛋白和E7蛋白主要通过灭活p53通路和pRb通路,从多个水平提高端粒酶的表达和活性,使细胞逃过细胞复制衰老而继续增殖,实现细胞永生化.综述人乳头瘤病毒癌基因E6和E7的最新研究进展,探讨未来研究的趋势和研究方向.%Immortalized cell is the ideal cell model for studying cell proliferation, differentiation, apoptosis and aging. At present, a variety of methods for cell immortalization has been established, among them HPV oncogenes E6 and E7 are widely used in cell immortalization. The key role of oncoproteins E6 and E7 in cell immortalization is to inactivate p53 and pRb pathways, to improve telomerase expression and activity at multiple levels, which allow cells to escape from replicate Senescence and proliferate, and accomplish cell immortalization. This article reviews the advance in research of HPV oncoprotein-induced cell immortalization, and discusses the ongoing trends and future directions.

  15. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

    Science.gov (United States)

    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  16. Probabilistic immortality of Cu damascene interconnects

    Science.gov (United States)

    Hau-Riege, Stefan P.

    2002-02-01

    We have studied electromigration short-line effects in Cu damascene interconnects through experiments on lines of various lengths L, stressed at a variety of current densities j, and embedded in different dielectric materials. We observed two modes of resistance evolution: Either the resistance of the lines remains constant for the duration of the test, so that the lines are considered immortal, or the lines fail due to abrupt open-circuit failure. The resistance was not observed to gradually increase and then saturate, as commonly observed in Al-based interconnects, because the barrier is too thin and resistive to serve as a redundant current path should voiding occur. The critical stress for void nucleation was found to be smaller than 41 MPa, since voiding occurred even under the mildest test conditions of j=2 MA/cm2 and L=10.5 μm at 300 °C. A small fraction of short Cu lines failed even at low current densities, which deems necessary a concept of probabilistic immortality rather than deterministic immortality. Experiments and modeling suggest that the probability of immortality is described by (jL2/B), where B is the effective elastic modulus of the metallization scheme. By contrast, the immortality of Al-based interconnects with shunt layers is described by (jL) if no voids nucleate, and (jL/B) if voids do nucleate. Even though the phenomenology of short-line effects differs for Al- and Cu-based interconnects, the immortality of interconnects of either materials system can be explained by the phenomena of nucleation barriers for void formation and void-growth saturation. The differences are due solely to the absence of a shunt layer and the low critical stress for void nucleation in the case of Cu.

  17. Otto Rank and man's urge to immortality.

    Science.gov (United States)

    Goldwert, M

    1985-04-01

    Otto Rank, one of Sigmund Freud's original followers, posited the existence of an "urge to immortality" as man's deepest drive. In his Psychology and the Soul, Rank traced the desire for immortality through four historical eras, with particular emphasis on the creativity of the hero and the artist. By the end of his life, Rank had not only repudiated orthodox psychoanalysis and developed then abandoned a psychology of the will, he had moved "beyond psychology" to a religious view of history and the nature of man.

  18. Antioxidant and Anti-Inflammatory Effects of Selected Natural Compounds Contained in a Dietary Supplement on Two Human Immortalized Keratinocyte Lines

    Directory of Open Access Journals (Sweden)

    Elena Fasano

    2014-01-01

    Full Text Available Several advantages may derive from the use of dietary supplements containing multiple natural antioxidants and/or anti-inflammatory agents. At present, however, there is scarce information on the properties and potential of combined supplements. To fill the gap, the antioxidant and anti-inflammatory activities exerted by a combination of seven natural components (coenzyme Q10, krill oil, lipoic acid, resveratrol, grape seed oil, α-tocopherol, and selenium contained in a dietary supplement used for the prevention of skin disorders were investigated in vitro. Each component was administered, alone or in combination, to human keratinocytes, and the inhibition of Reactive Oxygen Species production and lipid peroxidation as well as the ability to reduce inflammatory cytokine secretion and to modulate Nuclear Factor-κB pathway was evaluated. The combination exhibited high antioxidant activity and in specific conditions the combination’s efficiency was higher than that of the most powerful components administered individually. Moreover, the combination showed remarkable anti-inflammatory properties. It reduced more efficiently than each component the secretion of Monocyte Chemoattractant Protein-1, a crucial cytokine for the development of chronic inflammation in skin, and inhibited Nuclear Factor-κB molecular pathway. Overall, our findings suggest that the combined formulation may have the potential to powerfully inhibit oxidative stress and inflammation at skin level.

  19. The intrabody targeting of hTERT attenuates the immortality of cancer cells.

    Science.gov (United States)

    Zhu, Xiangying; Yang, Nan; Cai, Jianguo; Yang, Guimei; Liang, Shenghua; Ren, Daming

    2010-01-01

    hTERT (human telomerase reverse transcriptase) plays a key role in the process of cell immortalization. Overexpression of hTERT has been implicated in 85% of malignant tumors and offers a specific target for cancer therapy. In this paper, we describe an effective approach using a single-chain variable fragment (scFv) intrabody derived from monoclonal hybridoma directed against hTERT to attenuate the immortalization of human uterine cervix and hepatoma cells. The scFv we constructed had a high affinity to hTERT, and specifically neutralized over 70% of telomere synthesis activity, thereby inhibiting the viability and proliferation of the cancer cells. Our results indicate that this anti-hTERT intrabody is a promising tool to target hTERT and intervene in the immortalization process of cancer cells.

  20. Characterization of neurons from immortalized dental pulp stem cells for the study of neurogenetic disorders

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    Nora Urraca

    2015-11-01

    Full Text Available A major challenge to the study and treatment of neurogenetic syndromes is accessing live neurons for study from affected individuals. Although several sources of stem cells are currently available, acquiring these involve invasive procedures, may be difficult or expensive to generate and are limited in number. Dental pulp stem cells (DPSCs are multipotent stem cells that reside deep the pulp of shed teeth. To investigate the characteristics of DPSCs that make them a valuable resource for translational research, we performed a set of viability, senescence, immortalization and gene expression studies on control DPSC and derived neurons. We investigated the basic transport conditions and maximum passage number for primary DPSCs. We immortalized control DPSCs using human telomerase reverse transcriptase (hTERT and evaluated neuronal differentiation potential and global gene expression changes by RNA-seq. We show that neurons from immortalized DPSCs share morphological and electrophysiological properties with non-immortalized DPSCs. We also show that differentiation of DPSCs into neurons significantly alters gene expression for 1305 transcripts. Here we show that these changes in gene expression are concurrent with changes in protein levels of the transcriptional repressor REST/NRSF, which is known to be involved in neuronal differentiation. Immortalization significantly altered the expression of 183 genes after neuronal differentiation, 94 of which also changed during differentiation. Our studies indicate that viable DPSCs can be obtained from teeth stored for ≥72 h, these can then be immortalized and still produce functional neurons for in vitro studies, but that constitutive hTERT immortalization is not be the best approach for long term use of patient derived DPSCs for the study of disease.

  1. Staphylococcus aureus alpha-toxin mediates general and cell type-specific changes in metabolite concentrations of immortalized human airway epithelial cells.

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    Philipp Gierok

    Full Text Available Staphylococcus aureus alpha-toxin (Hla is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using 1H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o- under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml of recombinant Hla (rHla in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD+. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o- cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.

  2. Activation of p53, inhibition of telomerase activity and induction of estrogen receptor beta are associated with the anti-growth effects of combination of ovarian hormones and retinoids in immortalized human mammary epithelial cells

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    Smith-Schneider Sallie

    2005-03-01

    Full Text Available Abstract Background A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P. However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line. Results We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA or all-trans-retinoic acid (ATRA, and strongly inhibited by N-(4-hydroxyphenyl retinamide (HPR. The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments. Conclusion Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that

  3. Education as Immortality: Toward the Rehabilitation of an Ideal.

    Science.gov (United States)

    Blacker, David

    1998-01-01

    Observes that immortality remains an important animating ideal for teaching and learning, despite being long neglected as theological or egoistic. Makes the case that the role of immortality in pedagogy has a long history in Western thought. Argues that individuals should recognize and address ways that longing for immortality shapes educators'…

  4. Towards Maturity in 1 Peter: Freedom, Holiness, Immortality

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    Paul B. Decock

    2016-04-01

    Full Text Available Growth towards maturity is dependent on the presence of freedom, holiness and immortality. These are presented as divine qualities that are utterly lacking in human beings. However, while human beings are ignorant and weak, sinful and mortal the addressees of 1 Peter1 are reminded that they have also been begotten anew by the imperishable seed of God’s Word, the Good News of Jesus Christ in order to share immortal life. This article looks first at human beings who as God’s creatures are ‘flesh’, but are also enabled to acknowledge their ‘fleshly’ state, to appreciate (‘desire’ and ‘taste’ (2:2–3 the Gospel and to submit to God. The second part considers the saving role of Christ as the powerful yet rejected ‘stone’ placed and offered by God as the model and means to transcend the ‘flesh’ in the flesh (4:1–2. A final part focuses on the new birth and the growth process in which the fleshly desires and ways of living give way to a manner of life, which is a witness to God’s saving power.

  5. Malignancy without immortality? Cellular immortalization as a possible late event in melanoma progression.

    Science.gov (United States)

    Soo, Julia K; Mackenzie Ross, Alastair D; Kallenberg, David M; Milagre, Carla; Heung Chong, W; Chow, Jade; Hill, Lucy; Hoare, Stacey; Collinson, Rebecca S; Hossain, Mehnaz; Keith, W Nicol; Marais, Richard; Bennett, Dorothy C

    2011-06-01

    Cell senescence is a permanent growth arrest following extended proliferation. Cultured cancer cells including metastatic melanoma cells often appear immortal (proliferate indefinitely), while uncultured benign nevi (moles) show senescence markers. Here, with new explantation methods, we investigated which classes of primary pigmented lesions are typically immortal. Nevi yielded a few proliferating cells, consistent with most nevus cells being senescent. No nevus culture (0/28) appeared immortal. Some thin and thick melanoma cultures proved immortal under these conditions, but surprisingly few (4/37). All arrested cultures displayed three senescence markers in some cells: β-galactosidase, nuclear p16, and heterochromatic foci/aggregates. However, melanoma cultures also showed features of telomeric crisis (arrest because of ultrashort telomeres). Moreover, crisis markers including anaphase bridges were frequent in uncultured vertical growth-phase (VGP) melanomas. Conversely, all immortal melanoma cultures expressed telomerase reverse transcriptase and telomerase, showing aneuploidy. The findings suggest that primary melanomas are typically precrisis, with immortalization/telomere maintenance as a late event. 2011 John Wiley & Sons A/S.

  6. 舌癌永生化细胞系细胞淋巴道转移裸鼠模型的建立%Establishment of lymphatic metastasis of immortalized human tongue carcinoma cell line in nude mouse

    Institute of Scientific and Technical Information of China (English)

    邹维艳; 严海芹; 孙美群; 尹海燕; 周艳梅; 李徽徽; 王俊斌

    2012-01-01

    Objective: To establish a nude mice model of lymphatic metastasis using immortalized human tongue carcinoma cell line. Methods: Lymphatic metastasis nude mice models were established by injecting carcinoma cells into the nail pad. The lymph nodes were collected 4 weeks later. The detection methods included H-E staining, immunohistochemistry stain of EMA and the purification and culture of tongue squamous cell carcinoma cells in vitro. Results: The lymphatic metastasis rate was 88% in Tca8113-Ml group and 60% in Tca8113 group. There were lymphatic metastasis carcinomal cells in the lymph node of the tongue carcinoma metastasis model. In purification and culture, the cells of epithelium like growth were observed in vitro. Conclusion: Injecting Tca8113-Ml cells into the nail pad can readily and stably establish a high rate of the lymphatic metastasis in an animal model. The model was regarded as a basis study of the lymphatic metastasis mechanisms.%目的:建立人舌癌细胞Tca8113-Ml裸鼠淋巴结转移模型,研究分析其转移的特性,为舌癌转移的研究提供动物模型.方法:4~6周龄裸鼠随机分为生理盐水对照组、TcaS113-Ml组、Tca8113组,通过足垫注射舌癌细胞,建立淋巴道转移模型.观察各组裸鼠淋巴结转移情况,进行H-E染色、上皮膜抗原(EMA)免疫组织化学显色及淋巴结舌癌细胞纯化培养.结果;淋巴结有散在或灶性鳞状上皮细胞转移,体外纯化培养的癌细胞呈上皮样生长.Tca8113-Ml组淋巴结转移率为88%,Tca8113组仅为60%.结论:足垫注射舌癌永生化细胞系Tca8113-Ml细胞系细胞建立淋巴道转移模型具有简单、稳定、转移率高等特点,为舌癌淋巴道转移的机制研究提供了一个良好动物模型.

  7. Establishment and characterization of a telomerase immortalized porcine luteal cells.

    Science.gov (United States)

    Zhang, Liang; Huang, Yong; Wang, Zhenyu; Luo, Xiaomao; Zhang, Hongling; Du, Qian; Chang, Lingling; Zhao, Xiaomin; Tong, Dewen

    2017-05-01

    Luteal cells play a crucial role in pregnancy through secreting progesterone to maintain pregnancy and support of fetus. However, low cellular yields and inability to passage primary porcine luteal cells (PLCs) in vitro limit the luteal cell study. Therefore, developing an immortalized porcine luteal cell line is necessary for studying luteal cells activity and function in different diseases. In this study, primary PLCs were obtained from gilts at day 30 to day 50 of gestation and immortalized by human telomerase reverse transcriptase (hTERT). The porcine corpus luteal cell line (hTERT-PLCs) expressed hTERT gene steady, maintained high hTERT activity and normal karyotype. The phase contrast microscope and transmission electron microscope observation showed primary PLCs and hTERT-PLCs were polygonal and exhibited abundant mitochondria, smooth endoplasmic reticulum and lipid droplets. 3β hydroxysteroid dehydrogenase (3βHSD) and Oil-Red-O staining showed that hTERT-PLCs at passage 30 and 50 were similar to primary PLCs. The hTERT-PLCs expressed steroidogenesis-related proteins, enzymes and receptors, such as steroidogenic acute regulatory protein, P450 cholesterol side-chain cleavage, 3βHSD, 20αHSD, luteinizing hormone receptor, progesterone receptor, prolactin receptor, estrogen receptorα/β, as well as primary PLCs. Consequently, hTERT-PLCs could secret progesterone and exhibited similar responses to luteinizing hormone and prostaglandin F2α as primary PLCs. In addition, the hTERT-PLCs did not show neoplastic transformation or anchorage independent growth. In summary, we developed an immortalized porcine luteal cell line which maintained its originally morphological, biological and functional characteristics. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Immunohistochemical study of hepatocyte, cholangiocyte and stem cell markers of hepatocellular carcinoma: the second report: relationship with tumor size and cell differentiation.

    Science.gov (United States)

    Kumagai, Arisa; Kondo, Fukuo; Sano, Keiji; Inoue, Masafumi; Fujii, Takeshi; Hashimoto, Masaji; Watanabe, Masato; Soejima, Yurie; Ishida, Tsuyoshi; Tokairin, Takuo; Saito, Koji; Sasajima, Yuko; Takahashi, Yoshihisa; Uozaki, Hiroshi; Fukusato, Toshio

    2016-07-01

    The purpose of this study is to investigate whether ordinary hepatocellular carcinomas (HCCs) show positivity of stem/progenitor cell markers and cholangiocyte markers during the process of tumor progression. Ninety-four HCC lesions no larger than 8 cm from 94 patients were immuno-histochemically studied using two hepatocyte markers (Hep par 1 and α-fetoprotein), five cholangiocyte markers (cytokeratin CK7, CK19, Muc1, epithelial membrane antigen and carcinoembryonic antigen) and three hepatic stem/progenitor cell markers (CD56, c-Kit and EpCAM). The tumors were classified into three groups by tumor size: S1, tumors were also classified according to tumor differentiation: well, moderately and poorly differentiated. The relationship between the positive ratios of these markers, tumor size and tumor differentiation was examined. The positive ratios of cholangiocyte markers tended to be higher in larger sized and more poorly differentiated tumors (except for CK7). The positive ratios of stem/progenitor cell markers tended to be higher in larger sized and more poorly differentiated tumors (except for c-Kit). Ordinary HCC can acquire the characteristic of positivity of cholangiocyte and stem/progenitor cell markers during the process of tumor progression. © 2016 The Authors. Journal of Hepato-Biliary-Pancreatic Sciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Hepato-Biliary-Pancreatic Surgery.

  9. Achieving immortality in the C. elegans germline.

    Science.gov (United States)

    Smelick, Chris; Ahmed, Shawn

    2005-01-01

    Germline immortality is a topic that has intrigued theoretical biologists interested in aging for over a century. The germ cell lineage can be passed from one generation to the next, indefinitely. In contrast, somatic cells are typically only needed for a single generation and are then discarded. Germ cells may, therefore, harbor rejuvenation mechanisms that enable them to proliferate for eons. Such processes are thought to be either absent from or down-regulated in somatic cells, although cell non-autonomous forms of rejuvenation are formally possible. A thorough description of mechanisms that foster eternal youth in germ cells is lacking. The mysteries of germline immortality are being addressed in the nematode Caenorhabditis elegans by studying mutants that reproduce normally for several generations but eventually become sterile. The mortal germline mutants probably become sterile as a consequence of accumulating various forms of heritable cellular damage. Such mutants are abundant, indicating that several different biochemical pathways are required to rejuvenate the germline. Thus, forward genetics should help to define mechanisms that enable the germline to achieve immortality.

  10. Comparison of repair of DNA double-strand breaks in identical sequences in primary human fibroblast and immortal hamster-human hybrid cells harboring a single copy of human chromosome 11

    Science.gov (United States)

    Fouladi, B.; Waldren, C. A.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    2000-01-01

    We have optimized a pulsed-field gel electrophoresis assay that measures induction and repair of double-strand breaks (DSBs) in specific regions of the genome (Lobrich et al., Proc. Natl. Acad. Sci. USA 92, 12050-12054, 1995). The increased sensitivity resulting from these improvements makes it possible to analyze the size distribution of broken DNA molecules immediately after the introduction of DSBs and after repair incubation. This analysis shows that the distribution of broken DNA pieces after exposure to sparsely ionizing radiation is consistent with the distribution expected from randomly induced DSBs. It is apparent from the distribution of rejoined DNA pieces after repair incubation that DNA ends continue to rejoin between 3 and 24 h postirradiation and that some of these rejoining events are in fact misrejoining events, since novel restriction fragments both larger and smaller than the original fragment are generated after repair. This improved assay was also used to study the kinetics of DSB rejoining and the extent of misrejoining in identical DNA sequences in human GM38 cells and human-hamster hybrid A(L) cells containing a single human chromosome 11. Despite the numerous differences between these cells, which include species and tissue of origin, levels of TP53, expression of telomerase, and the presence or absence of a homologous chromosome for the restriction fragments examined, the kinetics of rejoining of radiation-induced DSBs and the extent of misrejoining were similar in the two cell lines when studied in the G(1) phase of the cell cycle. Furthermore, DSBs were removed from the single-copy human chromosome in the hamster A(L) cells with similar kinetics and misrejoining frequency as at a locus on this hybrid's CHO chromosomes.

  11. Novel isolation and biochemical characterization of immortalized fibroblasts for tissue engineering vocal fold lamina propria.

    Science.gov (United States)

    Chen, Xia; Thibeault, Susan L

    2009-06-01

    Tissue regeneration of the vocal fold lamina propria extracellular matrix (ECM) will be facilitated by the use of suitable vocal fold fibroblast (VFF) cell lines in appropriate model systems. Primary human VFFs (hVFFs) were steadily transduced by a retroviral vector containing human telomerase reverse transcriptase (hTERT) gene; immortalized cells grew and divided vigorously for more than 120 days. Biochemical characterization of the six transduced lines included, at different time points, expression of hTERT, telomerase activity, telomere lengths, and transcript levels of ECM constituents. Telomere lengths of the transfected lines were elongated and stable. Gene expression levels of collagen Ialpha1, collagen Ialpha2, collagen VIalpha3, elastin, and fibronectin were measured between the transduced cell clones and the primary hVFFs to verify transcription. Absence of inter- and intraspecies contamination was confirmed with DNA fingerprinting and karyotype analysis. Cell morphology, growth, and transcription expression were examined on 2D scaffolds-collagen, fibronectin, and hyaluronic acid. Immortalized hVFFs demonstrated normal attachment and spread on 2D scaffolds. Collagen Ialpha1, collagen Ialpha2, collagen VIalpha3, elastin, and fibronectin transcript expression was measured from immortalized hVFFs, for all surfaces. This is the first report of immortalization and biochemical characterization of hVFFs, providing a novel and invaluable tool for tissue regeneration applications in the larynx.

  12. THE IDEA OF IMMORTALITY IN THE THEOCENTRICAL IDEOLOGICAL SYSTEMS AND IN A RELIGIOUS WORLDVIEW

    Directory of Open Access Journals (Sweden)

    Ekaterina Vadimovna Shiryaeva

    2015-01-01

    Full Text Available The article addresses the idea of human immortality, reflected in different ways in different forms of religious outlook, analyzes cultural, historical and individualpersonal traits of the existence of this idea. It is proved that individual- personal characteristics of the subject of religious consciousness have crucial significance for the predominance of soft or rigid forms of retribution beyond the grave. The article makes comparison of Christianity, Islam and Buddhism and Western and Orthodox versions of immortality within the Christian religion. Detailed attention is paid to the ideas of the immortality in Russian philosophy (the heritage of N.F. Fedorov, N.A.Berdyaev, S.L. Frank, Lev Shestov.It is concluded that the moral reasons for rejecting the death by Russian thinkers are in their special attention to the value of the bodily-spiritual unity of the human personality.It is stated that two main social functions performed by religion as a social institution are repressive and compensatory functions; the latter exist and interact as a dialectical contradiction. The author considers the typical approaches to the problem of death and immortality by Orthodox theologians of the 19th and 20th centuries; it is noted that in their works there is a tendency towards decreasing the repressive component in the models of the underworld retribution. At the same time, the disciplinary function of the official church religiosity is important, as the church retains its influence mainly through the implementation of this function.It is concluded that the perceptions of the human postmortem existence reflect the national-cultural and subjective-personal features of understanding the justice as an ethical category, as well as the fact that the idea of immortality in religious consciousness takes different forms not only in accordance with the type of religion but rather with the type of personal attitude to the problem of death.

  13. Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

    Science.gov (United States)

    Rahman, Nurul Adhwa; Rasil, Alifah Nur'ain Haji Mat; Meyding-Lamade, Uta; Craemer, Eva Maria; Diah, Suwarni; Tuah, Ani Afiqah; Muharram, Siti Hanna

    2016-07-01

    Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier.

  14. Aging and longevity in the simplest animals and the quest for immortality.

    Science.gov (United States)

    Petralia, Ronald S; Mattson, Mark P; Yao, Pamela J

    2014-07-01

    Here we review the examples of great longevity and potential immortality in the earliest animal types and contrast and compare these to humans and other higher animals. We start by discussing aging in single-celled organisms such as yeast and ciliates, and the idea of the immortal cell clone. Then we describe how these cell clones could become organized into colonies of different cell types that lead to multicellular animal life. We survey aging and longevity in all of the basal metazoan groups including ctenophores (comb jellies), sponges, placozoans, cnidarians (hydras, jellyfish, corals and sea anemones) and myxozoans. Then we move to the simplest bilaterian animals (with a head, three body cell layers, and bilateral symmetry), the two phyla of flatworms. A key determinant of longevity and immortality in most of these simple animals is the large numbers of pluripotent stem cells that underlie the remarkable abilities of these animals to regenerate and rejuvenate themselves. Finally, we discuss briefly the evolution of the higher bilaterians and how longevity was reduced and immortality lost due to attainment of greater body complexity and cell cycle strategies that protect these complex organisms from developing tumors. We also briefly consider how the evolution of multiple aging-related mechanisms/pathways hinders our ability to understand and modify the aging process in higher organisms. Published by Elsevier B.V.

  15. Aging and longevity in the simplest animals and the quest for immortality

    Science.gov (United States)

    Petralia, Ronald S.; Mattson, Mark P.; Yao, Pamela J.

    2014-01-01

    Here we review the examples of great longevity and potential immortality in the earliest animal types and contrast and compare these to humans and other higher animals. We start by discussing aging in single-celled organisms such as yeast and ciliates, and the idea of the immortal cell clone. Then we describe how these cell clones could become organized into colonies of different cell types that lead to multicellular animal life. We survey aging and longevity in all of the basal metazoan groups including ctenophores (comb jellies), sponges, placozoans, cnidarians (hydras, jellyfish, corals and sea anemones) and myxozoans. Then we move to the simplest bilaterian animals (with a head, three body cell layers, and bilateral symmetry), the two phyla of flatworms. A key determinant of longevity and immortality in most of these simple animals is the large numbers of pluripotent stem cells that underlie the remarkable abilities of these animals to regenerate and rejuvenate themselves. Finally, we discuss briefly the evolution of the higher bilaterians and how longevity was reduced and immortality lost due to attainment of greater body complexity and cell cycle strategies that protect these complex organisms from developing tumors. We also briefly consider how the evolution of multiple aging-related mechanisms/pathwayshinders our ability to understand and modify the aging process in higher organisms. PMID:24910306

  16. Determination of acute lethal and chronic lethal dose thresholds of valproic acid using 3D spheroids constructed from the immortal human hepatocyte cell line HepG2/C3A

    DEFF Research Database (Denmark)

    Fey, S. J.; Wrzesinski, K.

    2013-01-01

    Valproic acid (VPA) is a broad-spectrum antiepileptic drug that is now used commonly for several other neurological and psychiatric indications. While VPA is usually well tolerated, on rare occasions, it has been associated with severe, and sometimes, fatal liver injuries. These complications may...... describe here a culture system based on 3D spheroid culture of immortal hepatocytes which can determine the toxicity of valproic acid (or structurally or functionally related molecules) in vitro. The spheroids were used to follow changes in ATP production, glucose uptake and adenylate kinase following...... also arise due to acute VPA overdose. As a branched chain carboxylic acid, VPA is readily metabolized in the liver via glucuronic acid conjugation, mitochondrial β- and cytosolic ω-oxidation to produce multiple metabolites, and it is probably some of these metabolites that are involved in its toxicity...

  17. Senescence and immortality in hepatocellular carcinoma.

    Science.gov (United States)

    Ozturk, Mehmet; Arslan-Ergul, Ayca; Bagislar, Sevgi; Senturk, Serif; Yuzugullu, Haluk

    2009-12-01

    Cellular senescence is a process leading to terminal growth arrest with characteristic morphological features. This process is mediated by telomere-dependent, oncogene-induced and ROS-induced pathways, but persistent DNA damage is the most common cause. Senescence arrest is mediated by p16(INK4a)- and p21(Cip1)-dependent pathways both leading to retinoblastoma protein (pRb) activation. p53 plays a relay role between DNA damage sensing and p21(Cip1) activation. pRb arrests the cell cycle by recruiting proliferation genes to facultative heterochromatin for permanent silencing. Replicative senescence that occurs in hepatocytes in culture and in liver cirrhosis is associated with lack of telomerase activity and results in telomere shortening. Hepatocellular carcinoma (HCC) cells display inactivating mutations of p53 and epigenetic silencing of p16(INK4a). Moreover, they re-express telomerase reverse transcriptase required for telomere maintenance. Thus, senescence bypass and cellular immortality is likely to contribute significantly to HCC development. Oncogene-induced senescence in premalignant lesions and reversible immortality of cancer cells including HCC offer new potentials for tumor prevention and treatment.

  18. Immortalized cells and one oncogene in malignant transformation: old insights on new explanation

    Science.gov (United States)

    2011-01-01

    Background Nearly thirty years ago, it was first shown that malignant transformation with single oncogene necessarily requires the immortal state of the cell. From that time this thesis for the cells of human origin was not disproved. The basic point which we want to focus on by this short communication is the correct interpretation of the results obtained on the widely used human embryonic kidney 293 (HEK293) cells. Results Intensive literature analysis revealed an increasing number of recent studies discovering new oncogenes with non-overlapping functions. Since the 1970s, dozens of oncogenes have been identified in human cancer. Cultured cell lines are often used as model systems in these experiments. In some investigations the results obtained on such cells are interpreted by the authors as a malignant transformation of normal animal or even normal human cells (as for example with HEK293 cells). However, when a cell line gains the ability to undergo continuous cell division, the cells are not normal any more, they are immortalized cells. Nevertheless, the authors consider these cells as normal human ones, what is basically incorrect. Moreover, it was early demonstrated that the widely used human embryonic kidney 293 (HEK293) cells have a relationship to neurons. Conclusions Thus, the experiments with established cell lines reinforce the notion that immortality is an essential requirement for malignant transformation that cooperates with other oncogenic changes to program the neoplastic state and substances under such investigation should be interpreted as factors which do not malignantly transform normal cells alone, but possess the ability to enhance the tumorigenic potential of already immortalized cells. PMID:21605454

  19. Cosmological immortality: how to eliminate aging on a universal scale.

    Science.gov (United States)

    Vidal, Clement

    2014-01-01

    The death of our universe is as certain as our individual death. Some cosmologists have elaborated models which would make the cosmos immortal. In this paper, I examine them as cosmological extrapolations of immortality narratives that civilizations have developed to face death anxiety. I first show why cosmological death should be a worry, then I briefly examine scenarios involving the notion of soul or resurrection on a cosmological scale. I discuss in how far an intelligent civilization could stay alive by engaging in stellar, galactic and universal rejuvenation. Finally, I argue that leaving a cosmological legacy via universe making is an inspiring and promising narrative to achieve cosmological immortality.

  20. Conjecture: Can continuous regeneration lead to immortality? Studies in the MRL mouse.

    Science.gov (United States)

    Heber-Katz, Ellen; Leferovich, John; Bedelbaeva, Khamilia; Gourevitch, Dmitri; Clark, Lise

    2006-01-01

    A particular mouse strain, the MRL mouse, has been shown to have unique healing properties that show normal replacement of tissue without scarring. The serendipitous discovery that the MRL mouse has a profound capacity for regeneration in some ways rivaling the classic newt and axolotl species raises the possibility that humans, too, may have an innate regenerative ability. We propose this mouse as a model for continuous regeneration with possible life-extending properties. We will use the classical "immortal" organism, the hydra, for comparison and examine those key phenotypes that contribute to their immortality as they are expressed in the MRL mouse versus control mouse strains. The phenotypes to be examined include the rate of proliferation and the rate of cell death, which leads to a continual turnover in cells without an increase in mass.

  1. Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell

    Institute of Scientific and Technical Information of China (English)

    Zhong-Ying Shen; Li-Yan Xu; En-Min Li; Wei-Jia Cai; Min-Hua Chen; Jian Shen; Yi Zeng

    2002-01-01

    AIM: To search for the biomarker of cellular immortalization,the telomere length, telomerase activity and its subunits incultured epithelial cells of human fetal esophagus in theprocess of immortalization.METHODS: The transgenic cell line of human fetalesophageal epithelium (SHEE) was established with E6 E7genes of bt man papillomavirus (HPV) type 18 in ourlaboratory. Morphological phenotype of cultured SHEE cellsfrom the 6th to 30th passages, was examined by phasecontrast microscopy, the telomere length was assayed bySouthern blot method, and the activity of telomerase wasanalyzed by telomeric repeat amplification protocol (TRAP).Expressions of subunits of telomerase, hTR and hTERT,were assessed by RT-PCR. DNA content in cell cycle wasdetected by flow cytometry. The cell apoptosis wasexamined by electron microscopy (EM) and TUNEL label.RESULTS: SHEE cells from the 6 th to 10 th passagesshowed cellular proliferation with a good differentiation.From the 12 th to the 16 th passages, many senescent andapoptotic cells appeared, and the telomere length sharplyshortened from 23 kb to 17 kb without expression of hTERTand telornerase activity. At the 20 th passage, SHEE cellsovercame the senescence and apoptosis and restored theirproliferative activity with expression of telomerase andhTERT at low levels, but the telomere length shortenedcontinuously to the lowest of 3 kb. After the 30 th passagecells proliferation was restored by increment of cells at S andG2M phase in the cell cycle end telomerase activity expressedat high levels and with maintenance of telomere length.CONCLUSION: At the early stage of SHEE cells, telomeresare shortened without expression of telomerase and hTERTcausing cellular senescence and cell death. From the 20 thto the 30 th passages, the activation of telomerase andmaintenance of telomere length show a progressive processfor immortalization of esophageal epithelial cells. Theexpression of telomerase may constitute a biomarker fordetection of

  2. Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

    Science.gov (United States)

    Härle-Bachor, C; Boukamp, P

    1996-06-25

    Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

  3. Cilia containing 9 + 2 structures grown from immortalized cells

    Institute of Scientific and Technical Information of China (English)

    Ming Zhang; Jose G Assouline

    2007-01-01

    Cilia depend on their highly differentiated structure, a 9 + 2 arrangement, to remove particles from the lung and to transport reproductive cells. Immortalized cells could potentially be of great use in cilia research. Immortalization of cells with cilia structure containing the 9 + 2 arrangement might be able to generate cell lines with such cilia structure. However, whether immortalized cells can retain such a highly differentiated structure remains unclear. Here we demonstrate that (1) using E1a gene transfection, tracheal cells are immortalized; (2) interestingly, in a gel culture the immortalized cells form spherical aggregations within which a lumen is developed; and (3) surprisingly, inside the aggregation, cilia containing a 9 + 2 arrangement grow from the cell's apical pole and protrude into the lumen. These results may influence future research in many areas such as understanding the mechanisms of cilia differentiation, cilia generation in other existing cell lines, cilia disorders, generation of other highly differentiated structures besides cilia using the gel culture,immortalization of other ciliated cells with the E1a gene, development of cilia motile function, and establishment of a research model to provide uniform ciliated cells.

  4. The Quest for Immortality: Science at the Frontiers of Aging

    Energy Technology Data Exchange (ETDEWEB)

    Olshansky, S. Jay (University of Illinois at Chicago); Carnes, Bruce A. (University of Chicago)

    2001-10-24

    For more than a decade, Dr. Olshansky and Dr. Carnes have been working in the field of aging to identify the biological attributes of species that influence their length of life. Amid countless claims by scientists and the lay population that human life expectancy will continue to rise indefinitely in the future, Olshansky and Carnes have stood fast in their conviction that such claims are without any scientific basis. In their book The Quest for Immortality, S. Jay Olshansky and Bruce A. Carnes provide a comprehensive account of the real science of aging, from a historical overview of religious and philosophical thinking about aging and death, to the biology behind the changes we see in the mirror, to the promising but frightening future of genetic engineering. They demystify the complex language and concepts of science, explain how to distinguish real science from media hype in order to help readers make informed health decisions, and offer a powerfully optimistic vision of aging and health in the twenty-first century.

  5. The singularity of being: Lacan and the immortal within.

    Science.gov (United States)

    Ruti, Mari

    2010-12-01

    Drawing on the work of Eric Santner, Slavoj Žižek, and Alenka Zupančič, this paper constructs a theory of subjective singularity from a Lacanian perspective. It argues that, unlike the "subject" (who comes into existence as a result of symbolic prohibition), or the "person" (who is aligned with the narcissistic conceits of the imaginary), the singular self emerges in response to a galvanizing directive arising from the real. This directive summons the individual to a "character" beyond his or her social and intersubjective investments. Consequently, singularity expresses the individual's nonnegotiable distinctiveness, eccentricity, or idiosyncrasy at the same time as it prevents both symbolic and imaginary closure. It opens to layers of rebelliousness that indicate that there are components of human life that exceed the realm of normative sociality. Indeed, insofar as singularity articulates something about the "undead" pulse of jouissance, it connects the individual to a paradoxical kind of immortality. This does not mean that the individual will not die, but rather that he or she is capable of "transcendent" experiences, such as heightened states of creativity, that (always momentarily) reach "outside" the parameters of mortal life. Such experiences allow the individual to feel "real" in ways that fend off symbolic abduction and psychic death.

  6. hTERT-immortalized ovine microglia propagate natural scrapie isolates.

    Science.gov (United States)

    Muñoz-Gutiérrez, Juan F; Schneider, David A; Baszler, Timothy V; Greenlee, Justin J; Nicholson, Eric M; Stanton, James B

    2015-02-16

    Ex vivo propagation of natural prion isolates (i.e., propagated solely in the natural host) is crucial for the characterization and study of transmissible spongiform encephalopathies (TSEs). Several well-established, prion-permissive cell culture systems are available; however, only a few cell lines are permissive to natural prion isolates and these cells are not pathophysiologically relevant (e.g., renal epithelium and fibroblast-like cells). Therefore, a pathophysiologically relevant cell line derived from a natural TSE host could be used for propagation of natural prion isolates. In this study, ovine brain macrophages (microglia) were immortalized by transfection with the human telomerase reverse transcriptase (hTERT) gene to identify cell lines (hTERT-microglia) permissive to natural scrapie prion isolates. Following transfection, hTERT-microglia were passaged up to 100 times and their lifespan was significantly longer compared to parental cells (Fisher's exact test, Pscrapie isolates (i.e., derived from brain homogenates of sheep infected with scrapie). Prion infectivity and partial protease resistance of the prion protein were maintained in hTERT-microglia. Comparisons between scrapie-permissive and non-permissive hTERT-microglia sublines revealed that overall quantity of the normal cellular prion protein was not associated with prion permissiveness. The use of hTERT-microglia in future TSE studies may be more germane to the characterization of the cellular and subcellular pathophysiology of natural scrapie prion isolates and to investigate host-specific factors involved in prion replication.

  7. SV40TAg和Cre/loxP系统诱导的正常人可逆性转化黑素细胞在豚鼠体内的存活能力和复色实验%Survival and melanogenic potential of reversibly immortalized human melanocytes mediated by SV40T antigen gene and Cre/loxP system in Guinea pigs

    Institute of Scientific and Technical Information of China (English)

    王鹰; 曾志华; 杨希川; 郝飞; 钟白玉

    2010-01-01

    目的 研究SV40TAg基因和Cre/loxP系统在体外诱导正常人黑素细胞的可逆性转化以及转化细胞在豚鼠体内的存活能力和复色效果.方法 用Cre-ER~(T2)病毒上清感染经SV40TAg基因转化的正常人黑素细胞(MCT),诱导Cre重组酶表达(MCTC);过氧化氢法建立黄褐色雄性豚鼠白癜风动物模型,按黑素细胞移植方法进行原代黑素细胞和MCTC移植,观察MCTC在豚鼠体内的存活能力及复色效果.结果 MCTC细胞移植实验结果显示,1个月左右移植区即可见色素沉着,连续观察3个月,可见0.5~1 cm的黑色斑片或褐色斑片形成,移植成功率为82.5%,原代黑素细胞移植成功率为76.7%.病理观察移植区有明显黑素沉积,部分毛囊内也可见黑素沉积.结论 联合应用SV40TAg基因和Cre/loxP系统可以成功地诱导具有良好生物学安全性的人源性可逆性永生化黑素细胞,具有与原代黑素细胞相似的在体存活率,具备在体黑素合成功能,可以达到良好的复色效果.%Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of

  8. A method for the immortalization of newborn mouse skin keratinocytes

    Directory of Open Access Journals (Sweden)

    Brianna O Hammiller

    2015-07-01

    Full Text Available Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers, while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures, revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation.

  9. Variation, "evolution", immortality and genetic instabilities in tumour cells.

    Science.gov (United States)

    Bignold, L P

    2007-08-18

    The pathological characteristics of tumour cells often include variation of their histopathological features (i.e. "degrees of de-differentiation") between cases of the same tumour type and between different foci within individual tumours. Usually, only a few cell lines from tumours are immortal. Currently, somatic mutation, replicative infidelity of DNA and aneuploidy are suggested as alternative mechanisms of genomic disturbance underlying tumours. Nevertheless, apart from Hansemann's ideas of "anaplasia" and "de-differentiation" (proposed in the 1890s), and supposed "evolutionary themes" in cancer cell biology, little has been published concerning how histopathologic variation and immortality in tumour cells might arise. This paper reviews applications of the concepts of "variation" to tumours, including concepts of "evolution" and "cellular Darwinism". It is proposed that combinations of somatic mutation, DNA replicative infidelity and aneuploidy may explain the variabilities in tumours, and provide immortality in occasional tumour cells. A possible model involves (i) an initial somatic mutation causing reduced replicative fidelity of DNA, which could be variable in intensity, and thus give rise to variations between cases; (ii) a phase of replicative infidelity of DNA causing daughter cells lines to develop various abnormalities to different degrees, and hence provide for variation between areas of the same tumour. As a last event (iii) occasional asymmetric chromosomal distributions (aneuploidy) might "refresh" the ability of a daughter cell to replicate DNA faithfully causing them to become immortal. Thus extensively mutant and variable, hyperploid, and occasionally immortal cells might arise.

  10. Stepwise DNA Methylation Changes Are Linked to Escape from Defined Proliferation Barriers and Mammary Epithelial Cell Immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Novak, Petr; Jensen, Taylor J.; Garbe, James C.; Stampfer, Martha R.; Futscher, Bernard W.

    2009-04-20

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16(INK4A) expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending on how stasis was overcome. A second step coincides with immortalization and results in hundreds of additional DNA methylation changes regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that mayprove useful in breast cancer risk assessment.

  11. TRANSPLANTATION AND POTENTIAL IMMORTALITY OF MAMMALIAN TISSUES

    Science.gov (United States)

    Loeb, Leo

    1926-01-01

    1. Serial transplantation of tumors made it possible in 1901 and following years to draw the conclusion that various mammalian tissues have potential immortality. Serial transplantations of normal tissues did not succeed at first, because the homoioreaction on the part of the lymphocytes and connective tissue of the host injures the transplant. 2. In continuation of these experiments we found that cartilage of the rat can be transplanted serially to other rats at least for a period of 3 years. At the end of that time great parts of the transplanted cartilage and perichondrium are alive. 3. Not only the cartilage of young rats can be homoiotransplanted, but also the cartilage of very old rats which are nearing the end of life. By using such animals we have been able to obtain cartilage and perichondrium approaching an age of 6 years which is almost double the average age of a rat. 4. We found that cartilage can be homoiotransplanted more readily than other tissues for the following reasons: (a) While in principle the homoioreaction towards cartilage is the same as against other tissues, cartilage elicits this reaction with less intensity; (b) cartilage is better able to resist the invasion of lymphocytes and connective tissue than the majority of other tissues; (c) a gradual adaptation between transplant and host seems to take place in the case of cartilage transplantation, as a result of which the lymphocytic reaction on the part of the host tissue decreases progressively the longer the cartilage is kept in the strange host. 5. At time of examination we not only found living transplanted cartilage tissue, but also perichondrial tissue, which in response to a stimulus apparently originating in the necrotic central cartilage, had been proliferating and replacing it. These results suggest that it may perhaps be possible under favorable conditions to keep cartilage alive indefinitely through serial transplantations. 6. At the same time these experiments permit the

  12. Measuring an Individual's Investment in the Future: Symbolic Immortality, Sensation Seeking, and Psychic Numbness.

    Science.gov (United States)

    Mathews, Robert C.; Mister, Rena D.

    1988-01-01

    Operationalized Lifton's constructs of symbolic immortality and developed instrument to measure individual's needs for symbolic immortality in Lifton's five modes (biological, religious, nature, creative, experiential) in study which also examined age effects on needs for symbolic immortality and relation between sensation seeking and symbolic…

  13. Locus of Control and Level of Conflict as Correlates of Immortality Orientation.

    Science.gov (United States)

    O'Dowd, William

    1985-01-01

    Assessed the orientation of 14 male professors toward immortality as a psychological motive. Results showed a generally low conscious concern with immortality issues; however, respondents who have accepted some sort of immortality show a more internal locus of control and better adjustment. (JAC)

  14. Symbolic Immortality in Ordinary Contexts: Impediments to the Nuclear Era.

    Science.gov (United States)

    Schmitt, Raymond L.

    1982-01-01

    Lifton's writings indicate that fear of nuclear holocaust has severely impaired and threatens to negate traditional modes of symbolic immortality in America. Lifton's research, however, has been limited to extreme contexts. Data were triangulated in four distinctive American contexts. Found substantial negative evidence of Lifton's suspicions in…

  15. The French Academy: Arbitrator of Taste, Order, Genius--Immortality.

    Science.gov (United States)

    Buzash, Michael D.

    The French Academy is the oldest of the scholarly societies of France. Its ideals and preferences of order, genius, and immortality have influenced the schools, conservatories, universities, and archives and the intellectual and artistic tastes of the time. Its foundation was laid by nine lettered, well-educated laymen and ecclesiastics around…

  16. Construction of human telomerase reverse transcriptase-immortalized rat bone marrow mesenchemal stem cell strains%构建人端粒酶反转录酶修饰的永生化大鼠骨髓间充质干细胞株

    Institute of Scientific and Technical Information of China (English)

    刘慧萍; 钟晓龙; 赵晴; 黄文起; 安珂

    2015-01-01

    BACKGROUND:Because of convenient source, multi-lineage differentiation and low immunogenicity, bone marrow mesenchymal stem cels are the ideal cel type to serve as vectors of transgenic cels in pain management. However, the replicative senescence and smal amount of cels obtained from the bone marrow restrict the application of bone marrow mesenchymal stem cels in pain research. OBJECTIVE:To construct human telomerase reverse transcriptase (hTERT)-immortalized rat bone marrow mesenchymal stem cels as transgenic celular vectors for pain therapy. METHODS:Bone marrow mesenchymal stem cels were obtained from whole rat bone marrow, and then transfected with a lentivirus containing the hTERT (pLV-Puro-EF1α-hTERT) folowed by puromycin selection. hTERT expression and telomerase activity in these transfected cels were determined by RT-PCR and TRAP. Morphological changes, capacity of cel growth and multi-lineage differentiation, chromosome karyotype and tumorigenicity were observed in vitro. Moreover, the expression of cel surface molecule, Nestin, MHC-I and MHC-II in transfected cels were also detected by flow cytometry and immunocytochemistry. RESULTS AND CONCLUSION: The bone marrow mesenchymal stem cels geneticaly modified by hTERT could be cultured and passaged through 30 generations in vitro. Compared to the primary and negative transfected cels, the hTERT-modified bone marrow mesenchymal stem cels showed higher expression of hTERT mRNA, telomerase activity and cel proliferation. Most of transfected cels stayed at G2/M and S stages. The proliferation index of the transfected cels were increased dramaticaly. The positive rates of CD29, CD44 and CD90 were over 70%, but the positive rates of CD34 and CD45 were less than 5%. Transfected cels were positive for Nestin in the cytoplasm, but negative for MHC-1 and MHC-11. In addition, this cel line continued to exhibit the characteristics of fibroblastic bone marrow mesenchymal stem cels, including phenotype, differentiation

  17. Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines

    Directory of Open Access Journals (Sweden)

    Kamalidehghan Behnam

    2012-10-01

    Full Text Available Abstract Background Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research. Methods Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping. Results MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER+, PR+, HER2+ and (ER+, PR-, HER2+, respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER-, PR-, HER2+ for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52–58 chromosomes per cell. Conclusions Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER+/PR+/HER2+ and ER+/PR-/HER2+ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.

  18. Stressed SIRT7: facing a crossroad of senescence and immortality.

    Science.gov (United States)

    Liu, Jun-Ping; Chen, Ruping

    2015-06-01

    SIRT7 with coenzyme NAD catalyzes protein de-acetylation. In stress response, SIRT7 regulates protein folding in mitochondria with unknown mechanisms. Decreases in SIRT7 entrain hematopoietic stem cell senescence, but increasing SIRT7 causes elevation of hematopoietic stem cell regenerative function. We discuss the recent findings on SIRT7 and its binding proteins, NRF1 and GABPβ1, in decision making between the choices of inducing cell aging and immortality. © 2015 Wiley Publishing Asia Pty Ltd.

  19. Koschei the immortal and anti-aging drugs.

    Science.gov (United States)

    Blagosklonny, M V

    2014-12-04

    In Slavic folklore, Koschei the Immortal was bony, thin and lean. Was his condition caused by severe calorie restriction (CR)? CR deactivates the target of rapamycin pathway and slows down aging. But the life-extending effect of severe CR is limited by starvation. What if Koschei's anti-aging formula included rapamycin? And was rapamycin (or another rapalog) combined with commonly available drugs such as metformin, aspirin, propranolol, angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors.

  20. Koschei the immortal and anti-aging drugs

    Science.gov (United States)

    Blagosklonny, M V

    2014-01-01

    In Slavic folklore, Koschei the Immortal was bony, thin and lean. Was his condition caused by severe calorie restriction (CR)? CR deactivates the target of rapamycin pathway and slows down aging. But the life-extending effect of severe CR is limited by starvation. What if Koschei's anti-aging formula included rapamycin? And was rapamycin (or another rapalog) combined with commonly available drugs such as metformin, aspirin, propranolol, angiotensin II receptor blockers and angiotensin-converting enzyme inhibitors. PMID:25476900

  1. Efficient immortalization of primary nasopharyngeal epithelial cells for EBV infection study.

    Directory of Open Access Journals (Sweden)

    Yim Ling Yip

    Full Text Available Nasopharyngeal carcinoma (NPC is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection

  2. Establishment of Inducible Immortalized Human γδT Cell Lines with hTERT Genes by Electrotransfection%hTERT基因电转染人γδT细胞建立可诱导的永生化细胞系

    Institute of Scientific and Technical Information of China (English)

    庞永胜; 何维; 王浩; 崔莲仙

    2012-01-01

    Inducible immortalized human γδT cell lines with exogenous hTERT genes and pTet-on plasmids were established by electrotransfection. Human PBMC were isolated from healthy donors and amplified in vitro. After being amplified for 7 days,human γδT cells were purified by using magnetic beads. The human γδT cells with a higher purity were eletrotransfected with exogenous hTERT genes and pTet-on plasmids. Identifications of these human γδT cells were performed after being treated with Doxycycline for 24 hours. In positive control groups, there were 37. 5% ± 0. 9860%(program T-23)and 30. 5%±0. 5590% (program T-20) human γδT cells expressing GFP when cultured for 4. 5 hours after electrotransfection. The results of identifications with PCR and RT-PCR indicated that there were hTERT genes in the genome DNA, and there were transcripts of hTERT genes in RNA of human γδT cells. Program T-23 was the first choice to get a higher efficiency of electrotransfection when compared with program T-20. To induce the expression of hTERT genes effectively,the concentration of Doxycycline should be 600ng/mL. Using the donor's serum to culture γδT cells may help them to survive after electrotransfection. These data suggest that human γδT cells can be electrotransfected -with exogenous hTERT genes and the hTERT genes integrated into the genome can be transcribed after being induced by Doxycycline.%利用电击将外源基因hTERT与pTet-on调控质粒共转染人γδT细胞,建立可诱导的永生化人γδT细胞系.分离人PBMC,体外刺激增殖后经磁珠分选纯化,然后对其进行外源hTERT基因和pTet-on调控质粒的共同电转染,并加入强力霉素诱导目的基因表达.电转染程序T 23和T-20的效率分别为37.5%±0.9860%和30.5%±0.5590%.经鉴定,外源hTERT基因能够整合人γδT细胞基因组中并在诱导后表达.程序T-23的转染效率更高,强力霉素的有效诱导浓度是600ng/mL,志愿者本人的血清更利于电转后细胞的存活.

  3. The use of hTERT-immortalized cells in tissue engineering

    DEFF Research Database (Denmark)

    Kassem, Moustapha; Abdallah, Basem; Yu, Zentao

    2004-01-01

    The use of human telomerase reverse transcriptase (hTERT)-immortalized cells in tissue engineering protocols is a potentially important application of telomere biology. Several human cell types have been created that overexpress the hTERT gene with enhanced telomerase activity, extended life span...... and maintained or even improved functional activities. Furthermore, some studies have employed the telomerized cells in tissue engineering protocols with very good results. However, high telomerase activity allows extensive cell proliferation that may be associated with genomic instability and risk for cell...... transformation. Thus, safety issues should be studied carefully before using the telomerized tissues in the clinic. Alternatively, the development of conditional or intermittent telomerase activation protocols is needed....

  4. HTLV-1 Rex is required for viral spread and persistence in vivo but is dispensable for cellular immortalization in vitro.

    Science.gov (United States)

    Ye, Jianxin; Silverman, Lee; Lairmore, Michael D; Green, Patrick L

    2003-12-01

    Human T-cell leukemia virus type 1 (HTLV-1) is associated with leukemia/lymphoma and neurologic disorders. Although the viral transcriptional activator Tax is the critical viral oncoprotein, Rex, which regulates the expression of the viral structural and enzymatic genes, is essential for efficient viral replication. Herein, we investigate the contribution of Rex in HTLV-1 immortalization of primary T cells in vitro and viral survival in an infectious rabbit animal model. A Rex-deficient HTLV-1 (HTLVRex-) was constructed and characterized for viral gene expression, protein production, and immortalization capacity. Cells transiently transfected with the HTLVRex- proviral clone produced low detectable levels of p19 Gag. 729HTLVRex- stable transfectants produced functional Tax, but undetectable levels of Rex or p19 Gag. Coculture of irradiated 729HTLVRex- cells with peripheral blood mononuclear cells (PBMCs) resulted in sustained interleukin-2 (IL-2)-dependent growth of primary T lymphocytes. These cells carried the HTLVRex- genome and expressed tax/rex mRNA but produced no detectable Rex or p19 Gag. Rabbits inoculated with irradiated 729HTLVRex- cells or 729HTLVRex- cells transiently transfected with a Rex cDNA expression plasmid failed to become persistently infected or mount a detectable antibody response to the viral gene products. Together, our results provide the first direct evidence that Rex and its function to modulate viral gene expression and virion production is not required for in vitro immortalization by HTLV-1. However, Rex is critical for efficient infection of cells and persistence in vivo.

  5. Step-wise DNA methylation changes are linked to escape from defined proliferation barriers and mammary epithelial cell immortalization

    Science.gov (United States)

    Novak, P; Jensen, TJ; Garbe, JC; Stampfer, MR; Futscher, BW

    2009-01-01

    The timing and progression of DNA methylation changes during carcinogenesis are not completely understood. To develop a timeline of aberrant DNA methylation events during malignant transformation, we analyzed genome-wide DNA methylation patterns in an isogenic human mammary epithelial cell (HMEC) culture model of transformation. To acquire immortality and malignancy, the cultured finite lifespan HMEC must overcome two distinct proliferation barriers. The first barrier, stasis, is mediated by the retinoblastoma protein and can be overcome by loss of p16 INK4A expression. HMEC that escape stasis and continue to proliferate become genomically unstable before encountering a second more stringent proliferation barrier, telomere dysfunction due to telomere attrition. Rare cells that acquire telomerase expression may escape this barrier, become immortal, and develop further malignant properties. Our analysis of HMEC transitioning from finite lifespan to malignantly transformed showed that aberrant DNA methylation changes occur in a stepwise fashion early in the transformation process. The first aberrant DNA methylation step coincides with overcoming stasis, and results in few to hundreds of changes, depending upon how stasis was overcome. A second step coincides with immortalization, and results in hundreds of additional DNA methylation changes, regardless of the immortalization pathway. A majority of these DNA methylation changes are also found in malignant breast cancer cells. These results show that large-scale epigenetic remodeling occurs in the earliest steps of mammary carcinogenesis, temporally links DNA methylation changes and overcoming cellular proliferation barriers, and provides a bank of potential epigenetic biomarkers that may prove useful in breast cancer risk assessment. PMID:19509227

  6. Brain Prostheses as a Dynamic System (Immortalizing the Human Brain?)

    CERN Document Server

    Astakhov, Vadim

    2007-01-01

    Interest in development of brain prostheses, which might be proposed to recover mental functions lost due to neuron-degenerative disease or trauma, requires new methods in molecular engineering and nanotechnology to build artificial brain tissues. We develop a Dynamic Core model to analyze complexity of damaged biological neural network as well as transition and recovery of the system functionality due to changes in the system environment. We provide a method to model complexity of physical systems which might be proposed as an artificial tissue or prosthesis. Delocalization of Dynamic Core model is developed to analyze migration of mental functions in dynamic bio-systems which undergo architecture transition induced by trauma. Term Dynamic Core is used to define a set of causally related functions and Delocalization is used to describe the process of migration. Information geometry and topological formalisms are proposed to analyze information processes. A holographic model is proposed to construct dynamic e...

  7. Recovery of important physiological functions in 3D culture of immortal hepatocytes

    DEFF Research Database (Denmark)

    Wrzesinski, Krzysztof; Fey, S. J.

    2011-01-01

    hepatocytes using the classical approaches (in “2D”) and using a system which leads to the generation of spheroids of cells held in suspension (“3D”). Both approaches gave rise to cultures where the large majority of cells were viable, produced similar amounts of ATP, incorporated similar amounts......It is widely expected that cells grown in 3D environments (in suspension, on scaffolds etc.) will be superior to growing cells in classical 2D culture flasks. These expectations include the belief that cells grown in 3D culture will possess physiological characteristics that resemble more closely...... to grow human liver cells in ‘3 dimensional’ cultures so that they behave very similar to the liver in our bodies. By growing the immortal hepatocytes in specially designed bioreactors they form small pieces of ‘pseudotissue’ which exhibit several of the functions seen in the adult liver. We have grown...

  8. The pursuit of immortality: from the ego to the soul.

    Science.gov (United States)

    Miller, Lisa; Miller, Kenneth; Haught, John; Murphy, Nancey

    2011-10-01

    Moderated by Lisa Miller from Newsweek, evolutionary biologist Kenneth Miller (Brown University) and theologians John Haught (Georgetown University) and Nancey Murphy (Fuller Theological Seminary) discuss the questions Are we immortal? Do our souls exist beyond our bodies? and What scientific evidence is there for mystical experience? from a cultural, historical, and scientific perspective. The following is an edited transcript of the discussion that occurred March 23, 2011, 7:00-8:15 PM, at the New York Academy of Sciences in New York City. © 2011 New York Academy of Sciences.

  9. The Immortality of the Soul: History of a Political Argument

    Directory of Open Access Journals (Sweden)

    Miguel Saralegui

    2014-08-01

    Full Text Available The paper examines a fundamental and little studied issue of political theology: the relationship between the defense of the immortality of the soul and the preservation of public order. It carries out a historical survey of authors such as Pietro Pomponazzi and Thomas More, who resort to political reasons in order to defend it, and David Hume, who feels that there are no political grounds to support such a relationship. Finally, the article analyzes the arguments proposed to show that its efficacy is conditioned by the historical situation.

  10. DNA asymmetry in stem cells - immortal or mortal?

    Science.gov (United States)

    Yadlapalli, Swathi; Yamashita, Yukiko M

    2013-09-15

    The immortal strand hypothesis proposes that stem cells retain a template copy of genomic DNA (i.e. an 'immortal strand') to avoid replication-induced mutations. An alternative hypothesis suggests that certain cells segregate sister chromatids non-randomly to transmit distinct epigenetic information. However, this area of research has been highly controversial, with conflicting data even from the same cell types. Moreover, historically, the same term of 'non-random sister chromatid segregation' or 'biased sister chromatid segregation' has been used to indicate distinct biological processes, generating a confusion in the biological significance and potential mechanism of each phenomenon. Here, we discuss the models of non-random sister chromatid segregation, and we explore the strengths and limitations of the various techniques and experimental model systems used to study this question. We also describe our recent study on Drosophila male germline stem cells, where sister chromatids of X and Y chromosomes are segregated non-randomly during cell division. We aim to integrate the existing evidence to speculate on the underlying mechanisms and biological relevance of this long-standing observation on non-random sister chromatid segregation.

  11. Dying To Teach: The Educator's Search for Immortality. Advances in Contemporary Educational Thought Series, Volume 18.

    Science.gov (United States)

    Blacker, David J.

    This book offers both an intellectual history and a sustained argument for the inescapability of education's immortality agenda. It seeks a Hegelian synthesis of common ideas about living in one's students by giving them something of oneself, developing the image of immortality as an a-temporal, mythic, meaning-making, relational enterprise.…

  12. Protective effect of N-acetyl cysteine against chemical hypoxia-induced injury to an immortal human skin keratinocyte line HaCaT%N-乙酰半胱氨酸对化学性缺氧引起HaCaT细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    张美芬; 杨春涛; 杨战利; 孟金兰; 曾凡钦; 韩艳芳; 陈培熹; 冯鉴强

    2010-01-01

    Objective To estimate the influences of N-acetyl cysteine (NAC) on a chemical hypoxiamimetic agent CoCl2 induced-injury to, and expressions of inflammatory factors by, an immortal human skin keratinocyte line HaCaT. Methods HaCaT cells were treated with CoCl2 of 2000 μmol/L for 4 hours to set up a chemical hypoxia-induced cell model of skin injury. NAC of various concentrations ( 1000, 2000, 3000 μmol/L)was used to pretreat HaCaT cells for 2 hours prior to the establishment of cell model. After these treatments,cell viability was detected by cell counting kit 8 (CCK-8), the levels of interleukin 6 and 8 (IL-6 and -8) and tumor necrosis factor α (TNF-α) in culture supernatant by ELISA kits, mitochondrial membrane potential (MMP) by rhodamine 123 (Rh123) staining and photofluorography, intracellular reduced glutathione (GSH)content by glutathione detection kit. Results An obvious decline was observed in HaCaT cell viability after pretreatment with various concentrations of NAC for 2 hours. The treatment with CoCl2 of 2000 μmol/L for 4 hours induced an elevation in the supernatant levels of IL-6, IL-8 and TNF-α and a decrease in GSH content and MMP, while the pretreatment with NAC for 2 hours retarded the CoCl2-induced increase in IL-6 and IL-8 levels as well as decrease in GSH content and MMP. Conclusion The reactive oxygen species (ROS) scavenger NAC can protect against CoCl2-induced injury to and inflammatory reaction in HaCaT cells, which may be associated with a decrement in oxidative stress.%目的 探讨N-乙酰半胱氨酸(NAC)能否保护人皮肤永生化角质形成细胞(HaCaT)对抗化学性低氧模拟剂氯化钴(CoCl2)诱导的损伤及对炎症因子的影响.方法 用2000 μmol/L CoCl2处理HaCaT细胞,建立化学性低氧诱导皮肤细胞损伤的实验模型.应用CCK-8比色法检测细胞存活率;ELISA试剂盒检测细胞培养基中IL-6、IL-8和TNF-α的水平;罗丹明123(Rh123)染色/荣光显微镜照相术检测线粒体膜电

  13. Aging and the germ line: where mortality and immortality meet.

    Science.gov (United States)

    Jones, D Leanne

    2007-01-01

    Germ cells are highly specialized cells that form gametes, and they are the only cells within an organism that contribute genes to offspring. Germline stem cells (GSCs) sustain gamete production, both oogenesis (egg production) and spermatogenesis (sperm production), in many organisms. Since the genetic information contained within germ cells is passed from generation to generation, the germ line is often referred to as immortal. Therefore, it is possible that germ cells possess unique strategies to protect and transmit the genetic information contained within them indefinitely. However, aging often leads to a dramatic decrease in gamete production and fecundity. In addition, single gene mutations affecting longevity often have a converse effect on reproduction. Recent studies examining age-related changes in GSC number and activity, as well as changes to the stem cell microenvironment, provide insights into the mechanisms underlying the observed reduction in gametogenesis over the lifetime of an organism.

  14. Aging and immortality in a cell proliferation model.

    Science.gov (United States)

    Antal, T; Blagoev, K B; Trugman, S A; Redner, S

    2007-10-07

    We investigate a model of cell division in which the length of telomeres within a cell regulates its proliferative potential. At each division, telomeres undergo a systematic length decrease as well as a superimposed fluctuation due to exchange of telomere DNA between the two daughter cells. A cell becomes senescent when one or more of its telomeres become shorter than a critical length. We map this telomere dynamics onto a biased branching-diffusion process with an absorbing boundary condition whenever any telomere reaches the critical length. Using first-passage ideas, we find a phase transition between finite lifetime and immortality (infinite proliferation) of the cell population as a function of the influence of telomere shortening, fluctuations, and cell division.

  15. Application of spontaneously immortalized odontoblast cells in tooth regeneration.

    Science.gov (United States)

    Arany, Szilvia; Kawagoe, Masami; Sugiyama, Toshihiro

    2009-03-27

    Here, we report on the first attempt to bioengineer tooth using a spontaneously immortalized mesenchymal cell line. To assess the odontogenic potential of this cell line, odontoblast-lineage cells (OLC) were re-associated with competent dental epithelium isolated from E14.5 mice. A novel three-dimensional organ germ culture method was applied to nurture the constructs in vitro. Additionally, recombinants were transplanted under the kidney capsule in host animals for 2 weeks. Transplants developed into tooth tissues in one-third of the cases. OLC-derived GFP-positive cells could be identified in mineralizing tooth germs by immunohistochemistry. OLCs were capable of intercellular and cell-matrix communication, thus they eventually differentiated into functional odontoblasts. In summary, we managed to utilize OLCs for dental mesenchyme substitution in tooth regeneration experiments. Therefore, our spontaneously transformed cell line proved its potential for future complex, tooth developmental and bioengineering studies.

  16. Immortalized Rat Astrocyte Strain Genetically Modified by Rat Preprogalanin Gene

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    To construct an immortalized rat astrocyte strain genetically modified by rat preprogalanin gene (IAST/GAL) and detect its galanin (GAL) expression and secretion, a cDNA fragment of rat GAL in plasmid of pBS KS(+)-GAL was inserted into eukaryotic expression vector pcDNA3.1(+) by DNA recombinant technology, then the restriction enzyme digestion and DNA sequencing were carried out to evaluate the recombinant. The pcDNA3.1 (+)-GAL and pcDNA3.1 (+) construct were transfected into immortalized rat astrocyte strain (IAST) by lipofectamine and the population of cells which stably integrated the construct was selected with 600 μg/mL G418. Individual clones were screened and expanded into clonal cell strains. Detection of Neo gene was used to validate the success of the transfection. Immunocytochemical staining, RT-PCR and radioimmunoassay were used to detect the expression and secretion level of GAL. The recombinant had been successfully constructed by restriction enzyme digestion and DNA sequencing. Detection of Neo gene showed that the pcDNA3.1 (+)-GAL and pcDNA3.1 (+) have been successfully transfected into IAST. After selection by using G418, IAST/GAL and IAST/Neo cell strains were obtained.IAST/GAL, IAST/Neo and IAST were immunostained positively for GAL, but the GAL average optical density of IAST/GAL was significantly higher than that of IAST/Neo and IAST (P<0.01). The level of GAL mRNA expression and the supernatant concentration of GAL in cultured IAST/GAL were significantly higher than those of IAST and IAST/Neo (P<0.01), but no significant differences were found between the IAST and IAST/Neo (P>0.05). It was concluded that IAST/GAL strain was constructed successfully and it might provide a basis for the further study of pain therapy.

  17. Circadian-independent cell mitosis in immortalized fibroblasts.

    Science.gov (United States)

    Yeom, Mijung; Pendergast, Julie S; Ohmiya, Yoshihiro; Yamazaki, Shin

    2010-05-25

    Two prominent timekeeping systems, the cell cycle, which controls cell division, and the circadian system, which controls 24-h rhythms of physiology and behavior, are found in nearly all living organisms. A distinct feature of circadian rhythms is that they are temperature-compensated such that the period of the rhythm remains constant (approximately 24 h) at different ambient temperatures. Even though the speed of cell division, or growth rate, is highly temperature-dependent, the cell-mitosis rhythm is temperature-compensated. Twenty-four-hour fluctuations in cell division have also been observed in numerous species, suggesting that the circadian system is regulating the timing of cell division. We tested whether the cell-cycle rhythm was coupled to the circadian system in immortalized rat-1 fibroblasts by monitoring cell-cycle gene promoter-driven luciferase activity. We found that there was no consistent phase relationship between the circadian and cell cycles, and that the cell-cycle rhythm was not temperature-compensated in rat-1 fibroblasts. These data suggest that the circadian system does not regulate the cell-mitosis rhythm in rat-1 fibroblasts. These findings are inconsistent with numerous studies that suggest that cell mitosis is regulated by the circadian system in mammalian tissues in vivo. To account for this discrepancy, we propose two possibilities: (i) There is no direct coupling between the circadian rhythm and cell cycle but the timing of cell mitosis is synchronized with the rhythmic host environment, or (ii) coupling between the circadian rhythm and cell cycle exists in normal cells but it is disconnected in immortalized cells.

  18. Establishment and culture optimization of a new type of pituitary immortalized cell line

    Energy Technology Data Exchange (ETDEWEB)

    Kokubu, Yuko [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Asashima, Makoto [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Life Science Center of TARA, The University of Tsukuba, Ibaraki-ken 305-8577 (Japan); Kurisaki, Akira, E-mail: akikuri@hotmail.com [Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tsukuba, Ibaraki 305-8562 (Japan); Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8562 (Japan)

    2015-08-07

    The pituitary gland is a center of the endocrine system that controls homeostasis in an organism by secreting various hormones. The glandular anterior pituitary consists of five different cell types, each expressing specific hormones. However, their regulation and the appropriate conditions for their in vitro culture are not well defined. Here, we report the immortalization of mouse pituitary cells by introducing TERT, E6, and E7 transgenes. The immortalized cell lines mainly expressed a thyrotroph-specific thyroid stimulating hormone beta (Tshb). After optimization of the culture conditions, these immortalized cells proliferated and maintained morphological characteristics similar to those of primary pituitary cells under sphere culture conditions in DMEM/F12 medium supplemented with N2, B27, basic FGF, and EGF. These cell lines responded to PKA or PKC pathway activators and induced the expression of Tshb mRNA. Moreover, transplantation of the immortalized cell line into subcutaneous regions and kidney capsules of mice further increased Tshb expression. These results suggest that immortalization of pituitary cells with TERT, E6, and E7 transgenes is a useful method for generating proliferating cells for the in vitro analysis of pituitary regulatory mechanisms. - Highlights: • Mouse pituitary cell lines were immortalized by introducing TERT, E6, and E7. • The immortalized cell lines mainly expressed thyroid stimulating hormone beta. • The cell lines responded to PKA or PKC pathway activators, and induced Tshb.

  19. Immortalized gingival fibroblasts as a cytotoxicity test model for dental materials.

    Science.gov (United States)

    Illeperuma, Rasika P; Park, Young J; Kim, Jin M; Bae, Jung Y; Che, Zhong M; Son, Hwa K; Han, Mi R; Kim, Kwang M; Kim, Jin

    2012-03-01

    In vitro cytotoxicity test is an initial step to identify the harmful effects of new dental materials. Aim of this study was to develop a stable human cell line derived from normal gingival fibroblasts (hNOF) and to assess its feasibility in in vitro cytotoxicity testing. Immortalized human gingival fibroblasts (hTERT-hNOF) were successfully established with human telomerase reverse transcriptase gene transfection, preserving its phenotypical characteristics, replicative potential and biological properties. Utilizing standard cytotoxicity test modeling and dental materials, hTERT-hNOF were evaluated for their feasibility in cytotoxicity testing, compared with hNOF and L929 cells. Similar pattern of cytotoxic response was observed among hNOF, hTERT-hNOF and L929 cells. Cytotoxicity response of hTERT-hNOF was significantly similar to hNOF, moreover hTERT-hNOF and hNOF were found to be more sensitive towards the tested dental materials compared to L929 cells. This study suggested that hTERT-hNOF is an effective cytotoxic test model for dental materials.

  20. Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

    Directory of Open Access Journals (Sweden)

    Colo Anna

    2008-06-01

    Full Text Available Abstract Background Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45 is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA using specific oligonucleotides and nuclear protein extracts. Results We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7, SOD2 (superoxide dismutase 2, 100P (S100 calcium binding protein P, PI3 (protease inhibitor 3, skin-derived, CSTA (cystatin A, RARRES1 (retinoic acid receptor responder 1, and LXN (latexin. The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion This is the first in

  1. The conserved mitochondrial gene distribution in relatives of Turritopsis nutricula, an immortal jellyfish

    National Research Council Canada - National Science Library

    Devarapalli, Pratap; Kumavath, Ranjith N; Barh, Debmalya; Azevedo, Vasco

    2014-01-01

    Turritopsis nutricula (T. nutricula) is the one of the known reported organisms that can revert its life cycle to the polyp stage even after becoming sexually mature, defining itself as the only immortal organism in the animal kingdom...

  2. Selling space colonization and immortality: A psychosocial, anthropological critique of the rush to colonize Mars

    Science.gov (United States)

    Slobodian, Rayna Elizabeth

    2015-08-01

    Extensive media coverage regarding the proposal to send four people to Mars by 2025 has exploded recently. Private enterprise has taken the reins to venture into space, which has typically only been reserved for government agencies. I argue, that with this new direction comes less regulation, raising questions regarding the ethics of sending people into outer space to colonize Mars within a decade. Marketers selling colonization to the public include perspectives such as biological drives, species survival, inclusiveness and utopian ideals. I challenge these narratives by suggesting that much of our desire to colonize space within the next decade is motivated by ego, money and romanticism. More specifically, I will examine the roles that fear and stories of immortality play within selling space and how those stories are marketed. I am passionate about space and hope that one day humanity will colonize other worlds, but the rush to settle is dangerous and careless. I assert that humanity should first gain more experience and knowledge before colonizing outer space, using this research to mitigate the risk to astronauts and proceed with careful consideration for the lives of potential astronauts.

  3. TP53-dependent chromosome instability is associated with transient reductions in telomere length in immortal telomerase-positive cell lines

    Science.gov (United States)

    Schwartz, J. L.; Jordan, R.; Liber, H.; Murnane, J. P.; Evans, H. H.

    2001-01-01

    Telomere shortening in telomerase-negative somatic cells leads to the activation of the TP53 protein and the elimination of potentially unstable cells. We examined the effect of TP53 gene expression on both telomere metabolism and chromosome stability in immortal, telomerase-positive cell lines. Telomere length, telomerase activity, and chromosome instability were measured in multiple clones isolated from three related human B-lymphoblast cell lines that vary in TP53 expression; TK6 cells express wild-type TP53, WTK1 cells overexpress a mutant form of TP53, and NH32 cells express no TP53 protein. Clonal variations in both telomere length and chromosome stability were observed, and shorter telomeres were associated with higher levels of chromosome instability. The shortest telomeres were found in WTK1- and NH32-derived cells, and these cells had 5- to 10-fold higher levels of chromosome instability. The primary marker of instability was the presence of dicentric chromosomes. Aneuploidy and other stable chromosome alterations were also found in clones showing high levels of dicentrics. Polyploidy was found only in WTK1-derived cells. Both telomere length and chromosome instability fluctuated in the different cell populations with time in culture, presumably as unstable cells and cells with short telomeres were eliminated from the growing population. Our results suggest that transient reductions in telomere lengths may be common in immortal cell lines and that these alterations in telomere metabolism can have a profound effect on chromosome stability. Copyright 2000 Wiley-Liss, Inc.

  4. The Piwi-piRNA pathway: road to immortality.

    Science.gov (United States)

    Sturm, Ádám; Perczel, András; Ivics, Zoltán; Vellai, Tibor

    2017-10-01

    Despite its medical, social, and economic significance, understanding what primarily causes aging, that is, the mechanisms of the aging process, remains a fundamental and fascinating problem in biology. Accumulating evidence indicates that a small RNA-based gene regulatory machinery, the Piwi-piRNA pathway, represents a shared feature of nonaging (potentially immortal) biological systems, including the germline, somatic cancer stem cells, and certain 'lower' eukaryotic organisms like the planarian flatworm and freshwater hydra. The pathway primarily functions to repress the activity of mobile genetic elements, also called transposable elements (TEs) or 'jumping genes', which are capable of moving from one genomic locus to another, thereby causing insertional mutations. TEs become increasingly active and multiply in the genomes of somatic cells as the organism ages. These characteristics of TEs highlight their decisive mutagenic role in the progressive disintegration of genetic information, a molecular hallmark associated with aging. Hence, TE-mediated genomic instability may substantially contribute to the aging process. © 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  5. GSK3 Inhibitor-BIO Regulates Proliferation of Immortalized Pancreatic Mesenchymal Stem Cells (iPMSCs)

    Science.gov (United States)

    Cao, Hui; Chu, Yuankui; Lv, Xiao; Qiu, Pubin; Liu, Chao; Zhang, Huiru; Li, Dan; Peng, Sha; Dou, Zhongying; Hua, Jinlian

    2012-01-01

    Background The small molecule 6-bromoindirubin-30-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, is a pharmacological agent known to maintain self-renewal in human and mouse embryonic stem cells (ESCs). However, the precise role of GSK3 in immortalized pancreatic mesenchymal stem cells (iPMSCs) growth and survival is not completely understood at present. Results To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effect of BIO on iPMSCs. We found that the inactivation of GSK3 by BIO can robustly stimulate iPMSCs proliferation and mass formation as shown by QRT-PCR, western blotting, 5-Bromo-2-deoxyuridine (BrdU) immunostaining assay and tunel assay. However, we did not find the related roles of BIO on β cell differentiation by immunostaining, QRT-PCR assay, glucose-stimulated insulin release and C-peptide content analysis. Conclusions These results suggest that BIO plays a key role in the regulation of cell mass proliferation and maintenance of the undifferentiated state of iPMSCs. PMID:22384031

  6. Aging in bacteria, immortality or not-a critical review.

    Science.gov (United States)

    Gómez, José M G

    2010-12-01

    Bacteria were traditionally thought to have a symmetrical binary fission without a clear distinction between soma and germ-line, being thus considered as immortal biological entities. Yet it has been recently described that bacteria also undergo replicative aging (RA). That is, they exhibit finite replicative abilities under good conditions to growth. The apparently initial indistinguishability of sibling cells after cytokinesis is broken. After division, the daughter cell that inherits the "old" pole present in the "mother cell" progressively exhibits a decline in its proliferative capacity with increasing cell pole age. This is a clear hallmark and phenotypic manifestation of a bona fide RA phenomenon in toto. While the exact molecular mechanism(s) underlying to this lost of replicative potential are not yet fully understood, the "old pole cell" is considered as an aging parent that in a repeatedly manner is able to produce rejuvenated offspring which inherit a resetting of the biological clock. On the order hand, bacteria exhibit in addition to this "mandatory" RA the dubbed conditional senescence (CS). CS is defined as a decline in cellular viability observed in arrested-growing bacteria populations, a phenomenon apparently not related to RA under growing active conditions. To understand bacterial aging, it is necessary to put it within the sociality-multicellularity framework. This is a new conceptual paradigm that expresses the natural reality of the bacterial world. From this more ecological perspective these bacterial aging phenomena probably should represent an insurance/bethedging anticipative survival strategy. This is underpinned in a self-generation of an appropriate level of populational phenotypic diversity. That is, bacterial aging could be considered a communitarian adaptive response to cope with different environmental stresses and threats. I have highlighted the necessity to construct an integrative conceptual framework to achieve a unified view

  7. Expectations of immortality: dam safety management into the next millennium

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, M.D. [Tonkin and Taylor International Ltd., Auckland, (New Zealand)

    1999-07-01

    Topics concerning the problems associated with older and aging dams are considered including: what can be done to extent the lifetime of an old dam, the decision to decommission a dam based on a value judgment that the risk of maintaining the dam is too great for society's acceptance, the possibility of change in the level of risk tolerance with time in a technological environment, traditional surveillance methods used by dam owners in the Y2K situation, and the unreality of dam immortality. Trends and means for preserving older dams for their owner's purposes are outlined, as well as their lifetime compared to that of the downstream systems they serve. Despite the fact that we live in a throwaway society, dam owners cannot just leave their dam asset when they are through with using it. Someone has to maintain the dam, or ensure that it is safely decommissioned when the owner is finished with it. On a worldwide scale the available pool of experienced dam engineers is shrinking. This problem needs to be addressed by a shift towards operating and dam safety management skills based on a firm awareness of dam design principles. A shift in society's expectations has occurred such that dam designers and owners must now recognize the impact a dam can have both on its natural and social environments. Because of the increasing emphasis on paying attention to the impacts of people's activities on the planet, engineers more than anyone else must have a significant influence in that direction. 9 refs.

  8. A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality.

    Science.gov (United States)

    Buckley, Bethany A; Burkhart, Kirk B; Gu, Sam Guoping; Spracklin, George; Kershner, Aaron; Fritz, Heidi; Kimble, Judith; Fire, Andrew; Kennedy, Scott

    2012-09-20

    Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys 9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.

  9. Immortalized bovine mammary epithelial cells express stem cell markers and differentiate in vitro.

    Science.gov (United States)

    Hu, Han; Zheng, Nan; Gao, Haina; Dai, Wenting; Zhang, Yangdong; Li, Songli; Wang, Jiaqi

    2016-08-01

    The bovine mammary epithelial cell is a secretory cell, and its cell number and secretory activity determine milk production. In this study, we immortalized a bovine mammary epithelial cell line by SV40 large T antigen gene using a retrovirus based on Chinese Holstein primary mammary epithelial cells (CMEC) cultured in vitro. An immortalized bovine mammary epithelial cell line surpassed the 50-passage mark and was designated the CMEC-H. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line, but the cell line never induced tumor formation in nude mice. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44 and p63) cell markers. The induced expression of milk proteins, αS1 -casein, β-casein, κ-casein, and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells. The established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.

  10. Upregulation of the cell-cycle regulator RGC-32 in Epstein-Barr virus-immortalized cells.

    Directory of Open Access Journals (Sweden)

    Sandra N Schlick

    Full Text Available Epstein-Barr virus (EBV is implicated in the pathogenesis of multiple human tumours of lymphoid and epithelial origin. The virus infects and immortalizes B cells establishing a persistent latent infection characterized by varying patterns of EBV latent gene expression (latency 0, I, II and III. The CDK1 activator, Response Gene to Complement-32 (RGC-32, C13ORF15, is overexpressed in colon, breast and ovarian cancer tissues and we have detected selective high-level RGC-32 protein expression in EBV-immortalized latency III cells. Significantly, we show that overexpression of RGC-32 in B cells is sufficient to disrupt G2 cell-cycle arrest consistent with activation of CDK1, implicating RGC-32 in the EBV transformation process. Surprisingly, RGC-32 mRNA is expressed at high levels in latency I Burkitt's lymphoma (BL cells and in some EBV-negative BL cell-lines, although RGC-32 protein expression is not detectable. We show that RGC-32 mRNA expression is elevated in latency I cells due to transcriptional activation by high levels of the differentially expressed RUNX1c transcription factor. We found that proteosomal degradation or blocked cytoplasmic export of the RGC-32 message were not responsible for the lack of RGC-32 protein expression in latency I cells. Significantly, analysis of the ribosomal association of the RGC-32 mRNA in latency I and latency III cells revealed that RGC-32 transcripts were associated with multiple ribosomes in both cell-types implicating post-initiation translational repression mechanisms in the block to RGC-32 protein production in latency I cells. In summary, our results are the first to demonstrate RGC-32 protein upregulation in cells transformed by a human tumour virus and to identify post-initiation translational mechanisms as an expression control point for this key cell-cycle regulator.

  11. Construction and identification of immortalized rat astrocyte cell line expressing enkephalin

    Institute of Scientific and Technical Information of China (English)

    XU Ying; TIAN Yu-ke; TIAN Xue-bi; AN Ke; YANG Hui

    2007-01-01

    Objective: To provide a sound cell source for further ex-vivo gene therapy for chronic pain, we attempt to develop an immortalized rat astrocyte cell line that expresses enkephalin regulated by doxycycline.Methods: Retrovirus infection method was employed to develop an immortalized rat astrocyte cell line that could express enkephalin regulated by doxycycline. The hPPE gene expression level of immoralized astroyte cells (IAC)/hPPE was detected by RT-PCR, indirect immunofiuorescence staining and radioimmunoassay.Results: IAC carrying Tet-on system transfected with preproenkephalin gene could secrete enkephalin that was regulated by doxycycline in a dose-dependent manner and hPPE gene activation could be repeated in on-off-on cycles through administration or removal of doxycycline.Conclusion: An immortalized rat astrocyte cell line that secrete enkephalin under the control of doxycycline is established successfully, which provides a research basis for transgenic cell transplantation for analgesia.

  12. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents.

    Directory of Open Access Journals (Sweden)

    Melville B Vaughan

    Full Text Available BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent. METHODOLOGY/PRINCIPAL FINDINGS: Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras, were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63, basement-membrane formation (collagen IV, laminin-5, and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes. CONCLUSIONS/SIGNIFICANCE: The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

  13. Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

    Energy Technology Data Exchange (ETDEWEB)

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G., E-mail: chinnag@slu.edu

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.

  14. A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

    Science.gov (United States)

    Vrba, Lukas; Garbe, James C; Stampfer, Martha R; Futscher, Bernard W

    2015-01-01

    Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2′-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis. PMID

  15. Open-ended question: is immortality exclusively inherent to the germline?--A mini-review.

    Science.gov (United States)

    Lepperdinger, Günter

    2009-01-01

    All somatic cells are subject to aging. Germline links generations, and thus, pluripotent germ cells are considered potentially immortal. The current understanding how the germline escapes this otherwise inevitable phenomenon is outlined in this article. (c) 2008 S. Karger AG, Basel.

  16. Immortality of Prejudice in Striving Ubuntu: Case Studies of Community Managed Schools in Nepal

    Science.gov (United States)

    Rajbhandari, Mani Man Singh; Rajbhandari, Smriti

    2016-01-01

    The immortality of prejudice after the school management transfer has not been judged. This makes communities to take responsibility for schools further by compelling the government to mandate amendments of Community Managed Schools (CMS) Directives. The purpose was to explore the CMS enduring Ubuntu against immorality of prejudice, through…

  17. Precise conditional immortalization of mouse cells using tetracycline-regulated SV40 large T-antigen.

    Science.gov (United States)

    Anastassiadis, Konstantinos; Rostovskaya, Maria; Lubitz, Sandra; Weidlich, Stefanie; Stewart, A Francis

    2010-04-01

    Cellular immortalization provides a way for expansion and subsequent molecular characterization of rare cell types. Ideally, immortalization can be achieved by the reversible expression of immortalizing proteins. Here, we describe the use of conditional immortalization based on a modified tetracycline-regulated system for the expression of SV40 large T-antigen in embryonic stem (ES) cells and mice. The modified system relies on a codon improved reverse tetracycline transactivator (irtTA) fused to the ligand-binding domain (LBD) of the androgen receptor (irtTA-ABD) or of a mutated glucocorticoid receptor (irtTA-GBD*). Induction of T-antigen is conferred only after addition of two ligands, one to activate the LBD (mibolerone for irtTA-ABD or dexamethasone for irtTA-GBD*) and one to activate the tetracycline transactivator (doxycycline). In ES cells, changes in gene expression upon large T induction were limited and reversible upon deinduction. Similarly, expression of T-antigen was very tightly regulated in mice. We have isolated and expanded bone marrow mesenchymal stem cells that could be genetically manipulated and maintained their differentiation properties after several passages of expansion under conditions that induce the expression of large T-antigen. 2010 Wiley-Liss, Inc.

  18. Functionally Charged Polystyrene Particles Activate Immortalized Mouse Microglia (BV2): Cellular and Genomic Response

    Science.gov (United States)

    The effect of particle surface charge on the biological activation of immortalized mouse microglia (BV2) was examined. Same size (~850-950 nm) spherical polystyrene microparticles (SPM) with net negative (carboxyl, COOH-) or positive (dimethyl amino, CH3)2

  19. Establishment of spontaneously immortalized rat type 1 astroglial cell lines: the role of p53 in astroglial carcinogenesis.

    Science.gov (United States)

    Hiraga, S; Arita, N; Ohnishi, T; Izumoto, S; Taki, T; Higuchi, M; Iwaisako, K; Sakoda, S; Yamamoto, Y; Hayakawa, T

    1996-11-01

    We established five spontaneously immortalized cell lines using purified rat type 1 astroglia on a rigid transfer schedule. All the cell lines maintained their polygonal shape, regular pavement growth, low saturation density, positive glial fibrillary acidic protein expression, and serum requirements, while none were tumorigenic in nude mice. We then obtained a spontaneously transformed cell line by maintaining the cells for 6 months at a high cell density. Since alterations of the tumor suppressor p53 gene have been reported in the immortalization of some cell lines and in transformation of others, we characterized p53 in immortalized, spontaneously transformed, and 5 Nethyl-N-nitrosourea (ENU)-transformed cell lines. While each of the ENU-induced or the spontaneously transformed cell lines exhibited p53 gene mutations that resulted in amino acid alterations, no alterations in the p53 gene were observed in any of the immortalized cell lines. Thus, alterations of the p53 protein correlate more strongly with transformation than with immortalization of type 1 astroglia. Immortalization may be regulated by gene(s) other than p53. Spontaneously immortalized type 1 astroglial cell lines may provide a new tool to investigate an initial step of astroglial carcinogenesis.

  20. Milk as a symbol of immortality in the “Orphic” gold tablets from Thurii and Pelinna

    Directory of Open Access Journals (Sweden)

    Stian Sundell Torjussen

    2014-11-01

    Full Text Available This article offers an interpretation of the enigmatic “kid-in-milk” formula which appears in four of the “Orphic” gold tablets from Thurii and Pelinna. These tiny tablets accompanied the dead in their graves and contained texts of various lengths which were believed to help the deceased on his or her journey to the otherworld. Many see the tablets as Orphic texts, but this question has been highly debated during the last century. The four tablets in question, from two sites in southern Italy and Greece, tell how the deceased has suffered in life, but that he or she has attained immortality through initiation. The immortalization was referred to and summed up in the “kid-in-milk” formula, where, it is argued, milk was a direct reference to immortality. Thus milk in this eschatological context is a symbol of immortality which served as a focal point for both the text and the initates.

  1. Upregulation of CYP 450s expression of immortalized hepatocyte-like cells derived from mesenchymal stem cells by enzyme inducers

    Directory of Open Access Journals (Sweden)

    Limsrichamrern Somchai

    2011-09-01

    Full Text Available Abstract Background The strenuous procurement of cultured human hepatocytes and their short lives have constrained the cell culture model of cytochrome P450 (CYP450 induction, xenobiotic biotransformation, and hepatotoxicity. The development of continuous non-tumorous cell line steadily containing hepatocyte phenotypes would substitute the primary hepatocytes for these studies. Results 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. Conclusion 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.

  2. Cell-based assay for the detection of chemically induced cellular stress by immortalized untransformed transgenic hepatocytes

    Directory of Open Access Journals (Sweden)

    Vezzoni Paolo

    2004-03-01

    Full Text Available Abstract Background Primary hepatocytes, one of the most widely used cell types for toxicological studies, have a very limited life span and must be freshly derived from mice or even humans. Attempts to use stable cell lines maintaining the enzymatic pattern of liver cells have been so far unsatisfactory. Stress proteins (heat shock proteins, HSPs have been proposed as general markers of cellular injury and their use for environmental monitoring has been suggested. The aim of this work is to develop a bi-transgenic hepatocyte cell line in order to evaluate the ability of various organic and inorganic chemicals to induce the expression of the HSP70 driven reporter gene. We previously described transgenic mice (Hsp70/hGH secreting high levels of human Growth Hormone (hGH following exposure to toxic compounds in vivo and in vitro in primary cultures derived from different organs. In addition, we also reported another transgenic model (AT/cytoMet allowing the reproducible immortalization of untransformed hepatocytes retaining in vitro complex liver functions. Results The transgenic mouse line Hsp70/hGH was crossed with the AT/cytoMet transgenic strain permitting the reproducible immortalization of untransformed hepatocytes. From double transgenic animals we derived several stable hepatic cell lines (MMH-GH which showed a highly-differentiated phenotype as judged from the retention of epithelial cell polarity and the profile of gene expression, including hepatocyte-enriched transcription factors and detoxifying enzymes. In these cell lines, stresses induced by exposure to inorganic [Sodium Arsenite (NaAsO2 and Cadmium Chloride (CdCl2], and organic [Benzo(aPyrene (BaP, PentaChloroPhenol (PCP, TetraChloroHydroQuinone (TCHQ, 1-Chloro-2,4-DiNitro-Benzene (CDNB] compounds, specifically induced hGH release in the culture medium. Conclusions MMH-GH, an innovative model to evaluate the toxic potential of chemical and physical xenobiotics, provides a simple

  3. Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

    DEFF Research Database (Denmark)

    Vestentoft, Peter S; Jelnes, Peter; Hopkinson, Branden M

    2011-01-01

    biliary tree forms through several developmental stages involving an initial transition of primitive hepatocytes into cholangiocytes shaping the ductal plate followed by a process of maturation and remodeling where the intrahepatic biliary tree develops through an asymmetrical form of cholangiocyte...

  4. Illusions of Immortality: The Confrontation of Adolescence and AIDS.

    Science.gov (United States)

    New York State Dept. of Health, Albany.

    Acquired Immune Deficiency Syndrome (AIDS) is a potent and a present danger for teenagers, casting a dark shadow over their lives now and in the future. A small, but significant, number of teenagers will develop Human Immune Virus (HIV)-related illness before they turn 20; a far greater number will become infected with the virus during…

  5. Virus-Free Human Placental Cell Lines To Study Genetic Functions | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The National Institute of Child Health and Human Development's Section on Cellular Differentiation is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize immortalized virus-free human placental cell lines.The National Institute of Child Health and Human Development's Section on Cellular Differentiation is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize immortalized virus-free human placental cell lines.

  6. A germline-centric view of cell fate commitment, reprogramming and immortality.

    Science.gov (United States)

    Torres-Padilla, Maria-Elena; Ciosk, Rafal

    2013-02-01

    To ensure species continuity, the tantalising developmental plasticity of early embryonic cells, also called totipotency, must be transmitted to the offspring. This responsibility rests within the reproductive cell lineage: the germ line. At the recent EMBO/EMBL symposium 'Germline - Immortality through Totipotency', researchers discussed the mechanisms that establish and control totipotency, with an eye towards the mechanisms that may endow germ cells with the ability to propagate totipotency across generations.

  7. Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

    Science.gov (United States)

    Gardell, Alison M; Qin, Qin; Rice, Robert H; Li, Johnathan; Kültz, Dietmar

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.

  8. “Because I could not stop for Death-”:Belief in Immortality

    Institute of Scientific and Technical Information of China (English)

    袁飞

    2015-01-01

    One of the everlasting themes in poetry writing is “death”, with which Emily Dickinson is also obsessed in her careful observation and contemplation of life. This paper attempts to analyze one of her death poems: “Because I could not stop for Death-”. It shows the poet’s reflection on the inevitable fate of al living creatures in the universe and her firm belief in immortality thereafter.

  9. A Grand Challenge: Immortal Information and Through-Life Knowledge Management (KIM

    Directory of Open Access Journals (Sweden)

    Alexander Ball

    2006-11-01

    Full Text Available ‘Immortal information and through-life knowledge management: strategies and tools for the emerging product-service business paradigm’, is a Grand Challenge project involving eleven different UK universities and incorporating substantial industry collaboration. It is investigating a range of issues associated with the move towards a product-service paradigm in the engineering sector, in particular the long-term curation of digital data, learning from production and use, and appropriate governance and management techniques.

  10. The conserved mitochondrial gene distribution in relatives of Turritopsis nutricula, an immortal jellyfish

    OpenAIRE

    Devarapalli, Pratap; Kumavath, Ranjith N.; Barh, Debmalya; Azevedo, Vasco

    2014-01-01

    Turritopsis nutricula (T. nutricula) is the one of the known reported organisms that can revert its life cycle to the polyp stage even after becoming sexually mature, defining itself as the only immortal organism in the animal kingdom. Therefore, the animal is having prime importance in basic biological, aging, and biomedical researches. However, till date, the genome of this organism has not been sequenced and even there is no molecular phylogenetic study to reveal its close relatives. Here,...

  11. Transplantations and Cloning of an Immortal Cell Line from Rat SCN

    Science.gov (United States)

    1994-05-31

    8217 PON’SORJNG /MONITORING Department of the Air Force .~c~~ .. _... Aa.Ur Air Force Office of Scientific Research (AFOSR) al~I’~4 Chronobiology Program 94...number of neurons with a common genetic background that can be cultivated for primary SCN cultures and the uniformity of these preparations with...immortalized cells express SCN-like antigenic properties, further screening of the lines was conducted to determine whether these genetically -engineered

  12. Hopelessly mortal: The role of mortality salience, immortality and trait self-esteem in personal hope.

    Science.gov (United States)

    Wisman, Arnaud; Heflick, Nathan A

    2016-08-01

    Do people lose hope when thinking about death? Based on Terror Management Theory, we predicted that thoughts of death (i.e., mortality salience) would reduce personal hope for people low, but not high, in self-esteem, and that this reduction in hope would be ameliorated by promises of immortality. In Studies 1 and 2, mortality salience reduced personal hope for people low in self-esteem, but not for people high in self-esteem. In Study 3, mortality salience reduced hope for people low in self-esteem when they read an argument that there is no afterlife, but not when they read "evidence" supporting life after death. In Study 4, this effect was replicated with an essay affirming scientific medical advances that promise immortality. Together, these findings uniquely demonstrate that thoughts of mortality interact with trait self-esteem to cause changes in personal hope, and that literal immortality beliefs can aid psychological adjustment when thinking about death. Implications for understanding personal hope, trait self-esteem, afterlife beliefs and terror management are discussed.

  13. Three-dimensional culture system can induce expression of casein in immortalized bovine mammary epithelial cells.

    Science.gov (United States)

    Zhan, Kang; Lin, Miao; Liu, MingMei; Sui, YangNan; Babekir, Haitham Mohammed; Zhao, GuoQi

    2017-05-01

    Primary bovine mammary epithelial cells (BMECs) are not ideal models for long-term studies of lactation mechanisms because these cells in a monolayer culture system cannot be polarized to simulate the physiological functions in vitro. We investigate the effects of different culture models and karyotypes on casein expression in a three-dimensional (3D) culture system. The immortalized cells' karyotypes were analyzed at passages 10, 20, 30 and 40 to detect the effects of chromosome stability. Western blotting examined that whether or not the immortalized cells at passages 5, 10, 20, 30, 40 and 50 could induce expression of casein in a 3D culture system. The proper polarization of the acinar structures was monitored. BMECs were successfully immortalized. The cell karyotype at passage 30 remained at 60 chromosomes and the average value was 57.1 ± 0.40 after passage 40. The polarized protein's levels were up-regulated in 3D culture compared to 2D culture. Expression of αs1, β and κ-casein could be detectable in a passage range in 3D culture. Expression of αs2-casein was undetectable in all experimental groups. However, all casein expressions were barely detectable in traditional 2D culture system. Therefore, 3D culture system is an important tool for the long-term study of lactation mechanisms in vitro. © 2016 Japanese Society of Animal Science.

  14. Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

    Institute of Scientific and Technical Information of China (English)

    Shu-Long; Wang; Ge; Zhao; Wei; Zhu; Xiao-Meng; Dong; Ting; Liu; Yuan-Yuan; Li; Wen-Gang; Song; Yi-Qiang; Wang

    2015-01-01

    AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

  15. Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

    Directory of Open Access Journals (Sweden)

    Shu-Long Wang

    2015-01-01

    Full Text Available AIM: To investigate into the potential involvement of pyrin containing 3 gene (NLRP3, a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses. METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1 (HSV-1. Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40 (SV40-immortalized human corneal epithelial cell line were also examined. Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β. RESULTS: The NLRP3 activation induced by HSV-1 infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore, in the SV40-immortalized human corneal epithelial cells, NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium (known as an inhibitor of NLRP3 activation effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot. CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

  16. Retrovirus-Mediated Gene Transfer in Immortalization of Progenitor Hair Cell Lines in Newborn Rat

    Institute of Scientific and Technical Information of China (English)

    ZHANG Yuan; ZHAI Suo-qiang; SONG Wei; GUO Wei; ZHENG Gui-liang; HU Yin-yan

    2008-01-01

    Objective To present an experimental method that allows isolation of greater epithelial ridge (GER) and lesser epithelial ridge(LER) cells from postnatal rat cochleae using a combinatorial approach of enzymatic digestion and mechanical separation and to investigate a retrovirus-mediated gene transfer technique for its possibl utility in immortalization of the GER and LER cell lines, in an effort to establish an in vitro model system of hair cell differentiation. Methods GER and LER cells were dissected from postnatal rat cochleae and immortalized by transferring the SV40 large T antigen using a retrovirus. The established cell lines were confirmed through morphology observation, immunnocytochemical staining and RT-PCR analysis. The Hathl gene was transferred into the cell lines using adenovirus-mediated techniques to explore their potential to differentiate into hair cells. Results The established cell lines were stably maintained for more than 20 passages and displayed many features similar to primary GER and LER cells. They grew in patches and assumed a polygonal morphology. Immunostaining showed labeling by SV40 large T antigen and Islet1 (a specific marker for GER and LER). All passages of the cell lines expressed SV40 large T antigen on RT-PCR analysis. The cells also showed the capability to differenti-ate into hair cell-like cells when forced to express Hathl. Conclusion Retrovirus-mediated gene transfer can be used in establishing immortalized progenitor hair cell lines in newborn rat, which may provide an invaluable system for studying hair cell differentiation and regeneration for new treatment of sensory hearing loss caused by hair cell loss.

  17. Co-culture of Gastric Organoids and Immortalized Stomach Mesenchymal Cells.

    Science.gov (United States)

    Bertaux-Skeirik, Nina; Centeno, Jomaris; Feng, Rui; Schumacher, Michael A; Shivdasani, Ramesh A; Zavros, Yana

    2016-01-01

    Three-dimensional primary epithelial-derived gastric organoids have recently been established as an important tool to study gastric development, physiology, and disease. Specifically, mouse-derived fundic gastric organoids (mFGOs) co-cultured with Immortalized Stomach Mesenchymal Cells (ISMCs) reflect expression patterns of mature fundic cell types seen in vivo, thus allowing for long-term in vitro studies of gastric epithelial cell physiology, regeneration, and bacterial-host interactions. Here, we describe the development and culture of mFGOs, co-cultured with ISMCs.

  18. Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes

    Directory of Open Access Journals (Sweden)

    Lee Suk-Keun

    2007-09-01

    Full Text Available Abstract Background Interleukin-8 (IL-8 is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK and malignant human oral keratinocytes (HN12 cells with deferoxamine (DFO. Methods IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. Electrophoretic mobility shift assays was used to determine NF-κB binding activity. Phosphorylation and degradation of the I-κB were analyized by Western blot. Results IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1β to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IκB, and activation of NF-κB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione, and by pyrrolidine dithiocarbamate, an inhibitor of NF-κB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(PH oxidase. Conclusion This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions.

  19. Attempting immortality

    Energy Technology Data Exchange (ETDEWEB)

    Krull, W. [GKSS-Forschungszentrum Geesthacht GmbH (Germany)

    1995-12-01

    The world`s population of research reactors is growing old. Many have been adapted to serve new purposes over their lives, from testing materials for nuclear power programmes and supporting neutron physics experiments, to colouring gemstones, doping silicon and generating medical isotopes. In the first article of this survey of research reactor issues, Wilfried Krull from GKSS in Germany describes the effects on a reactor of supporting these changes in application as ``design ageing`` . Managing this and other symptoms of ageing to extend plant life is a key task for operators, and Krull discusses the efforts being made internationally to handle them. Eventually, terminal decline of one vital component can determine when a reactor has to be shutdown for refurbishment. For BR2 in Belgium, it was the beryllium matrix. Edgar Koonen from SCK-CEN explains work being done to replace it and extend reactor life for around 15 years. The Triga at the University of Austria`s Atomic Institute has already been running for over 30 years and Helmuth Bock, of the Institute, credits its good health to thorough routine maintenance procedures and custom-made tools for the work. The HANARO reactor in Korea achieved first criticality in February this year - one of the few new research reactors to have started up in recent years. Seong-yun Kim of KAERI describes its design and uses, including medical isotope production. About 80% of medical Mo-99 comes from the 38-year-old NRU in Canada. Russell Ball from B and W Nuclear Environmental Services describes a conceptual reactor for dedicated Mo-99 production in the last article of the survey. (UK).

  20. Immortal Melody

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    China celebrates 50 years of the Butterfly Lovers violin concerto The legend of Liang Shanbo and Zhu Yingtai, known as China’s Romeo and Juliet, has moved generations of Chinese. The doleful and romantic story of

  1. Immortality and the base of multicellular life: Lessons from cnidarian stem cells.

    Science.gov (United States)

    Watanabe, Hiroshi; Hoang, Van Thanh; Mättner, Robert; Holstein, Thomas W

    2009-12-01

    Cnidarians are phylogenetically basal members of the animal kingdom (>600 million years old). Together with plants they share some remarkable features that cannot be found in higher animals. Cnidarians and plants exhibit an almost unlimited regeneration capacity and immortality. Immortality can be ascribed to the asexual mode of reproduction that requires cells with an unlimited self-renewal capacity. We propose that the basic properties of animal stem cells are tightly linked to this archaic mode of reproduction. The cnidarian stem cells can give rise to a number of differentiated cell types including neuronal and germ cells. The genomes of Hydra and Nematostella, representatives of two major cnidarian classes indicate a surprising complexity of both genomes, which is in the range of vertebrates. Recent work indicates that highly conserved signalling pathways control Hydra stem cell differentiation. Furthermore, the availability of genomic resources and novel technologies provide approaches to analyse these cells in vivo. Studies of stem cells in cnidarians will therefore open important insights into the basic mechanisms of stem cell biology. Their critical phylogenetic position at the base of the metazoan branch in the tree of life makes them an important link in unravelling the common mechanisms of stem cell biology between animals and plants.

  2. Activation of Holliday junction recognizing protein involved in the chromosomal stability and immortality of cancer cells.

    Science.gov (United States)

    Kato, Tatsuya; Sato, Nagato; Hayama, Satoshi; Yamabuki, Takumi; Ito, Tomoo; Miyamoto, Masaki; Kondo, Satoshi; Nakamura, Yusuke; Daigo, Yataro

    2007-09-15

    We identified a novel gene HJURP (Holliday junction-recognizing protein) whose activation seemed to play a pivotal role in the immortality of cancer cells. HJURP was considered a possible downstream target for ataxia telangiectasia mutated signaling, and its expression was increased by DNA double-strand breaks (DSB). HJURP was involved in the homologous recombination pathway in the DSB repair process through interaction with hMSH5 and NBS1, which is a part of the MRN protein complex. HJURP formed nuclear foci in cells at S phase and those subjected to DNA damage. In vitro assays implied that HJURP bound directly to the Holliday junction and rDNA arrays. Treatment of cancer cells with small interfering RNA (siRNA) against HJURP caused abnormal chromosomal fusions and led to genomic instability and senescence. In addition, HJURP overexpression was observed in a majority of lung cancers and was associated with poor prognosis as well. We suggest that HJURP is an indispensable factor for chromosomal stability in immortalized cancer cells and is a potential novel therapeutic target for the development of anticancer drugs.

  3. Reduced insulin/IGF-1 signaling restores germ cell immortality to Caenorhabditis elegans Piwi mutants.

    Science.gov (United States)

    Simon, Matt; Sarkies, Peter; Ikegami, Kohta; Doebley, Anna-Lisa; Goldstein, Leonard D; Mitchell, Jacinth; Sakaguchi, Aisa; Miska, Eric A; Ahmed, Shawn

    2014-05-08

    Defects in the Piwi/piRNA pathway lead to transposon desilencing and immediate sterility in many organisms. We found that the C. elegans Piwi mutant prg-1 became sterile after growth for many generations. This phenotype did not occur for RNAi mutants with strong transposon-silencing defects and was separable from the role of PRG-1 in transgene silencing. Brief periods of starvation extended the transgenerational lifespan of prg-1 mutants by stimulating the DAF-16/FOXO longevity transcription factor. Constitutive activation of DAF-16 via reduced daf-2 insulin/IGF-1 signaling immortalized prg-1 strains via RNAi proteins and histone H3 lysine 4 demethylases. In late-generation prg-1 mutants, desilencing of repetitive segments of the genome occurred, and silencing of repetitive loci was restored in prg-1; daf-2 mutants. This study reveals an unexpected interface between aging and transgenerational maintenance of germ cells, where somatic longevity is coupled to a genome-silencing pathway that promotes germ cell immortality in parallel to the Piwi/piRNA system. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Development of cancer-initiating cells and immortalized cells with genomic instability

    Science.gov (United States)

    Yoshioka, Ken-ichi; Atsumi, Yuko; Nakagama, Hitoshi; Teraoka, Hirobumi

    2015-01-01

    Cancers that develop after middle age usually exhibit genomic instability and multiple mutations. This is in direct contrast to pediatric tumors that usually develop as a result of specific chromosomal translocations and epigenetic aberrations. The development of genomic instability is associated with mutations that contribute to cellular immortalization and transformation. Cancer occurs when cancer-initiating cells (CICs), also called cancer stem cells, develop as a result of these mutations. In this paper, we explore how CICs develop as a result of genomic instability, including looking at which cancer suppression mechanisms are abrogated. A recent in vitro study revealed the existence of a CIC induction pathway in differentiating stem cells. Under aberrant differentiation conditions, cells become senescent and develop genomic instabilities that lead to the development of CICs. The resulting CICs contain a mutation in the alternative reading frame of CDKN2A (ARF)/p53 module, i.e., in either ARF or p53. We summarize recently established knowledge of CIC development and cellular immortality, explore the role of the ARF/p53 module in protecting cells from transformation, and describe a risk factor for genomic destabilization that increases during the process of normal cell growth and differentiation and is associated with the downregulation of histone H2AX to levels representative of growth arrest in normal cells. PMID:25815132

  5. Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2.

    Science.gov (United States)

    Wang, Feng; Wu, Li-An; Li, Wentong; Yang, Yuan; Guo, Feng; Gao, Qingping; Chuang, Hui-Hsiu; Shoff, Lisa; Wang, Wei; Chen, Shuo

    2013-09-01

    Odontogenesis is the result of the reciprocal interactions between epithelial-mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.

  6. Establishment and characterization of immortal hepatocytes derived from various transgenic mouse lines.

    Science.gov (United States)

    Klocke, Rainer; Gómez-Lechón, M José; Ehrhardt, Anja; Mendoza-Figueroa, Tomas; Donato, M Teresa; López-Revilla, Rubén; Castell, José V; Paul, Dieter

    2002-06-21

    The potential of three genetic changes introduced into mice by the transgenic or knockout technology aimed at immortalizing hepatocytes in vitro and concomitantly preserving their differentiated hepatic functions was analyzed. Six hepatocyte lines were isolated from neonatal and adult transgenic mice expressing either IgEGF (a secretable variant of hEGF) or SV40 T antigen in the liver and from neonatal and adult p53 knockout (KO) mice and have been subcultured >150 times in serum-free, arginine-deficient medium. Only in SV40 T antigen transgenic lines profiles of mRNAs encoding serum proteins, transcription factors, and liver-specific enzymes were similar to those found in livers and primary hepatocytes. Accordingly, these cells displayed basal and inducible expression of CYP proteins as well as testosterone metabolizing activities. Thus, either knockout of the p53 gene or expression of SV40 T antigen or of IgEGF imparts immortality to hepatocytes in vitro, but only SV40 T antigen expression is compatible with the concomitant long-term preservation of differentiated liver functions.

  7. Immortal Time Bias: A Frequently Unrecognized Threat to Validity in the Evaluation of Postoperative Radiotherapy

    Energy Technology Data Exchange (ETDEWEB)

    Park, Henry S. [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States); Gross, Cary P. [Cancer Outcomes Policy and Effectiveness Research Center, Yale University School of Medicine, New Haven, CT (United States); Makarov, Danil V. [Department of Urology, New York University School of Medicine, New York, NY (United States); Yu, James B., E-mail: james.b.yu@yale.edu [Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT (United States); Cancer Outcomes Policy and Effectiveness Research Center, Yale University School of Medicine, New Haven, CT (United States)

    2012-08-01

    Purpose: To evaluate the influence of immortal time bias on observational cohort studies of postoperative radiotherapy (PORT) and the effectiveness of sequential landmark analysis to account for this bias. Methods and Materials: First, we reviewed previous studies of the Surveillance, Epidemiology, and End Results (SEER) database to determine how frequently this bias was considered. Second, we used SEER to select three tumor types (glioblastoma multiforme, Stage IA-IVM0 gastric adenocarcinoma, and Stage II-III rectal carcinoma) for which prospective trials demonstrated an improvement in survival associated with PORT. For each tumor type, we calculated conditional survivals and adjusted hazard ratios of PORT vs. postoperative observation cohorts while restricting the sample at sequential monthly landmarks. Results: Sixty-two percent of previous SEER publications evaluating PORT failed to use a landmark analysis. As expected, delivery of PORT for all three tumor types was associated with improved survival, with the largest associated benefit favoring PORT when all patients were included regardless of survival. Preselecting a cohort with a longer minimum survival sequentially diminished the apparent benefit of PORT. Conclusions: Although the majority of previous SEER articles do not correct for it, immortal time bias leads to altered estimates of PORT effectiveness, which are very sensitive to landmark selection. We suggest the routine use of sequential landmark analysis to account for this bias.

  8. 姜黄素诱导人永生化表皮HaCaT细胞凋亡中Nucleophosmin的表达和定位变化%Expression and Localization of Nucleophosmin During Curcumin-induced Apoptosis in the Immortalized Human Epithelial HaCaT Cells

    Institute of Scientific and Technical Information of China (English)

    杨海波; 宋巍; 陈兰英; 李祺福; 于淮滨

    2013-01-01

    该文以姜黄素诱导处理前后的人永生化表皮HaCaT细胞为研究对象,对核仁磷酸蛋白(nucleophosmin,NPM)在核基质中存在、分布及其与凋亡相关基因产物在姜黄素处理前后HaCaT细胞中的共定位关系进行观察研究.Western blot结果显示,NPM存在于人永生化表皮HaCaT细胞核基质蛋白组分中,并在姜黄素处理后的细胞核基质中表达下调,免疫荧光显微镜观察显示,NPM定位在核基质上,经姜黄素处理后出现分布位置与表达水平的变化,激光共聚焦显微镜观察可见NPM与HaCaT细胞中凋亡相关基因Bax、Bcl-2、mtP53和Rb基因产物均存在共定位关系,但在姜黄素处理后细胞中其共定位分布区域出现变化.研究结果证实NPM是一种核基质蛋白,定位于核基质上,NPM在HaCaT细胞诱导凋亡过程中的表达分布及其与凋亡相关基因产物的共定位现象值得进一步探索和研究.%To explore the existence and distribution of nucleophosmin (NPM) in the nuclear matrix and its colocalization with the other apoptosis-related gene products following curcumin treatment in the human epithelial HaCaT cells,the nuclear matrix of HaCaT cells was extracted pre/post curcumin induced apoptosis.Western blot analysis showed that NPM existed in the fractions of nuclear matrix proteins and was down-regulated after curcumin treatment.The immunofluorescence observation revealed that NPM located in the nuclear matrix,curcumin treatment altered its expression level and distribution profile.The colocalization of NPM with the products of apoptosis-related repression genes,including Bax,Bcl-2,mtP53 and Rb,using laser scanning confocal microscopy,were evaluated,and substantial differences were observed following curcumin treatment.The results implied that NPM is a nuclear matrix protein,and the level of its expression and the colocalization with apoptosis-related gene products may play an important role during the apoptosis of HaCaT cells.

  9. A mutant RAS gene acts through protein kinase C to augment interleukin-3 dependent proliferation in a fastidious immortal myeloid cell line.

    Science.gov (United States)

    Boswell, H S; Harrington, M A; Burgess, G S; Nahreini, T L; Derigs, H G; Hodges, T D; English, D; Crean, C D; Gabig, T G

    1989-09-01

    The functional role of a mutant RAS gene in immortal myeloid cell proliferation was examined in a fastidious interleukin-3 (IL-3) dependent cell line (NFS/N1.H7) formed by forced proliferation in IL-3 of marrow cells of the NFS/N mouse. The NFS/N1.H7 cell line was strictly dependent upon IL-3 for growth, and the cell line could be activated by phorbol esters (PMA) to augment IL-3 dependent proliferation, but when pKC was downregulated, diminished IL-3 proliferative response resulted. Transfection (electroporation) of the T24 RAS-containing vector pAL8 to NFS/N1.H7 led to clones (H7 NeoRas.F3, H7 NeoRas.E2) that had incorporated the entire 6.6 Kb human mutant H-RAS genome. The mutant RAS-containing clones demonstrated greater proliferation than parent cells or cells containing a control (neo-resistance) vector over a range of suboptimal IL-3 does and in optimal IL-3 concentrations had a faster doubling rate than parent cells. The clone H7 NeoRas.F3 was studied biochemically, and found to constitutively form 3-fold more 3H-diacylglycerol than the parent cell line upon exposure to 3H-glycerol. PMA could partially repair the proliferative defect of NFS/N1.H7 compared to the RAS-expressor. These studies affirm a secondary, accelerating role for a mutant RAS gene product acting through pKC to promote clonal expansion of immortal myeloid cells stimulated by IL-3.

  10. Choline transport via choline transporter-like protein 1 in conditionally immortalized rat syncytiotrophoblast cell lines TR-TBT.

    Science.gov (United States)

    Lee, N-Y; Choi, H-M; Kang, Y-S

    2009-04-01

    Choline is an essential nutrient for phospholipids and acetylcholine biosynthesis in normal development of fetus. In the present study, we investigated the functional characteristics of choline transport system and inhibitory effect of cationic drugs on choline transport in rat conditionally immortalized syncytiotrophoblast cell line (TR-TBT). Choline transport was weakly Na(+) dependent and significantly influenced by extracellular pH and by membrane depolarization. The transport process of choline is saturable with Michaelis-Menten constants (K(m)) of 68microM and 130microM in TR-TBT 18d-1 and TR-TBT 18d-2 respectively. Choline uptake in the cells was inhibited by unlabeled choline and hemicholinium-3 as well as various organic cations including guanidine, amiloride and acetylcholine. However, the prototypical organic cation tetraethylammonium and cimetidine showed very little inhibitory effect of choline uptake in TR-TBT cells. RT-PCR revealed that choline transporter-like protein 1 (CTL1) and organic cation transporter 2 (OCT2) are expressed in TR-TBT cells. The transport properties of choline in TR-TBT cells were similar or identical to that of CTL1 but not OCT2. CTL1 was also detected in human placenta. In addition, several cationic drugs such as diphenhydramine and verapamil competitively inhibited choline uptake in TR-TBT 18d-1 with K(i) of 115microM and 55microM, respectively. Our results suggest that choline transport system, which has intermediate affinity and weakly Na(+) dependent, in TR-TBT seems to occur through a CTL1 and this system may have relevance with the uptake of pharmacologically important organic cation drugs.

  11. Shutoff of BZLF1 gene expression is necessary for immortalization of primary B cells by Epstein-Barr virus.

    Science.gov (United States)

    Yu, Xianming; McCarthy, Patrick J; Wang, Zhenxun; Gorlen, Daniel A; Mertz, Janet E

    2012-08-01

    The BZLF1 gene controls the switch between latent and lytic infection by Epstein-Barr virus (EBV). We previously reported that both the ZV and ZIIR elements within the BZLF1 promoter, Zp, are potent transcription silencers within the context of an intact EBV genome. We report here identification of another sequence element, ZV', which synergized with ZV in repressing Zp via binding ZEB1 or ZEB2. We then determined the phenotype of a variant of EBV strain B95.8 in which the ZV, ZV', and ZIIR elements were concurrently mutated. HEK293 cell lines infected with this triple mutant (tmt) virus spontaneously synthesized 6- to 10-fold more viral BZLF1, BRLF1, BMRF1, and BLLF1 RNAs, 3- to 6-fold more viral Zta, Rta, and EAD proteins, 3- to 5-fold more viral DNA, and 7- to 9-fold more infectious virus than did 293 cell lines latently infected with either the ZV ZV' double mutant (dmt) or ZIIR mutant (mt) virus. While ZV ZV' ZIIR tmt EBV efficiently infected human primary blood B cells in vitro, it was highly defective in immortalizing them. Instead of the nearly complete silencing of BZLF1 gene expression that occurs within 4 days after primary infection with wild-type EBV, the ZV ZV' ZIIR tmt-infected cells continued to synthesize BZLF1 RNA, with 90% of them dying within 9 days postinfection. BL41 cells infected with this "superlytic" virus also exhibited increased synthesis of BZLF1 and BMRF1 RNAs. Thus, we conclude that the ZV, ZV', and ZIIR silencing elements act synergistically to repress transcription from Zp, thereby tightly controlling BZLF1 gene expression, which is crucial for establishing and maintaining EBV latency.

  12. Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

    Science.gov (United States)

    Ganguly, Abira; Bhattacharjee, Chumki; Bhave, Madhura; Kailaje, Vaishali; Jain, Bhawik K; Sengupta, Isha; Rangarajan, Annapoorni; Bhattacharyya, Dibyendu

    2016-03-01

    Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

  13. Clinical features of familial adenomas polyps in Chinese and establishment of its immortal lymphocyte cell lines

    Institute of Scientific and Technical Information of China (English)

    Shan-Rong Cai; Su-Zhang Zhang; Shu Zheng

    2007-01-01

    AIM:To reserve the rare Chinese familial adenomas polyp (FAP) family resource and to investigate the clinical features of FAP in Chinese for its diagnosis.METHODS: Clinical features of patients with FAP were investigated. If there is any question, their medical records were verified. Blood sample was taken and lymphocyte immortal cell lines were established with modified EB-transformation methods. Congenital hypertrophy of retinal pigment epithelium (CHRPE) was checked by an experienced ophthalmologist.RESULTS: Twenty seven families including 21 classical FAP (CFAP) families, 3 attenuated FAP (AFAP) families,and 3 suspected AFAP families were investigated. A total of 116 lymphocyte immortal cell lines were established from 26 families. In all the FAP families, colorectal cancer occurred at the mean age of 42.84 years. Of the 16 families checked, 15 (93.75%) had CHRPE. The mean number of patients suffering from colorectal neoplasm was 3.14 in CFAP families and 2.0 in AFAP families (P < 0.01). The mean oldest age at diagnosis of FAP was 41.75 years in CFAP families, and 58.67 years in AFAP families, respectively (P < 0.01). Mean age of development of colorectal cancer was 42.23 in CFAP and 57.33 years old in AFAP (P < 0.01). Mean of the earliest age at diagnosis of FAP was 29.95 years in the FAP families with a positive family history and 46.80 years in the FAP families with a negative family history (P <0.01). The ratio of extra-intestinal tumors to colorectal neoplasms was different in the two kinds of families with positive and negative family history (P < 0.01).CONCLUSION: Additional use of ciclosporin will effectively improve to establish lymphocyte immortal cell lines with modified EB- transformation methods. In Chinese FAP, there was a high frequency of CHRPE, and a later age at diagnosis and a later age of development of colorectal cancer in AFAP. And earlier age at diagnosis in FAP with positive family history was also found that will help to

  14. MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny

    NARCIS (Netherlands)

    Horton, S J; Jaques, J; Woolthuis, C; van Dijk, J; Mesuraca, M; Huls, G; Morrone, G; Vellenga, E; Schuringa, J J

    The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of

  15. Glucose metabolite glyoxal induces senescence in telomerase-immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Larsen, Simon Asbjørn; Kassem, Moustapha; Rattan, Suresh

    2012-01-01

    Background Various by-products of the cellular metabolism, such as reactive carbonyl species (RCS) are potentially harmful to cells and tissues, and play a role in many physiological and pathological processes. Among various RCS is the highly reactive dicarbonyl glyoxal (GO), which is a natural p...

  16. MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny

    NARCIS (Netherlands)

    Horton, S J; Jaques, J; Woolthuis, C; van Dijk, J; Mesuraca, M; Huls, G; Morrone, G; Vellenga, E; Schuringa, J J

    2013-01-01

    The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of t

  17. Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.

    NARCIS (Netherlands)

    Satchell, S.C.; Tasman, C.H.; Singh, A.; Ni, L.; Geelen, J.M.; Ruhland, C.J. von; O'Hare, M.J.; Saleem, M.A.; Heuvel, L.P.W.J. van den; Mathieson, P.W.

    2006-01-01

    Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of c

  18. Romidepsin reduces histone deacetylase activity, induces acetylation of histones, inhibits proliferation, and activates apoptosis in immortalized epithelial endometriotic cells.

    Science.gov (United States)

    Imesch, Patrick; Fink, Daniel; Fedier, André

    2010-12-01

    Romidepsin inhibited HDAC activity, produced acetylation of the histone proteins, up-regulated p21, and down-regulated cyclins B1 and D1, resulting in proliferation inhibition and apoptosis activation in 11z immortalized epithelial endometriotic cells. Our findings provide evidence that endometriotic cells are sensitive to the epigenetic effects of romidepsin and suggest that endometriosis may be therapeutically targeted by romidepsin.

  19. Immortalization and characterization of mouse floxed Bmp2/4 osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Li-An [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Department of Pediatric Dentistry, School of Stomatology, The Fourth Military Medical University, Xi-an (China); Yuan, Guohua; Yang, Guobin [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Key Laboratory of Oral Biomedical Engineering Ministry of Education, Wuhan (China); Ortiz-Gonzalez, Iris [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Yang, Wuchen; Cui, Yong [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); MacDougall, Mary [Department of Oral/Maxillofacial Surgery, University of Alabama, Birmingham, AL (United States); Donly, Kevin J. [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States); Harris, Stephen [Department of Periodontics, Dental School, The University of Texas Health Science Center at San Antonio, TX (United States); Chen, Shuo, E-mail: chens0@uthscsa.edu [Department of Pediatric Dentistry, The University of Texas Health Science Center at San Antonio, TX (United States)

    2009-08-14

    Generation of a floxed Bmp2/4 osteoblast cell line is a valuable tool for studying the modulatory effects of Bmp2 and Bmp4 on osteoblast differentiation as well as relevant molecular events. In this study, primary floxed Bmp2/4 mouse osteoblasts were cultured and transfected with simian virus 40 large T-antigen. Transfection was verified by polymerase chain reaction (PCR) and immunohistochemistry. To examine the characteristics of the transfected cells, morphology, proliferation and mineralization were analyzed, expression of cell-specific genes including Runx2, ATF4, Dlx3, Osx, dentin matrix protein 1, bone sialoprotein, osteopontin, osteocalcin, osteonectin and collagen type I was detected. These results show that transfected floxed Bmp2/4 osteoblasts bypassed senescence with a higher proliferation rate, but retain the genotypic and phenotypic characteristics similar to the primary cells. Thus, we for the first time demonstrate the establishment of an immortalized mouse floxed Bmp2/4 osteoblast cell line.

  20. Anti-melanogenic effects of black, green, and white tea extracts on immortalized melanocytes.

    Science.gov (United States)

    Kim, Young Chul; Choi, So Young; Park, Eun Ye

    2015-01-01

    Tea contains polyphenols and is one of the most popular beverages consumed worldwide. Because most tyrosinase inhibitors that regulate melanogenesis are phenol/catechol derivatives, this study investigated the inhibitory effects of Camellia sinensis water extracts (CSWEs), including black tea, green tea, and white tea extracts, on melanogenesis using immortalized melanocytes. CSWEs inhibited melanin accumulation and melanin synthesis along with tyrosinase activity in a concentration-dependent manner. These inhibitory effects were superior to those of arbutin, a well-known depigmenting agent. The anti-melanogenic activity of black (fermented) tea was higher than that of a predominant tea catecholamine, epigallocatechin gallate. CSWEs, especially black tea extract, decreased tyrosinase protein levels in a concentration-dependent manner. These results suggest that the anti-melanogenic effect of CSWEs is mediated by a decrease in both tyrosinase activity and protein expression, and may be augmented by fermentation. Thus, CSWEs could be useful skin-whitening agents in the cosmetic industry.

  1. Time to Nadir PSA: Of Popes and PSA--The Immortality Bias.

    Science.gov (United States)

    Johnson, Skyler B; Jackson, William C; Murgic, Jure; Feng, Felix Y; Hamstra, Daniel A

    2015-10-01

    The objective of the study was to investigate prostate-specific antigen (PSA) nadir (nPSA) and time to nPSA (TnPSA) as prognostic variables for outcomes after definitive high-dose (>75 Gy) external beam radiation therapy (RT) without androgen deprivation therapy while correcting for immortal-time bias. nPSA and TnPSA were available for 410 patients. nPSA and TnPSA's impact on freedom from biochemical failure, freedom from metastasis, and prostate cancer-specific survival was assessed on univariate and multivariate analysis using Kaplan-Meier and Cox proportional hazards regression. Outcomes were also evaluated relative to the time since achieving nPSA and not relative to the time of RT, given the intrinsic time bias in TnPSA. Median nPSA was 0.7 ng/mL (interquartile range: 0.4 to 1.1), with a median TnPSA of 25.0 months (IQR: 15.0 to 40.0). On univariate analysis both nPSA and TnPSA were predictive of all endpoints: freedom from biochemical failure, freedom from metastasis, and prostate cancer-specific survival, as categorical (all Pimmortal-time bias the benefit of long TnPSA was mostly lost. On Cox proportional hazards, a TnPSA12 months from the time of RT the TnPSA was no longer prognostic. For dose-escalated RT a lower nPSA is prognostic, but the benefit of a long TnPSA is largely an immortal-time bias and that a longer TnPSA is not in and of itself a significant favorable factor except as compared with those with the shortest TnPSA of <12 months.

  2. Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization

    Institute of Scientific and Technical Information of China (English)

    Atsushi Masamune; Masahiro Satoh; Kazuhiro Kikuta; Noriaki Suzuki; Tooru Shimosegawa

    2003-01-01

    AIM: Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous immortalization.METHODS: PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift assay.Activation of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme immunoassay.RESULTS: Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution,and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely immortalized.SIPS cells retained morphological characteristics of primary,culture-activated PSCs. SIPS expressed α-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type Ⅰ collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1β activated nuclear factor-κB, activator protein-1, and MAP kinases.Interleukin-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP kinases.CONCLUSION: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.

  3. Aquaporin expression and cell volume regulation in the SV40 immortalized rat submandibular acinar cell line.

    Science.gov (United States)

    Hansen, Ann-Kristin; Galtung, Hilde Kanli

    2007-03-01

    The amount of aquaporins present and the cellular ability to perform regulatory volume changes are likely to be important for fluid secretions from exocrine glands. In this work these phenomena were studied in an SV40 immortalized rat submandibular acinar cell line. The regulatory cell volume characteristics have not previously been determined in these cells. Cell volume regulation following hyposmotic exposure and aquaporin induction was examined with Coulter counter methodology, radioactive efflux studies, fura-2 fluorescence, and polymerase chain reaction and Western blot techniques. Cell volume regulation was inhibited by the K(+) channel antagonists quinine and BaCl(2) and the Cl(-) channel blocker 5-nitro-2-(3-phenypropylamino)benzoic acid. A concomitant increase in cellular (3)H-taurine release and Ca(2+) concentration was also observed. Chelation of both intra- and extracellular Ca(2+) with EGTA and the Ca(2+) ionophore A23187 did not, however, affect cell volume regulation. Aquaporin 5 (AQP5) mRNA and protein levels were upregulated in hyperosmotic conditions and downregulated upon return to isosmotic solutions, but were reduced by the mitogen-activated ERK-activating kinase (MEK) inhibitor U0126. A 24-h MEK inhibition also diminished hyposmotically induced cell swelling and cell volume regulation. In conclusion, it was determined that regulatory volume changes in this immortalized cell line are due to KCl and taurine efflux. In conditions that increased AQP5 levels, the cells showed a faster cell swelling and a more complete volume recovery following hyposmotic exposure. This response could be overturned by MEK inhibition.

  4. Establishment of a Conditionally Immortalized Wilms Tumor Cell Line with a Homozygous WT1 Deletion within a Heterozygous 11p13 Deletion and UPD Limited to 11p15.

    Directory of Open Access Journals (Sweden)

    Artur Brandt

    Full Text Available We describe a stromal predominant Wilms tumor with focal anaplasia and a complex, tumor specific chromosome 11 aberration: a homozygous deletion of the entire WT1 gene within a heterozygous 11p13 deletion and an additional region of uniparental disomy (UPD limited to 11p15.5-p15.2 including the IGF2 gene. The tumor carried a heterozygous p.T41A mutation in CTNNB1. Cells established from the tumor carried the same chromosome 11 aberration, but a different, homozygous p.S45Δ CTNNB1 mutation. Uniparental disomy (UPD 3p21.3pter lead to the homozygous CTNNB1 mutation. The tumor cell line was immortalized using the catalytic subunit of human telomerase (hTERT in conjunction with a novel thermolabile mutant (U19dl89-97tsA58 of SV40 large T antigen (LT. This cell line is cytogenetically stable and can be grown indefinitely representing a valuable tool to study the effect of a complete lack of WT1 in tumor cells. The origin/fate of Wilms tumors with WT1 mutations is currently poorly defined. Here we studied the expression of several genes expressed in early kidney development, e.g. FOXD1, PAX3, SIX1, OSR1, OSR2 and MEIS1 and show that these are expressed at similar levels in the parental and the immortalized Wilms10 cells. In addition the limited potential for muscle/ osteogenic/ adipogenic differentiation similar to all other WT1 mutant cell lines is also observed in the Wilms10 tumor cell line and this is retained in the immortalized cells. In summary these Wilms10 cells are a valuable model system for functional studies of WT1 mutant cells.

  5. MIO-M1 cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics

    National Research Council Canada - National Science Library

    Lawrence, Jean M; Singhal, Shweta; Bhatia, Bhairavi; Keegan, David J; Reh, Thomas A; Luthert, Philip J; Khaw, Peng T; Limb, Gloria Astrid

    2007-01-01

    .... We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors...

  6. Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

    Energy Technology Data Exchange (ETDEWEB)

    Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

    1992-08-01

    Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

  7. Expression of a TGF-{beta} regulated cyclin-dependent kinase inhibitor in normal and immortalized airway epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Tierney, L.A.; Bloomfield, C.; Johnson, N.F. [and others

    1995-12-01

    Tumors arising from epithelial cells, including lung cancers are frequently resistant to factors that regulate growth and differentiation in normal in normal cells. Once such factor is transforming growth factor-{Beta} (TGF-{Beta}). Escape from the growth-inhibitory effects of TGF-{Beta} is thought to be a key step in the transformation of airway epithelial cells. most lung cancer cell lines require serum for growth. In contrast, normal human bronchial epithelial (NHBE) cells are exquisitely sensitive to growth-inhibitory and differentiating effects of TGF-{Beta}. The recent identification of a novel cyclin-dependent kinase inhibitor, p15{sup INK4B}, which is regulated by TGF-{Beta}, suggests a mechanism by which TGF-{Beta} mediates growth arrest in NHBE cells. The purpose of this study was two-fold: (1) to determine if p15{sup INK4B} is induced by TGF-{Beta} in NHBE cells or immortalized bronchial epithelial (R.1) cells and if that induction corresponds to a G1/S cell-cycle arrest; (2) to determine the temporal relationship between p15{sup INK4B} induction, cell-cycle arrest, and the phosphorylation state of the pRB because it is thought that p15{sup INK4B} acts indirectly by preventing phosphorylation of the RB gene product. In this study, expression of p15{sup INK4B} was examined in NHBE cells and R.1 cells at different time intervals following TGF-{Beta} treatment. The expression of this kinase inhibitor and its relationship to the cell and the pRb phosphorylation state were examined in cells that were both sensitive (NHBE) and resistant (R.1) to the effects of TGF-{Beta}. These results suggest that the cyclin-dependent kinase inhibitor, p15{sup INK4B}, is involved in airway epithelial cell differentiation and that loss or reduction of expression plays a role in the resistance of transformed or neoplastic cells to the growth-inhibitory effects of TGF-{Beta}.

  8. Inactivation of Sag/Rbx2/Roc2 E3 Ubiquitin Ligase Triggers Senescence and Inhibits Kras-Induced Immortalization

    Directory of Open Access Journals (Sweden)

    Mingjia Tan

    2015-01-01

    Full Text Available Our recent study showed that SAG/RBX2 E3 ubiquitin ligase regulates apoptosis and vasculogenesis by promoting degradation of NOXA and NF1, and co-operates with Kras to promote lung tumorigenesis by activating NFκB and mTOR pathways via targeted degradation of tumor suppressive substrates including IκB, DEPTOR, p21 and p27. Here we investigated the role of Sag/Rbx2 E3 ligase in cellular senescence and immortalization of mouse embryonic fibroblasts (MEFs and report that Sag is required for proper cell proliferation and KrasG12D-induced immortalization. Sag inactivation by genetic deletion remarkably suppresses cell proliferation by inducing senescence, which is associated with accumulation of p16, but not p53. Mechanistically, Sag deletion caused accumulation of Jun-B, a substrate of Sag-Fbxw7 E3 ligase and a transcription factor that drives p16 transcription. Importantly, senescence triggered by Sag deletion can be largely rescued by simultaneous deletion of Cdkn2a, the p16 encoding gene, indicating its causal role. Furthermore, KrasG12D-induced immortalization can also be abrogated by Sag deletion via senescence induction, which is again rescued by simultaneous deletion of Cdkn2a. Finally, we found that Sag deletion inactivates KrasG12D activity and block the MAPK signaling pathway, together with accumulated p16, to induce senescence. Taken together, our results demonstrated that Sag is a KrasG12D-cooperating oncogene required for KrasG12D-induced immortalization and transformation, and targeting SAG-SCF E3 ligase may, therefore, have therapeutic value for senescence-based cancer treatment.

  9. Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax

    Directory of Open Access Journals (Sweden)

    Kravetz Amanda J

    2004-01-01

    Full Text Available Abstract Background Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined. Results We found that immortalization occurred earlier and more frequently when the myogenic cells lacked the pro-apoptotic protein Bax. Furthermore, myogenesis was altered by Bax inactivation as Bax-null cells produced muscle colonies with more nuclei than wild-type cells, though a lower percentage of the Bax-null nuclei were incorporated into multinucleate myotubes. In vivo, both the fast and slow myofibers in Bax-null muscles had smaller cross-sectional areas than those in wild-type muscles. After immortalization, both Bax-null and Bax-positive myogenic cells expressed desmin, retained the capacity to form multinucleate myotubes, expressed p19ARF protein, and retained p53 functions. Expression of p16INK4a, however, was found in only about half of the immortalized myogenic cell lines. Conclusions Mouse myogenic cells can undergo spontaneous immortalization via a mechanism that can include, but does not require, loss of p16INK4a, and also does not require inactivation of p19ARF or p53. Furthermore, loss of Bax, which appears to be a downstream effector of p53, accelerates immortalization of myogenic cells and alters myogenesis.

  10. Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients

    Directory of Open Access Journals (Sweden)

    Magomet Aushev

    2017-09-01

    Full Text Available Epidermolysis bullosa simplex (EBS is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the KRT5 locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

  11. Shadows of the susceptible-infectious-susceptible immortality transition in small networks

    Science.gov (United States)

    Holme, Petter

    2015-07-01

    Much of the research on the behavior of the SIS model on networks has concerned the infinite size limit; in particular the phase transition between a state where outbreaks can reach a finite fraction of the population, and a state where only a finite number would be infected. For finite networks, there is also a dynamic transition—the immortality transition—when the per-contact transmission probability λ reaches 1. If λ <1 , the probability that an outbreak will survive by an observation time t tends to zero as t →∞ ; if λ =1 , this probability is 1. We show that treating λ =1 as a critical point predicts the λ dependence of the survival probability also for more moderate λ values. The exponent, however, depends on the underlying network. This fact could, by measuring how a vertex's deletion changes the exponent, be used to evaluate the role of a vertex in the outbreak. Our work also confirms an extremely clear separation between the early die-off (from the outbreak failing to take hold in the population) and the later extinctions (corresponding to rare stochastic events of several consecutive transmission events failing to occur).

  12. Generation and characterization of the first immortalized alpaca cell line suitable for diagnostic and immunization studies.

    Directory of Open Access Journals (Sweden)

    Valentina Franceschi

    Full Text Available Raising of alpacas as exotic livestock for wool and meat production and as companion animals is growing in importance in the United States, Europe and Australia. Furthermore the alpaca, as well as the rest of the camelids, possesses the peculiarity of producing single-chain antibodies from which nanobodies can be generated. Nanobodies, due to their structural simplicity and reduced size, are very versatile in terms of manipulation and bio-therapeutic exploitation. In fact the biotech companies involved in nanobody production and application continue to grow in number and size. Hence, the development of reagents and tools to assist in the further growth of this new scientific and entrepreneurial reality is becoming a necessity. These are needed mainly to address alpaca disease diagnosis and prophylaxis, and to develop alpaca immunization strategies for nanobody generation. For instance an immortalized alpaca cell line would be extremely valuable. In the present work the first stabilized alpaca cell line from alpaca skin stromal cells (ASSCs was generated and characterized. This cell line was shown to be suitable for replication of viruses bovine herpesvirus-1, bovine viral diarrhea virus and caprine herpesvirus-1 and the endocellular parasite Neospora caninum. Moreover ASSCs were easy to transfect and transduce by several methods. These two latter characteristics are extremely useful when recombinant antigens need to be produced in a host homologous system. This work could be considered as a starting point for the expansion of the biotechnologies linked to alpaca farming and industry.

  13. Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

    Energy Technology Data Exchange (ETDEWEB)

    Henderson, R.F.; Waide, J.J.; Lechner, J.F.

    1995-12-01

    A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

  14. Biodentine induces immortalized murine pulp cell differentiation into odontoblast-like cells and stimulates biomineralization.

    Science.gov (United States)

    Zanini, Marjorie; Sautier, Jean Michel; Berdal, Ariane; Simon, Stéphane

    2012-09-01

    Biodentine (Septodont, Saint Maur des Faussés, France), a new tricalcium silicate-based cement, has recently been commercialized and advertised as a bioactive material. Its clinical application and physical properties have been widely described, but, so far, its bioactivity and biological effect on pulp cells have not been clearly shown. Thus, the aim of this study was to evaluate the biological effect of Biodentine on immortalized murine pulp cells (OD-21). OD-21 cells were cultured with or without Biodentine. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) colorimetric assay after 2, 3, and 5 days of stimulation. The expression of several biomolecular markers was analyzed to screen differentiation pathways, both on a gene level with Real-time reverse transcription polymerase chain reaction and on a protein level by measuring alkaline phosphatase activity. Alizarin red staining was used to assess and quantify biomineralization. The expression patterns of several genes confirmed the differentiation of OD-21 cells into odontoblasts during the period of cell culture. Our results suggest that Biodentine is bioactive because it increased OD-21 cell proliferation and biomineralization in comparison with controls. Because of its bioactivity, Biodentine can be considered as a suitable material for clinical indications of dentin-pulp complex regeneration, such as direct pulp capping. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  15. Mapping of imprinted quantitative trait loci using immortalized F2 populations.

    Directory of Open Access Journals (Sweden)

    Yongxian Wen

    Full Text Available Mapping of imprinted quantitative trait loci (iQTLs is helpful for understanding the effects of genomic imprinting on complex traits in animals and plants. At present, the experimental designs and corresponding statistical methods having been proposed for iQTL mapping are all based on temporary populations including F2 and BC1, which can be used only once and suffer some other shortcomings respectively. In this paper, we propose a framework for iQTL mapping, including methods of interval mapping (IM and composite interval mapping (CIM based on conventional low-density genetic maps and point mapping (PM and composite point mapping (CPM based on ultrahigh-density genetic maps, using an immortalized F2 (imF2 population generated by random crosses between recombinant inbred lines or doubled haploid lines. We demonstrate by simulations that imF2 populations are very desirable and the proposed statistical methods (especially CIM and CPM are very powerful for iQTL mapping, with which the imprinting effects as well as the additive and dominance effects of iQTLs can be unbiasedly estimated.

  16. The conserved mitochondrial gene distribution in relatives of Turritopsis nutricula, an immortal jellyfish.

    Science.gov (United States)

    Devarapalli, Pratap; Kumavath, Ranjith N; Barh, Debmalya; Azevedo, Vasco

    2014-01-01

    Turritopsis nutricula (T. nutricula) is the one of the known reported organisms that can revert its life cycle to the polyp stage even after becoming sexually mature, defining itself as the only immortal organism in the animal kingdom. Therefore, the animal is having prime importance in basic biological, aging, and biomedical researches. However, till date, the genome of this organism has not been sequenced and even there is no molecular phylogenetic study to reveal its close relatives. Here, using phylogenetic analysis based on available 16s rRNA gene and protein sequences of Cytochrome oxidase subunit-I (COI or COX1) of T. nutricula, we have predicted the closest relatives of the organism. While we found Nemopsis bachei could be closest organism based on COX1 gene sequence; T. dohrnii may be designated as the closest taxon to T. nutricula based on rRNA. Moreover, we have figured out four species that showed similar root distance based on COX1 protein sequence.

  17. Short hairpin RNA screen indicates that Klotho beta/FGF19 protein overcomes stasis in human colonic epithelial cells.

    Science.gov (United States)

    Kim, Jinyong; Eskiocak, Ugur; Stadler, Guido; Lou, Zhenjun; Kuro-o, Makoto; Shay, Jerry W; Wright, Woodring E

    2011-12-16

    Normal human colonic epithelial cells (HCECs) are not immortalized by telomerase alone but also require CDK4. Some human cell types growth-arrest due to stress- or aberrant signaling-induced senescence (stasis). Stasis represents the consequences of growth conditions culture that are inadequate to maintain long-term proliferation. Overexpressed CDK4 titers out p16 and allows cells to ignore the growth arrest signals produced by stasis. To identify factors contributing to the inadequate culture environment, we used a 62,000-member shRNA library to knock down factors cooperating with human telomerase reverse transcriptase (hTERT) in the immortalization of HCECs. Knockdown of Klotho gamma (KLG; also known as KLPH and LCTL) allowed hTERT to immortalize HCECs. KLG is one isoform of the Klotho family of factors that coordinate interaction between different FGF ligands and the FGF receptor. We also found that knockdown of KLG induced another member of the Klotho family, Klotho beta (KLB). Induction of KLB was maintained and could activate ERK1/2 in immortalized cells. Supplementation of the culture medium with the KLB ligand FGF19 had a similar effect on hTERT-expressing HCECs as knockdown of KLG regarding both immortalization and down-regulation of the tumor suppressor Klotho alpha. Together, these data suggest that KLB is an important regulator in the immortalization of HCECs by facilitating FGF19 growth factor signaling.

  18. Differential response of primary and immortalized CD4+ T cells to Neisseria gonorrhoeae-induced cytokines determines the effect on HIV-1 replication.

    Directory of Open Access Journals (Sweden)

    Wendy N Dobson-Belaire

    Full Text Available To compare the effect of gonococcal co-infection on immortalized versus primary CD4(+ T cells the Jurkat cell line or freshly isolated human CD4(+ T cells were infected with the HIV-1 X4 strain NL4-3. These cells were exposed to whole gonococci, supernatants from gonococcal-infected PBMCs, or N. gonorrhoeae-induced cytokines at varying levels. Supernatants from gonococcal-infected PBMCs stimulated HIV-1 replication in Jurkat cells while effectively inhibiting HIV-1 replication in primary CD4(+ T cells. ELISA-based analyses revealed that the gonococcal-induced supernatants contained high levels of proinflammatory cytokines that promote HIV-1 replication, as well as the HIV-inhibitory IFNα. While all the T cells responded to the HIV-stimulatory cytokines, albeit to differing degrees, the Jurkat cells were refractory to IFNα. Combined, these results indicate that N. gonorrhoeae elicits immune-modulating cytokines that both activate and inhibit HIV-production; the outcome of co-infection depending upon the balance between these opposing signals.

  19. [SOUND CONTACTS] Transcendence and immortality in Busoni’s "Doktor Faust"

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    Antonella DiGiulio

    2015-04-01

    Full Text Available The categorization of the many musical and literary works inspired by the Faust legend is based on their two possible conclusions: Faust ends up in hell or is redeemed and attaints heaven. Busoni’s work is often categorized in terms of the redemptive form. I will analyze a third possibility: during the last part of the nineteenth century, philosophers conceptualized that man’s feelings of unhappiness came from his confinement into an eternal recurrence and that life doesn’t end with the death. Scholars tend to ignore the links between Doktor Faust and these philosophical thoughts because of Busoni’s explicit denial of such intentions. And yet Busoni wrote that an opera is the highest form of artistic expression, where music gives words to the unspoken. Therefore I will argue that Busoni characterizes Doktor Faust as a hybridization of the new philosophical theories with literature and music. Busoni’s depiction of the main character in this opera may not be intentional, but the composer summed up all the qualities of Nietzsche’s Übermensch, D’Annunzio’s Superuomo, and Pascoli’s fanciullino in Faust. Faust discovers his own transcendent power, which is independent from the material universe and yet belongs to nature’s laws that are beyond God and the evil. Doktor Faust’s attainment of immortality does not pray for divine redemption and wins out over everything: in this opera, Faust is neither redeemed nor damned. Faust becomes aware that death is nothing but the means that lead to rebirth: “ich, Faust, ein ewiger Wille”.

  20. A nuclear Argonaute promotes multi-generational epigenetic inheritance and germline immortality

    Science.gov (United States)

    Buckley, Bethany; Burkhart, Kirk; Gu, Sam Guoping; Spracklin, George; Kershner, Aaron; Fritz, Heidi; Kimble, Judith; Fire, Andrew; Kennedy, Scott

    2012-01-01

    Epigenetic information is frequently erased near the start of each new generation (1). In some cases, however, epigenetic information can be transmitted from parent to progeny (epigenetic inheritance) (2). A particularly striking example of epigenetic inheritance is dsRNA-mediated gene silencing (RNAi) in C. elegans, which can be inherited for more than five generations (3–8). To understand this process we conducted a genetic screen for animals defective for transmitting RNAi silencing signals to future generations. This screen identified the gene heritable RNAi defective (hrde)-1. hrde-1 encodes an Argonaute (Ago) that associates with small interfering (si)RNAs in germ cells of the progeny of animals exposed to dsRNA. In nuclei of these germ cells, HRDE-1 engages the Nrde nuclear RNAi pathway to direct H3K9me3 at RNAi targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed siRNAs, which direct nuclear gene silencing in germ cells. In hrde-1 or nuclear RNAi deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Ago HRDE-1 directs gene-silencing events in germ cell nuclei, which drive multi-generational RNAi inheritance and promote immortality of the germ cell lineage. We propose that C. elegans uses the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes. PMID:22810588

  1. Functional Characteristics of Reversibly Immortalized Hepatic Progenitor Cells Derived from Mouse Embryonic Liver

    Directory of Open Access Journals (Sweden)

    Yang Bi

    2014-10-01

    Full Text Available Background/Aims: Liver is a vital organ and retains its regeneration capability throughout adulthood, which requires contributions from different cell populations, including liver precursors and intrahepatic stem cells. To overcome the mortality of hepatic progenitors (iHPs in vitro, we aim to establish reversibly immortalized hepatic progenitor cells from mouse embryonic liver. Methods and Results: Using retroviral system to stably express SV40 T antigen flanked with Cre/LoxP sites, we establish a repertoire of iHP clones with varied differentiation potential. The iHP cells maintain long-term proliferative activity and express varied levels of progenitor markers (Pou5f1/Oct4 and Dlk and hepatocyte markers (AFP, Alb and ApoB. Five representative iHP clones express hepatic/pancreatic transcription factors HNF3α/Foxa1, HNF3β/Foxa2, and HNF4α/MODY1. Dexamethasone is shown to promote the expression of hepatocyte markers AFP and TAT, along with ICG-uptake and glycogen storage functions in the iHP clones. Cre-mediated removal of SV40 T antigen reverses the proliferative activity of iHP cells. When iHP cells are subcutaneously implanted in athymic nude mice, no tumor formation is observed for up to 8 weeks. Conclusions: We demonstrate that the established iHP cells are stable, reversible, and non-tumorigenic hepatic progenitor-like cells, which should be valuable for studying liver organogenesis, metabolic regulations, and hepatic lineage-specific differentiation.

  2. Production and Functional Characterization of Murine Osteoclasts Differentiated from ER-Hoxb8-Immortalized Myeloid Progenitor Cells.

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    Frank Zach

    Full Text Available In vitro differentiation into functional osteoclasts is routinely achieved by incubation of embryonic stem cells, induced pluripotent stem cells, or primary as well as cryopreserved spleen and bone marrow-derived cells with soluble receptor activator of nuclear factor kappa-B ligand and macrophage colony-stimulating factor. Additionally, osteoclasts can be derived from co-cultures with osteoblasts or by direct administration of soluble receptor activator of nuclear factor kappa-B ligand to RAW 264.7 macrophage lineage cells. However, despite their benefits for osteoclast-associated research, these different methods have several drawbacks with respect to differentiation yields, time and animal consumption, storage life of progenitor cells or the limited potential for genetic manipulation of osteoclast precursors. In the present study, we therefore established a novel protocol for the differentiation of osteoclasts from murine ER-Hoxb8-immortalized myeloid stem cells. We isolated and immortalized bone marrow cells from wild type and genetically manipulated mouse lines, optimized protocols for osteoclast differentiation and compared these cells to osteoclasts derived from conventional sources. In vitro generated ER-Hoxb8 osteoclasts displayed typical osteoclast characteristics such as multi-nucleation, tartrate-resistant acid phosphatase staining of supernatants and cells, F-actin ring formation and bone resorption activity. Furthermore, the osteoclast differentiation time course was traced on a gene expression level. Increased expression of osteoclast-specific genes and decreased expression of stem cell marker genes during differentiation of osteoclasts from ER-Hoxb8-immortalized myeloid progenitor cells were detected by gene array and confirmed by semi-quantitative and quantitative RT-PCR approaches. In summary, we established a novel method for the quantitative production of murine bona fide osteoclasts from ER-Hoxb8 stem cells generated from

  3. Oxidative DNA damage correlates with cell immortalization and mir-92 expression in hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Romilda Cardin

    2012-05-01

    Full Text Available Abstract Background MicroRNAs expression has been extensively studied in hepatocellular carcinoma but little is known regarding the relationship, if any, with inflammation, production of reactive oxygen species (ROS, host’s repair mechanisms and cell immortalization. This study aimed at assessing the extent of oxidative DNA damage (8-hydroxydeoxyguanosine - 8-OHdG in different phases of the carcinogenetic process, in relation to DNA repair gene polymorphism, telomeric dysfunction and to the expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death. Methods Tissue samples obtained either at surgery, [neoplastic (HCC and adjacent non-cancerous cirrhotic tissues (NCCT] at percutaneous or laparoscopic biopsy (patients with HCV or HBV-related hepatitis or patients undergoing cholecystectomy were analysed for 8-OHdG (HPLC-ED, OGG1 (a DNA repair gene polymorphism (PCR-RFLP, telomerase activity, telomere length (T/S, by RT-PCR, Taqman microRNA assay and Bad/Bax mRNA (RT-PCR. Fifty-eight samples from 29 HCC patients (obtained in both neoplastic and peritumoral tissues, 22 from chronic hepatitis (CH and 10 controls (cholecystectomy patients - CON were examined. Results Eight-OHdG levels were significantly higher in HCC and NCCT than in CH and CON (p=0.001. Telomerase activity was significantly higher in HCC than in the remaining subgroups (p=0.002; conversely T/S was significantly lower in HCC (p=0.05. MiR-199a-b, -195, -122, -92a and −145 were down-regulated in the majority of HCCs while miR-222 was up-regulated. A positive correlation was observed among 8-OHdG levels, disease stage, telomerase activity, OGG1 polymorphisms and ALT/GGT levels. In HCC, miR-92 expression correlated positively with telomerase activity, 8-OHdG levels and Bad/Bax mRNA. Conclusions The above findings confirm the accumulation, in the progression of chronic liver damage to HCC, of a ROS

  4. QTL analysis of Kernel-related traits in maize using an immortalized F2 population.

    Science.gov (United States)

    Zhang, Zhanhui; Liu, Zonghua; Hu, Yanmin; Li, Weihua; Fu, Zhiyuan; Ding, Dong; Li, Haochuan; Qiao, Mengmeng; Tang, Jihua

    2014-01-01

    Kernel size and weight are important determinants of grain yield in maize. In this study, multivariate conditional and unconditional quantitative trait loci (QTL), and digenic epistatic analyses were utilized in order to elucidate the genetic basis for these kernel-related traits. Five kernel-related traits, including kernel weight (KW), volume (KV), length (KL), thickness (KT), and width (KWI), were collected from an immortalized F2 (IF2) maize population comprising of 243 crosses performed at two separate locations over a span of two years. A total of 54 unconditional main QTL for these five kernel-related traits were identified, many of which were clustered in chromosomal bins 6.04-6.06, 7.02-7.03, and 10.06-10.07. In addition, qKL3, qKWI6, qKV10a, qKV10b, qKW10a, and qKW7a were detected across multiple environments. Sixteen main QTL were identified for KW conditioned on the other four kernel traits (KL, KWI, KT, and KV). Thirteen main QTL were identified for KV conditioned on three kernel-shape traits. Conditional mapping analysis revealed that KWI and KV had the strongest influence on KW at the individual QTL level, followed by KT, and then KL; KV was mostly strongly influenced by KT, followed by KWI, and was least impacted by KL. Digenic epistatic analysis identified 18 digenic interactions involving 34 loci over the entire genome. However, only a small proportion of them were identical to the main QTL we detected. Additionally, conditional digenic epistatic analysis revealed that the digenic epistasis for KW and KV were entirely determined by their constituent traits. The main QTL identified in this study for determining kernel-related traits with high broad-sense heritability may play important roles during kernel development. Furthermore, digenic interactions were shown to exert relatively large effects on KL (the highest AA and DD effects were 4.6% and 6.7%, respectively) and KT (the highest AA effects were 4.3%).

  5. Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

    Science.gov (United States)

    Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M; Yang, Liwei; LaRocque, Jeannine R; Hall, Julie; Miska, Eric A; Ahmed, Shawn

    2014-10-14

    Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing.

  6. Reversible immortalization of Nestin-positive precursor cells from pancreas and differentiation into insulin-secreting cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Pei; Li, Li; Qi, Hui [The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People' s Hospital), Jinan University, 518020 Shenzhen (China); Zhou, Han-xin [Department of General Surgery, First Hospital (Shenzhen Second People' s Hospital) of Shenzhen University, 518020 Shenzhen (China); Deng, Chun-yan [The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People' s Hospital), Jinan University, 518020 Shenzhen (China); Li, Fu-rong, E-mail: frli62@yahoo.com [The Clinical Medical Research Center, The Second Clinical Medical College (Shenzhen People' s Hospital), Jinan University, 518020 Shenzhen (China); Shenzhen Institution of Gerontology, 518020 Shenzhen (China)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer The NPPCs from mouse pancreas were isolated. Black-Right-Pointing-Pointer Tet-on system for SV40 large in NPPCs was used to get RINPPCs. Black-Right-Pointing-Pointer The RINPPCs can undergo at least 80 population doublings without senescence. Black-Right-Pointing-Pointer The RINPPCs can be induced to differentiate into insulin-producing cells. Black-Right-Pointing-Pointer The combination of GLP-1 and sodium butyrate promoted the differentiation process. -- Abstract: Pancreatic stem cells or progenitor cells posses the ability of directed differentiation into pancreatic {beta} cells. However, these cells usually have limited proliferative capacity and finite lifespan in vitro. In the present study, Nestin-positive progenitor cells (NPPCs) from mouse pancreas that expressed the pancreatic stem cells or progenitor cell marker Nestin were isolated to obtain a sufficient number of differentiated pancreatic {beta} cells. Tet-on system for SV40 large T-antigen expression in NPPCs was used to achieve reversible immortalization. The reversible immortal Nestin-positive progenitor cells (RINPPCs) can undergo at least 80 population doublings without senescence in vitro while maintaining their biological and genetic characteristics. RINPPCs can be efficiently induced to differentiate into insulin-producing cells that contain a combination of glucagon-like peptide-1 (GLP-1) and sodium butyrate. The results of the present study can be used to explore transplantation therapy of type I diabetes mellitus.

  7. Immortal Ring-Opening Polymerization of rac-Lactide Using Polymeric Alcohol as Initiator to Prepare Graft Copolymer

    Directory of Open Access Journals (Sweden)

    Na Liu

    2016-01-01

    Full Text Available In the presence of a small molecular protic initiator, immortal ring-opening polymerization (ROP of lactide (LA is a highly efficient strategy to synthesize polylactide in a controllable manner, while using polymeric alcohol as an initiator has been less investigated. A series of polymeric alcohols (PS–OH composed of styrene and 4.3%–18% hydroxyl functional styrene (diethyl(hydroxy(4-vinylphenylmethylphosphonate, St–OH were synthesized through reversible addition-fragmentation transfer (RAFT polymerization. Using PS–OH as an initiator, the immortal ROP of rac-LA was catalyzed by dibutylmagnesium (MgnBu2 under various ratios of monomer to hydroxyl group within PS–OH to generate polystyrene-g-polylactide (PS–g–PLA copolymers with different graft lengths. After thermal annealing at 115 °C, the PLA domain aggregated to nanospheres among the PS continuum. The size of the nanospheres, varying from 130.1 to 224.2 nm, was related to the graft density and length of PS–g–PLA. Nanoporous films were afforded through chemical etching of the PLA component.

  8. The tumorigenicity of immortalized cells differentiated from mouse embryonic stem cells%小鼠胚胎干细胞分化细胞永生化后致瘤性研究

    Institute of Scientific and Technical Information of China (English)

    沈干; 丛笑倩

    2012-01-01

    目的 研究小鼠胚胎干细胞(embryonic stem cells,ESC)诱导分化的血管内皮细胞永生化后的致瘤现象.方法 体外培养系中,将诱导的小鼠ESC的拟胚体分化为圆形细胞.通过脂质体将人端粒酶催化亚基(human telomerase reverse transcriptase,hTE RT)基因转染诱导分化中的圆形细胞,应用RT-PCR及免疫组织化学等方法分析、观察诱导分化的血管样结构的组成细胞和被hTERT基因转染的圆形细胞的形态和生物学特性.并将基因转染的细胞植入裸鼠皮下,观察其致瘤性.结果 携带hTERT基因、从ESC分化而来的圆形细胞TEC3,在体外可大量增殖,培养的前2 d(48 h),细胞数就增加了1倍多,至72 h细胞数增加了12倍,持续传代,端粒酶活性高表达,95%细胞表达Flk-1、CD34和vWF,并有管道化生长特性.将细胞植入裸鼠皮下后1周有肿瘤形成.结论 转染hTERT基因可使小鼠ESC分化细胞持续增殖,植入裸鼠体内具有致瘤性.%Objective To discuss the tumorigenicity of immortalized endothelial cells differentiated from embryonic stem cells. Methods The embryoid bodies (EB) formed in vitro from embryonic stem cells,were induced to differentiate into many “round cells” (the precursor of endothelial cells).These “ round cells” later formed the vascular tube-like structures. To immortalize these cells,human telomerase reverse transcriptase (hTERT) cDNA was transfected into “round cells” by lipofectine,RT-PCR and immunocytochemistry were used to evaluate the immortalized cells.And the tumorigenicity of these cells were evaluated by being injected into nude mice subcutaneously. Results 95% of these transfected cells expressed Flk-1,CD34 and vWF,and could proliferate in large quantity in vitro ( cell number was doubled in 2 days, and increased 12 times in 3 days), and were able to form tubular structures.Conclusions These results suggest that hTERT cDNA transfection can immortalize induced endothelial cells

  9. Perspectives on Immortality and Eternal Life in the Book of Wisdom and the Gospel of John: A Conceptual Analysis Based on Metaphorical Structuring

    NARCIS (Netherlands)

    Valentin, R.I.

    2016-01-01

    The similarities between the Fourth Gospel and wisdom literature have been addressed by many scholars. This study is also intended to continue along this line by looking at the particular link between the Wisdom of Solomon and the Gospel of John with a specific focus on the concepts of immortality

  10. Death anxiety and symbolic immortality in relatives at risk for familial amyloid polyneuropathy type I (FAP I, ATTR V30M).

    Science.gov (United States)

    Santos, Paula I; Figueiredo, Eurico; Gomes, Inês; Sequeiros, Jorge

    2010-12-01

    This study is an investigation of the impact of familial amyloid polyneuropathy type I (FAP I, ATTR V30M) on death anxiety and symbolic immortality. Templer and Drolet's scales were administered to 524 individuals: (1) 84 relatives at risk, (2) 92 relatives not at risk for FAP I; and (3) a control group (n = 348) with no known hereditary disease in their families. At-risk relatives had, on average, a higher score for death anxiety and a lower score for symbolic immortality, than either those not-at-risk or controls. There were no significant differences in scores on either measure for those not-at-risk versus controls. Being at risk increases death anxiety and threatens the sense of symbolic immortality and psychosocial wellbeing. This may be true for other serious hereditary disorders as well. Genetic counsellors should become familiar with these concepts, feel comfortable initiating discussions about death with their patients, and be able to identify and reinforce their patients' and family members' sense of symbolic immortality.

  11. CPM Is a Useful Cell Surface Marker to Isolate Expandable Bi-Potential Liver Progenitor Cells Derived from Human iPS Cells.

    Science.gov (United States)

    Kido, Taketomo; Koui, Yuta; Suzuki, Kaori; Kobayashi, Ayaka; Miura, Yasushi; Chern, Edward Y; Tanaka, Minoru; Miyajima, Atsushi

    2015-10-13

    To develop a culture system for large-scale production of mature hepatocytes, liver progenitor cells (LPCs) with a high proliferation potential would be advantageous. We have found that carboxypeptidase M (CPM) is highly expressed in embryonic LPCs, hepatoblasts, while its expression is decreased along with hepatic maturation. Consistently, CPM expression was transiently induced during hepatic specification from human-induced pluripotent stem cells (hiPSCs). CPM(+) cells isolated from differentiated hiPSCs at the immature hepatocyte stage proliferated extensively in vitro and expressed a set of genes that were typical of hepatoblasts. Moreover, the CPM(+) cells exhibited a mature hepatocyte phenotype after induction of hepatic maturation and also underwent cholangiocytic differentiation in a three-dimensional culture system. These results indicated that hiPSC-derived CPM(+) cells share the characteristics of LPCs, with the potential to proliferate and differentiate bi-directionally. Thus, CPM is a useful marker for isolating hiPSC-derived LPCs, which allows development of a large-scale culture system for producing hepatocytes and cholangiocytes.

  12. Myth about immortality in the contemporary pop culture: A case study about Toše Proeski

    Directory of Open Access Journals (Sweden)

    Stevanović Lada

    2013-01-01

    Full Text Available This paper explores the intersection of death and ideology in the sphere of popular culture, as exemplified by the afterlife destiny of Toše Proeski, a Macedonian pop singer whose huge popularity continued even after his death. His sudden, untimely death in his twenties had exploded as huge news, preoccupying all sorts of media. What I am interested in this paper is what has happened afterwards. In which way his popularity and his afterlife continue? Can we identify some kind of pop hero cult here, and is it possible to recognize common elements, or even a pattern according to which (newborn stars develop an afterlife destiny? Can we use this opportunity to discuss immortality myths? Do pop stars get monuments or are these reserved just for national heroes?

  13. Man should not let death attain the dominion of his thoughts: An Essay on Subjectivity, Self-Preservation and Immortality

    Directory of Open Access Journals (Sweden)

    Kasper Lysemose

    2010-12-01

    Full Text Available Mortality seems to have no place in the theories on subjectivity in Kant and Husserl. It is suggested that this entente cordiale is an expression of the shared principle at the heart of their philosophy, i.e. the principle of selfpreservation. Self-preservation is a principle that in a certain sense excludes mortality. It is argued that the primary sense of this exclusion is not theoretical but practical. Kant and Husserl are both endorsing the imperative that man should not let death attain the dominion of his thoughts (cf. Mann 1976, 600. The positive correlate to this is to be found in the demand that man should think of himself as if he was immortal. With special regard to Husserl the predicament that accompanies his relentless attempt to fulfill this demand is described as a sluice through which anthropology threatens to flow into the phenomenological enterprise.

  14. Detection of quantitative trait loci and heterotic loci for plant height using an immortalized F2 population in maize

    Institute of Scientific and Technical Information of China (English)

    TANG JiHua; MA XiQing; TENG WenTao; YAN JianBing; WU WeiRen; DAI JingRui; LI JianSheng

    2007-01-01

    A set of recombinant inbred lines (RIL) derived from Yuyu22, an elite hybrid widespread in China, was used to construct an immortalized F2 (IF2) population comprising 441 different crosses. Genetic linkage maps were constructed containing 10 linkages groups with 263 simple sequence repeat (SSR) molecular markers. Twelve and ten quantitative trait loci (QTL) were detected for plant height in the IF2 and RIL populations respectively, using the composite interval mapping method, and six same QTL were identified in the two populations. In addition, ten unique heterotic loci (HL) located on seven different chromosomes were revealed for plant height using the mid-parent heterosis as the input data. These HL explained 1.26%-8.41% of the genotypic variance in plant height heterosis and most expressed overdominant effects. Only three QTL and HL were located in the same chromosomal region, it implied that plant height and its heterosis might be controlled by two types of genetic mechanisms.

  15. Adaptive regulation of taurine and beta-alanine uptake in a human kidney cell line from the proximal tubule

    DEFF Research Database (Denmark)

    Jessen, H; Jacobsen, Christian

    1997-01-01

    1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine...

  16. Regulation of hTERT Expression and Function in Newly Immortalized p53(+) Human Mammary Epithelial Cell Lines

    Science.gov (United States)

    2008-06-01

    Cancer Cell 2006; 10:459-72. 9. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N, Vassiliou LV , Kolettas E, Niforou K, Zoumpourlis...Keshaviah A, Bae YK, Argani P, Marks J, Richardson A, Cooper A, Strausberg R, Riggins GJ, Schnitt S, Gabrielson E, Gelman R, Polyak K: Molecular markers in...3326. 70. Burstein HJ, Polyak K, Wong JS, Lester SC, Kaelin CM: Ductal Carci- noma in Situ of the Breast. New England Journal of Medicine 2004, 350

  17. Isolation and characterization of exosome from human embryonic stem cell-derived c-myc-immortalized mesenchymal stem cells

    NARCIS (Netherlands)

    Lai, Ruenn Chai; Yeo, Ronne Wee Yeh; Padmanabhan, Jayanthi; Choo, Andre; De Kleijn, Dominique P V; Lim, Sai Kiang

    2016-01-01

    Mesenchymal stem cells (MSC) are currently the cell type of choice in many cell therapy trials. The number of therapeutic applications for MSCs registered as product IND submissions with the FDA and initiation of registered clinical trials has increased substantially in recent years, in particular b

  18. Combination of immortalization and inducible death strategies to generate a human mesenchymal stromal cell line with controlled survival

    Directory of Open Access Journals (Sweden)

    Paul Bourgine

    2014-03-01

    By combining the opposite concepts of ‘induced-life’ and ‘inducible-death’, we generated a hMSCs line with defined properties and allowing for temporally controlled survival. The cell line represents a relevant tool for medical discovery in regenerative medicine and a potential means to address availability, standardization and safety requirements in cell & gene therapy. The concept of a hTERT-iCasp9 combination, here explored in the context of hMSCs, could be extended to other types of progenitor/stem cells.

  19. Micronucleus formation in human keratinocytes is dependent on radiation quality and tissue architecture.

    Science.gov (United States)

    Snijders, Antoine M; Mannion, Brandon J; Leung, Stanley G; Moon, Sol C; Kronenberg, Amy; Wiese, Claudia

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. © 2014 Wiley Periodicals, Inc.

  20. Molecular Mechanisms of Radiation-Induced Genomic Instability in Human Cells

    Energy Technology Data Exchange (ETDEWEB)

    Howard L. Liber; Jeffrey L. Schwartz

    2005-10-31

    There are many different model systems that have been used to study chromosome instability. What is clear from all these studies is that conclusions concerning chromosome instability depend greatly on the model system and instability endpoint that is studied. The model system for our studies was the human B-lymphoblastoid cell line TK6. TK6 was isolated from a spontaneously immortalized lymphoblast culture. Thus there was no outside genetic manipulation used to immortalize them. TK6 is a relatively stable p53-normal immortal cell line (37). It shows low gene and chromosome mutation frequencies (19;28;31). Our general approach to studying instability in TK6 cells has been to isolate individual clones and analyze gene and chromosome mutation frequencies in each. This approach maximizes the possibility of detecting low frequency events that might be selected against in mass cultures.

  1. Emerging of fractal geometry on surface of human cervical epithelial cells during progression towards cancer

    Science.gov (United States)

    Dokukin, M. E.; Guz, N. V.; Woodworth, C.D.; Sokolov, I.

    2015-01-01

    Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation. PMID:25844044

  2. Regard Death as Life-Concept of After Life in Stories of Immortals%视死如生--略论《搜神记》的死后世界观

    Institute of Scientific and Technical Information of China (English)

    李文智

    2016-01-01

    The Concept of afterlife in Stories of Immortals is not only reflects the wei-jin period religious complex situation,but also clearly reflects the Chinese original concept of afterlife and it`s evolution ,with human nature growing,the afterlife straight like the human version .Although the concept of afterlife has different expression,it is still the different expression of Chinese original concept of afterlife in the new era ,and shows no obvious foreign religious color.%《搜神记》的死后世界观不仅反映了魏晋时期宗教信仰的复杂状况,而且也明显地反映出中国原有死后世界观的嬗变情况,人性化不断增强,直似人间的翻版。《搜神记》对于死后世界观有不同表述,但所表现的仍然是中国本土原有的死后世界观在新的时代背景下承前启后的新阐释,并没有表现出明显的外来宗教色彩。

  3. Phenotype transformation of immortalized NCM460 colon epithelial cell line by TGF-β1 is associated with chromosome instability.

    Science.gov (United States)

    Huang, Chao; Wen, Bin

    2016-10-01

    Transforming growth factor-β1 (TGF-β1) within tumor microenvironment has a pivotal function in cancer initiation and tumorigenesis, and hence this study was to observe the malignant transformation induced by TGF-β1 in an immortalized colon epithelial cell line NCM460 for better understanding the mechanisms of colon carcinogenesis. Immortalized colon epithelial cell line NCM460 was used as the model of this study, and was treated with different concentrations of TGF-β1 for different time. Then, immunofluorescence was performed to observe the change of phenotype hallmarks including adherent junction protein E-cadherin, cytoskeleton protein vimentin, and tight junction marker ZO-1, western blotting analysis was performed to detect the expression of the above three markers and two transcription factors (Snail and Slug) involved in the transformation by TGF-β1. In addition, chromosome instability (CHI) including analysis of DNA-ploid was detected by flow cytometry. Our results revealed significant loss or reduction of ZO-1 and E-cadherin, and robust emergence of vimentin in the cell line NCM460 after a 15-, 20-, and 25-day treatment with 10 ng/ml TGF-β1. Interestingly, 20 and 25 days after stimulation with 5 ng/ml TGF-β1, expression of E-cadherin and ZO-1 revealed a pattern roughly similar to that of 10 ng/ml TGF-β1, especially, both expressions was vanished and vimentin expression was dramatically increased at days 25 after TGF-β1 stimulation. After a stimulation with 10 ng/ml TGF-β1 for 15, 20, and 25 days, the levels of Snail and Slug expression in the cells were significantly up-regulated, compared with the cells treated with TGF-β1 inhibitor LY364947, PBS or balnk control (P TGF-β1 after its stimulation for 15, 20, and 25 days. Very few mitotic cells with treatment of PBS for 15, 20 and 25 days were non-diploid whose DNA content was greater or less than 4 N, but these cells were significantly increased after exposure to TGF-β1 for 15, 20, and

  4. Retrieving Immortal Questions, Initiating Immortal Conversations

    Science.gov (United States)

    Duarte, Eduardo M.

    2012-01-01

    In his presidential address, which is included in this collection of papers, Kip Kline suggests that the time has arrived to redirect the work of philosophy of education away from the path of critical theory, and thus to depart from what he described as the discourse of "parrhesia." The author takes Kline's critique of critical philosophy of…

  5. Telomere,telomerase,cell of immortalization and aging%端粒、端粒酶与细胞衰老及永生化

    Institute of Scientific and Technical Information of China (English)

    滕勇; 胡蕴玉; 关玉成

    2008-01-01

    正常细胞体外培养时,表现为有限生长特性,经一定的细胞倍增次数后,失去了对促分裂因子刺激的反应,不可逆地失去增殖能力而停止分裂,细胞开始衰老的历程.目前认为染色体末端(端粒)的缺失会使细胞逐渐失去增殖能力,导致细胞的衰老和死亡.人端粒酶催化亚单位(hTERT)可以激活端粒酶的活性,延长染色体末端DNA,维持基因组的稳定.端粒、端粒酶、hTERT的发现为细胞衰老的研究提供了新的思路,同时也应用于永生化细胞系的建立,特别是在组织工程种子细胞生物性能研究和细胞库的建立中将发挥重要作用.%Normal cells have limited proliferation ability.After certain cycles of proliferation,they will lose the response ability to growth factors and finally cease division and start the course of aging. In current opinion,lacking of the terminal end of a chromosome(telomere)is the cause for cells to loss the proliferation ability and leads cells to aging and death.The human telomerase catalytic subunit 1(hTERT)can activate telomemse which prolong DNAs of the terminal end of chmmosome and help cells gain genomic stabilization.The discoveries of telomere,telomerase and hTERT provide new idea for studying of cell aging and the findings are also applied in the establishment of immortal cell line. Also they may play an important role in the studv of biological feature of seed cell in tissue engineering and the establishment of cell bank.

  6. A gene expression signature classifying telomerase and ALT immortalization reveals an hTERT regulatory network and suggests a mesenchymal stem cell origin for ALT

    DEFF Research Database (Denmark)

    Lafferty-Whyte, K; Cairney, C J; Will, M B

    2009-01-01

    Telomere length is maintained by two known mechanisms, the activation of telomerase or alternative lengthening of telomeres (ALT). The molecular mechanisms regulating the ALT phenotype are poorly understood and it is unknown how the decision of which pathway to activate is made at the cellular le......TERT in different tumour types and normal tissues. We also show evidence to suggest a novel mesenchymal stem cell origin for ALT immortalization in cell lines and mesenchymal tissues....

  7. Establishment of Immortalized BMP2/4 Double Knock-Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis.

    Science.gov (United States)

    Wu, Li-An; Wang, Feng; Donly, Kevin J; Baker, Andrew; Wan, Chunyan; Luo, Daoshu; MacDougall, Mary; Chen, Shuo

    2016-06-01

    Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock-out ((ko/ko)) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4(ko/ko) cells were verified by green immunofluorescence and PCR. BMP2/4(ko/ko) osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone-relate genes was reduced in the BMP2/4(ko/ko) cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4(ko/ko) osteoblasts as reflected by decreased Mmp-2 and Mmp-9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages.

  8. Rare Circulating Cells in Familial Waldenstrom Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization.

    Directory of Open Access Journals (Sweden)

    Maroulio Pertesi

    Full Text Available The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM. We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL, but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS, compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS (p = 10-7. The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV.

  9. Rare Circulating Cells in Familial Waldenström Macroglobulinemia Displaying the MYD88 L265P Mutation Are Enriched by Epstein-Barr Virus Immortalization.

    Science.gov (United States)

    Pertesi, Maroulio; Galia, Perrine; Nazaret, Nicolas; Vallée, Maxime; Garderet, Laurent; Leleu, Xavier; Avet-Loiseau, Hervé; Foll, Matthieu; Byrnes, Graham; Lachuer, Joel; McKay, James D; Dumontet, Charles

    2015-01-01

    The MYD88 L265P is a recurrent somatic mutation in neoplastic cells from patients with Waldenström Macroglobulinemia (WM). We identified the MYD88 L265P mutation in three individuals from unrelated families, but its presence did not explain the disease segregation within these WM pedigrees. We observed the mutation in these three individuals at high allele fractions in DNA extracted from EBV-immortalized Lymphoblastoid cell lines established from peripheral blood (LCL), but at much lower allele fractions in DNA extracted directly from peripheral blood, suggesting that this mutation is present in a clonal cell subpopulation rather than of germ-line origin. Furthermore, we observed that the MYD88 L265P mutation is enriched in WM families, detected in 40.5% of patients with familial WM or MGUS (10/22 WM, 5/15 MGUS), compared to 3.5% of patients with familial MM or MGUS (0/72 MM, 4/41 MGUS) (p = 10-7). The mutant allele frequency increased with passages in vitro after immortalization with Epstein-Barr virus (EBV) consistent with the MYD88 L265P described gain-of-function proposed for this mutation. The MYD88 L265P mutation appears to be frequently present in circulating cells in patients with WM, and MGUS, and these cells are amenable to immortalization by EBV.

  10. Multiparameter Analysis of Human Bone Marrow Stromal Cells Identifies Distinct Immunomodulatory and Differentiation-Competent Subtypes

    NARCIS (Netherlands)

    S. James (Sally); J. Fox (James); F. Afsari (Farinaz); J. Lee (Jennifer); S. Clough (Sally); C. Knight (Charlotte); J. Ashmore (James); P. Ashton (Peter); O. Preham (Olivier); M.J. Hoogduijn (Martin); R.D.A.R. Ponzoni (Raquel De Almeida Rocha); Y. Hancock; M. Coles (Mark); P.G. Genever (Paul)

    2015-01-01

    textabstractBone marrow stromal cells (BMSCs, also called bone-marrow-derived mesenchymal stromal cells) provide hematopoietic support and immunoregulation and contain a stem cell fraction capable of skeletogenic differentiation. We used immortalized human BMSC clonal lines for multi-level analysis

  11. Analysis of YeXiaoluan’ s Immortal Thoughts%浅析叶小鸾的游仙思想

    Institute of Scientific and Technical Information of China (English)

    钱丽君

    2016-01-01

    叶小鸾是明代较为出众的女性文人,其聪颖多才,小小年纪就凭借在吟诗填词上的悟性得到了家人的赞许;但她又生性敏感,时代背景与家庭氛围的熏染使其对俗世生活产生了颇多感悟,最终促使她形成了浓郁的游仙思想,其游仙思想中既蕴蓄着她对绝对自由的渴望,又包涵着她对众生平等的向往,还隐含着她对及时行乐的追求。%Ye Xiaoluan was a famous female writer lived in the Ming dynasty .When she was young , she got approval from the family by her virtue of the poetry writing .But family atmosphere and her personality prompted her to form an immortal thoughts . These ideas contained both her desire for the absolute freedom ,which is the yearning to the equality of all living things .The thought implied the pursuit of enjoying pleasure in good time .

  12. A contemplation in negation and emphasizes of outstanding philosophers of Tehran philosophy school in immortality of subject in substantial movement.

    Directory of Open Access Journals (Sweden)

    mohamad khosravi farsani

    2015-03-01

    Full Text Available Agha Ali and Jelveh ,two of outstanding of Tehran philosophy school, have reconsidered Sandra's opinions in two opposite sides. Agha Ali is the claimant of negation and ambiguity in most of Sandra's opinions while emphasizing on transcendent theosophy foundations ,and he tries to make these ambiguities clear .However , Jelveh follows Avicenna's way and has the idea of criticizing Sandra .This article has chosen the problem Of immortality of subject in substantial movement between all debates of these two philosophers of the same age.in this essay we are going to survey Agha Ali's claims in his new explantation in one hand ,and survey and criticize the new explanation of Jelveh in negation Of substantial movement .One of the consequences of surveying and criticizing these two philosophers is that we recognize Agha Ali has succeeded in making Sadra's opinions clear by presenting a new explanation of substantial movement subject. However ,Jelveh has merged Sadra's opinions with Avicenna's instead of debilitating Sadra's opinions and stabilizing Avicenna's opinions.

  13. MRG-1, an autosome-associated protein, silences X-linked genes and protects germline immortality in Caenorhabditis elegans.

    Science.gov (United States)

    Takasaki, Teruaki; Liu, Zheng; Habara, Yasuaki; Nishiwaki, Kiyoji; Nakayama, Jun-Ichi; Inoue, Kunio; Sakamoto, Hiroshi; Strome, Susan

    2007-02-01

    MRG15, a mammalian protein related to the mortality factor MORF4, is required for cell proliferation and embryo survival. Our genetic analysis has revealed that the Caenorhabditis elegans ortholog MRG-1 serves similar roles. Maternal MRG-1 promotes embryo survival and is required for proliferation and immortality of the primordial germ cells (PGCs). As expected of a chromodomain protein, MRG-1 associates with chromatin. Unexpectedly, it is concentrated on the autosomes and not detectable on the X chromosomes. This association is not dependent on the autosome-enriched protein MES-4. Focusing on possible roles of MRG-1 in regulating gene expression, we determined that MRG-1 is required to maintain repression in the maternal germ line of transgenes on extrachromosomal arrays, and of several X-linked genes previously shown to depend on MES-4 for repression. MRG-1 is not required for PGCs to acquire transcriptional competence or for the turn-on of expression of several PGC-expressed genes (pgl-1, glh-1, glh-4 and nos-1). By contrast to this result in PGCs, MRG-1 is required for ectopic expression of those germline genes in somatic cells lacking the NuRD complex component MEP-1. We discuss how an autosome-enriched protein might repress genes on the X chromosome, promote PGC proliferation and survival, and influence the germ versus soma distinction.

  14. Comparative QTL analysis of maize seed artificial aging between an immortalized F2 population and its corresponding RILs

    Institute of Scientific and Technical Information of China (English)

    Bin Wang; Zhanhui Zhang; Zhiyuan Fu; Zonghua Liu; Yanmin Hu; Jihua Tang

    2016-01-01

    Seed aging decreases the quality and vigor of crop seeds, thereby causing substantial agricultural and economic losses in crops. To identify genetic differences in seed aging between homozygotes and heterozygotes in maize, the seeds of a set of recombinant inbred lines (RILs) and an immortalized F2 (IF2) population were subjected to artificial aging treatments for 0, 2, 3, and 4 days under 45 ºC and 85%relative humidity and seed vigor was then evaluated in a field experiment. Seed vigor of all entries tested decreased sharply with longer aging treatment and seed vigor decreased more slowly in heterozygotes than in homozygotes. Forty-nine QTL were detected for four measured seed vigor traits in the RIL (28 QTL) and IF2 (21 QTL) populations. Only one QTL, qGP5, was detected in both populations, indicating that the genes involved in anti-aging mechanisms differed between inbred lines and hybrids. Several QTL were identified to be responsible for multiple seed vigor traits simultaneously in the RIL and IF2 populations under artificial aging conditions. These QTL may include major genes for seed vigor or seed aging. QTL qVI4b and qGE3a detected in the RIL population coincided with genes ZmLOX1 and ZmPLD1 in the same respective chromosomal regions. These QTL would be useful for screening for anti-aging genes in maize breeding.

  15. "IMMORTALITY OF MY SOUL...": THE GENESIS OF PUSHKIN'S POEM EXEGI MONUMENTUM (I HAVE ERECTED A MONUMENT TO MYSELF

    Directory of Open Access Journals (Sweden)

    Lyudmila Vasilyevna Stebeneva

    2013-11-01

    Full Text Available Pushkin's overcoming of death is represented by two aspects of his work: stoical Christian acceptance of death and identification of immortality of a soul with creative heritage. A special role in understanding of this issue is given to the Kamennoostrovsky cycle of poems. Choosing the semantic counter-poles of the cycle, Pushkin opposes absolute spiritual death (Italian Imitation against redemptional death which introduces people to Eternal life (Temporal power. Poet’s final contemplation of his past ideals in the poem From Pindemonti shows the alteration of the ideological emphasis in his artistic consciousness: An antique vector is transformed into pure aesthetics, whereas his fear of death is relieved by Christian values. These ideas reach their apotheosis in the poem Exegi Monumentum (I have erected a monument to myself, which accomplishes Pushkin’s spiritual quest of his final years, though it is not a part of his Kamennoostrovsky cycle of poems. It has been pointed out that Pushkin's bequest/manifesto themes and motifs first appeared in his Lyceum works and can be traced throughout all his creative periods.

  16. Conophylline Promotes the Proliferation of Immortalized Mesenchymal Stem Cells Derived from Fetal Porcine Pancreas (iPMSCs)

    Institute of Scientific and Technical Information of China (English)

    ZHANG Hui-ru; HUA Jin-lian; LI Dan; CAO Hui; L Xiao; CHU Yuan-kui; BAI Yao-fu; JIN Ya-ping; PENG Sha; DOU Zhong-ying

    2013-01-01

    Conophylline, is a bis (indole) alkaloid consisting of two pentacyclic aspidosperma skeletons, isolated from Tabernaemontana divaricata, which has been found to induce b-cell differentiation in rat pancreatic acinar carcinoma cells and in cultured rat pancreatic tissue. However, the precise role of conophylline in the growth and survival of immortalized pancreatic mesenchymal stem cells (iPMSCs) derived from fetal porcine pancreas were not understood at present. To determine whether this molecule is involved in controlling the proliferation of iPMSCs, we examined the effects of conophylline on iPMSCs. We found that conophylline can robustly stimulate iPMSCs proliferation, even promote their potential differentiation into islet-like clusters analyzed by cell counting, morphology, RT-PCR and real-time PCR, Western blotting, glucose-stimulated insulin release and insulin content analysis. The effects of conophylline were inhibited by LY294002, which is the inhibitor of the PI3K pathway. These results suggest that conophylline plays a key role in the regulation of cell mass proliferation, maintenance of the undifferentiated state of iPMSCs and also promotes iPMSCs differentiated into insulin-producing cells.

  17. Intracellular Ca2+ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line.

    Science.gov (United States)

    Aure, Marit H; Røed, Asbjørn; Galtung, Hilde Kanli

    2010-06-01

    The water channel aquaporin 5 (AQP5) seems to play a key role in salivary fluid secretion and appears to be critical in the cell volume regulation of acinar cells. Recently, the cation channel transient potential vanilloid receptor 4 (TRPV4) was shown to be functionally connected to AQP5 and also to cell volume regulation in salivary glands. We used the Simian virus 40 (SV40) immortalized cell line SMG C10 from the rat submandibular salivary gland to investigate the effect of ATP and the neurotransmitter analogue carbachol on Ca(2+) signalling and cell volume regulation, as well as the involvement of TRPV4 in the responses. We used fura-2-AM imaging, cell volume measurements, and western blotting. Both carbachol and ATP increased the concentration of intracellular Ca(2+), but no volume changes could be measured. Inhibition of TRPV4 with ruthenium red impaired both ATP- and carbachol-stimulated Ca(2+) signals. Peak Ca(2+) signalling during hyposmotic exposure was significantly decreased following inhibition of TRPV4, while the cells' ability to volume regulate appeared to be unaffected. These results show that in the SMG C10 cells, simulation of nervous stimulation did not induce cell swelling, although the cells had intact volume regulatory mechanisms. Furthermore, even though Ca(2+) signals were not needed for this volume regulation, TRPV4 seems to play a role during ATP and carbachol stimulation.

  18. Side population rather than CD133+ cells distinguishes enriched tumorigenicity in hTERT-immortalized primary prostate cancer cells

    Directory of Open Access Journals (Sweden)

    Wolcott Karen

    2011-09-01

    Full Text Available Abstract Background Subpopulations of cancer cells with the capacity of generating solid tumors have been characterized. In various cancer types, including prostate cancer cells, a side population (SP and CD133-expressing cells have been proposed as containing a population cancer cells with stem-like ability. Therefore the aim of this work was to determine, in prostate cancer cell lines, the frequency and tumorigenic potential of SP and CD133+ cells. Results In vitro 2D colony-forming assay and sphere-forming assay, Flow cytometry analysis and magnetic cell sorting were utilized to sort CD133+, CD133- and Side population (SP cells. Our findings indicate that CD44 and integrin α-6 are uniformly expressed in the hTERT cell lines; however, CD133 is expressed only in a small population (in vitro and in vivo. Additionally, for the hTERT cells, SP rather than CD133 expression showed an 8-fold enhanced tumorigenic potential. The data suggest that SP cells, rather than those with CD133 marker, contain the rare population of CSC capable of producing prostate tumors. Conclusion Collectively, our data suggest that although CD133 is expressed only in a small population of hTERT-immortalized prostate cancer cells, it is not likely to be associated with stem cells, as CD133- and CD133+ cells exhibited similar tumorigenicity. However, SP isolated cells, appear to be enriched with tumorigenic stem-like cells capable of generating palpable tumors.

  19. Comparative QTL analysis of maize seed artificial aging between an immortalized F2 population and its corresponding RILs

    Institute of Scientific and Technical Information of China (English)

    Bin Wang; Zhanhui Zhang; Zhiyuan Fu; Zonghua Liu; Yanmin Hu; Jihua Tang

    2016-01-01

    Seed aging decreases the quality and vigor of crop seeds,thereby causing substantial agricultural and economic losses in crops.To identify genetic differences in seed aging between homozygotes and heterozygotes in maize,the seeds of a set of recombinant inbred lines(RILs) and an immortalized F2(IF2) population were subjected to artificial aging treatments for 0,2,3,and 4 days under 45℃ and 85%relative humidity and seed vigor was then evaluated in a field experiment.Seed vigor of all entries tested decreased sharply with longer aging treatment and seed vigor decreased more slowly in heterozygotes than in homozygotes.Forty-nine QTL were detected for four measured seed vigor traits in the RIL(28QTL) and IF2(21 QTL) populations.Only one QTL,qGP5,was detected in both populations,indicating that the genes involved in anti-aging mechanisms differed between inbred lines and hybrids.Several QTL were identified to be responsible for multiple seed vigor traits simultaneously in the RIL and IF2 populations under artificial aging conditions.These QTL may include major genes for seed vigor or seed aging.QTL qVI4 b and qGE3 a detected in the RIL population coincided with genes ZmLOX1 and ZmPLD1 in the same respective chromosomal regions.These QTL would be useful for screening for anti-aging genes in maize breeding.

  20. Transcriptional regulation of proteoglycan 4 by 17β-estradiol in immortalized baboon temporomandibular joint disc cells.

    Science.gov (United States)

    McDaniel, Jennifer S; Akula Suresh Babu, Ramya; Navarro, Mary M; LeBaron, Richard G

    2014-04-01

    Temporomandibular joint disorders (TMDs) affect a significant portion of the population of the USA, with the majority of those seeking treatment being women of childbearing age. Owing to this striking sexual dimorphism it has been postulated that sex hormones play a role in the maintenance of normal temporomandibular joint (TMJ) function. Proteoglycan 4 (PRG4) is a secreted lubricating molecule required for maintaining low frictional levels within articular joints; however, its role in the TMJ is not well characterized. In this study we describe the development of immortalized baboon cells isolated from specific regions of the TMJ disc and their use in the investigation of PRG4 expression and localization patterns in the TMJ. We identified conserved estrogen response elements within the 5' flanking region of the PRG4 gene of several species, and found that treatment of baboon TMJ disc cells with estrogen led to reduced PRG4 promoter activity and reduced expression of PRG4 mRNA in vitro. The observed negative regulation of PRG4 by estrogen could lead to increased friction and degradation of joint components over time. This study, for the first time, provides evidence of the regulatory potential of estrogen on PRG4 gene expression and suggests a novel etiology for the gender disparity observed among TMD patients.

  1. Hepatocellular carcinoma-derived exosomes promote motility of immortalized hepatocyte through transfer of oncogenic proteins and RNAs.

    Science.gov (United States)

    He, Mian; Qin, Hao; Poon, Terence C W; Sze, Siu-Ching; Ding, Xiaofan; Co, Ngai Na; Ngai, Sai-Ming; Chan, Ting-Fung; Wong, Nathalie

    2015-09-01

    Exosomes are increasingly recognized as important mediators of cell-cell communication in cancer progression through the horizontal transfer of RNAs and proteins to neighboring or distant cells. Hepatocellular carcinoma (HCC) is a highly malignant cancer, whose metastasis is largely influenced by the tumor microenvironment. The possible role of exosomes in the interactions between HCC tumor cell and its surrounding hepatic milieu are however largely unknown. In this study, we comprehensively characterized the exosomal RNA and proteome contents derived from three HCC cell lines (HKCI-C3, HKCI-8 and MHCC97L) and an immortalized hepatocyte line (MIHA) using Ion Torrent sequencing and mass spectrometry, respectively. RNA deep sequencing and proteomic analysis revealed exosomes derived from metastatic HCC cell lines carried a large number of protumorigenic RNAs and proteins, such as MET protooncogene, S100 family members and the caveolins. Of interest, we found that exosomes from motile HCC cell lines could significantly enhance the migratory and invasive abilities of non-motile MIHA cell. We further demonstrated that uptake of these shuttled molecules could trigger PI3K/AKT and MAPK signaling pathways in MIHA with increased secretion of active MMP-2 and MMP-9. Our study showed for the first time that HCC-derived exosomes could mobilize normal hepatocyte, which may have implication in facilitating the protrusive activity of HCC cells through liver parenchyma during the process of metastasis.

  2. Slugging their way to immortality: driving mammary epithelial cells into a stem cell-like state.

    Science.gov (United States)

    Soady, Kelly; Smalley, Matthew J

    2012-09-10

    Delineating the molecular factors that define and maintain the mammary stem cell state is vital for understanding normal development and tumourigenesis. A recent study by Guo and colleagues identifies two master transcriptional regulators of mammary stem cells, Slug and Sox9, ectopic expression of which confers stem cell attributes on differentiated mammary epithelial cells. Slug and Sox9 expression was also shown to determine in vivo metastatic potential of human breast cancer cell lines. Understanding these factors in the context of normal lineage differentiation is an important step toward elucidating the mammary epithelial cell hierarchy and the origins of cancer stem cells.

  3. Reversal of informational entropy and the acquisition of germ-like immortality by somatic cells.

    Science.gov (United States)

    Kyriazis, Marios

    2014-01-01

    We live within an increasingly technological, information-laden environment for the first time in human evolution. This subjects us (and will continue to subject us in an accelerating fashion) to an unremitting exposure to 'meaningful information that requires action'. Directly dependent upon this new environment are novel evolutionary pressures, which can modify existing resource allocation mechanisms and may eventually favour the survival of somatic cells (particularly neurons) at the expense of germ line cells. In this theoretical paper I argue that persistent, structured information-sharing in both virtual and real domains, leads to increased biological complexity and functionality, which reflects upon human survival characteristics. Certain biological immortalisation mechanisms currently employed by germ cells may thus need to be downgraded in order to enable somatic cells to manage these new energy demands placed by our modern environment. Relevant concepts from a variety of disciplines such as the evolution of complex adaptive systems, information theory, digital hyper-connectivity, and cell immortalisation will be reviewed. Using logical, though sometimes speculative arguments, I will attempt to describe a new biology. A biology not driven by sex and reproduction but by information and somatic longevity.

  4. Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells.

    Science.gov (United States)

    Boonkaew, Benjawan; Kempf, Margit; Kimble, Roy; Cuttle, Leila

    2014-12-01

    A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver(®) and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver(®) showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

  5. Escape from Evil? Notes on Capacity, Tragedy, Coding and Non-Destructive Immortality Projects

    Directory of Open Access Journals (Sweden)

    Ronnie Lippens

    2015-09-01

    Full Text Available Like James Hardie-Bick’s (2015 contribution this paper elaborates on the importance for socio-legal studies of Ernest Becker’s (1924-1974 work on the destructive consequences of human … all too human forms of death denial. With Becker, it makes an attempt to think through the question, ‘Is it possible, in the light of the human condition, to escape evil?’ Terminally ill by the early 1970s, Becker himself was left no time to complete his thoughts on this matter. Focussing on the point where Becker left the discussion when he died in 1974 we make an attempt here, still with an eye on the aforementioned question, to explore the work of a number of philosophers whose work we know Becker was familiar with: Benedictus de Spinoza, Henri Bergson, and Albert Camus. In their work, it is argued here, one could find elements towards thinking through the question of evil, and the (impossibility of escaping it. Al igual que el artículo de James Hardie-Bick (2015, este artículo profundiza en la importancia que tiene para los estudios sociojurídicos el trabajo de Ernest Becker (1924-1974 sobre las consecuencias destructivas del ser humano… y la tan humana negación de la muerte. Con Becker, se realiza un intento de reflexionar sobre esta pregunta: “Es posible, a la luz de la condición humana, escapar del mal? Enfermo terminal a comienzos de los años 70, el propio Becker no tuvo tiempo de completar sus pensamientos sobre este asunto. Centrándonos en el punto en el que Becker dejó el debate, cuando murió en 1974, y sin perder de vista esta pregunta, se intenta analizar el trabajo de una serie de filósofos, con el que estaba familiarizado Becker: Benedictus de Spinoza, Henri Bergson y Albert Camus. Se defiende que en su trabajo se pueden encontrar elementos que hacen pensar sobre la cuestión del mal, y la (imposibilidad de escapar de él.DOWNLOAD THIS PAPER FROM SSRN: http://ssrn.com/abstract=2619429

  6. Cellular immortality in brain tumours: an integration of the cancer stem cell paradigm.

    Science.gov (United States)

    Rahman, Ruman; Heath, Rachel; Grundy, Richard

    2009-04-01

    Brain tumours are a diverse group of neoplasms that continue to present a formidable challenge in our attempt to achieve curable intervention. Our conceptual framework of human brain cancer has been redrawn in the current decade. There is a gathering acceptance that brain tumour formation is a phenotypic outcome of dysregulated neurogenesis, with tumours viewed as abnormally differentiated neural tissue. In relation, there is accumulating evidence that brain tumours, similar to leukaemia and many solid tumours, are organized as a developmental hierarchy which is maintained by a small fraction of cells endowed with many shared properties of tissue stem cells. Proof that neurogenesis persists throughout adult life, compliments this concept. Although the cancer cell of origin is unclear, the proliferative zones that harbour stem cells in the embryonic, post-natal and adult brain are attractive candidates within which tumour-initiation may ensue. Dysregulated, unlimited proliferation and an ability to bypass senescence are acquired capabilities of cancerous cells. These abilities in part require the establishment of a telomere maintenance mechanism for counteracting the shortening of chromosomal termini. A strategy based upon the synthesis of telomeric repeat sequences by the ribonucleoprotein telomerase, is prevalent in approximately 90% of human tumours studied, including the majority of brain tumours. This review will provide a developmental perspective with respect to normal (neurogenesis) and aberrant (tumourigenesis) cellular turnover, differentiation and function. Within this context our current knowledge of brain tumour telomere/telomerase biology will be discussed with respect to both its developmental and therapeutic relevance to the hierarchical model of brain tumourigenesis presented by the cancer stem cell paradigm.

  7. Transfection of normal human bronchial epithelial cells with the bcl-2 oncogene

    Energy Technology Data Exchange (ETDEWEB)

    Kennedy, C.H.; Kenyon, K.D.; Tesfaigzi, J. [and others

    1995-12-01

    In vitro, studies examining the transformation of virus-immortalized human bronchial epithelial (HBE) cells after exposure to chemical and physical carcinogens have contributed to our understanding of the mechanisms that underlie the development of lung cancer. Virus-immortalized HBE cells have been used because of both the limited life span of normal human bronchial epithelial (NHBE) cells in culture (approximately 30-35 population doublins) and their resistance to in vitro malignant transformation. For example, human papillomavirus (HPV)-immortalized HBE cells have been used to study the genetic changes that occur after exposure to {alpha}-particles in vitro. Although this model may prove to be useful for studying the 18% or less of bronchogenic carcinomas found to contain HPV sequences, it is not an appropriate model for studying the majority of lung epithelial malignancies in which HPV DNA is not detected. This view is supported by the fact that HPV-immortalized cell lines commonly exhibit aneuploidy. This results of this study suggest that: (1) NHBE cells can be transiently transfected with the pCMV{Beta} vector; and (2) the antibiotic hygromycin-resistant transfected cells.

  8. Activism as a Heroic Quest for Symbolic Immortality: An Existential Perspective on Collective Action

    Directory of Open Access Journals (Sweden)

    Julia Elad-Strenger

    2016-03-01

    Full Text Available Excellent research exists on the conditions that generate political and social activism. Yet a central issue has remained perplexing: how does the personal need to stand out as unique and heroic interact with the concern for the positive image of the group, and the desire to protect and bolster its status, goals and shared values, in propelling collective action? Inspired by existential theory and research, this paper proposes an existential perspective on activism that identifies the human desire for a sense of meaning and significance as an important motivation underlying individuals' choice to engage in collective action. This study outlines an integrative model of collective action, combining insights from existential psychology with insights from the social identity perspective, to bridge together needs and concerns associated with both personal identity and group identity into a single model of collective action through the concept of death-anxiety buffering mechanisms. This model suggests that collective action is an effective means to satisfy existential needs through bolstering and protecting group interests and values on the one hand, and realizing the activist's heroism project on the other. Suggestions for future research are discussed.

  9. Balancing Benefits and Risks of Immortal Data: Participants' Views of Open Consent in the Personal Genome Project.

    Science.gov (United States)

    Zarate, Oscar A; Brody, Julia Green; Brown, Phil; Ramirez-Andreotta, Mónica D; Perovich, Laura; Matz, Jacob

    2016-01-01

    An individual's health, genetic, or environmental-exposure data, placed in an online repository, creates a valuable shared resource that can accelerate biomedical research and even open opportunities for crowd-sourcing discoveries by members of the public. But these data become "immortalized" in ways that may create lasting risk as well as benefit. Once shared on the Internet, the data are difficult or impossible to redact, and identities may be revealed by a process called data linkage, in which online data sets are matched to each other. Reidentification (re-ID), the process of associating an individual's name with data that were considered deidentified, poses risks such as insurance or employment discrimination, social stigma, and breach of the promises often made in informed-consent documents. At the same time, re-ID poses risks to researchers and indeed to the future of science, should re-ID end up undermining the trust and participation of potential research participants. The ethical challenges of online data sharing are heightened as so-called big data becomes an increasingly important research tool and driver of new research structures. Big data is shifting research to include large numbers of researchers and institutions as well as large numbers of participants providing diverse types of data, so the participants' consent relationship is no longer with a person or even a research institution. In addition, consent is further transformed because big data analysis often begins with descriptive inquiry and generation of a hypothesis, and the research questions cannot be clearly defined at the outset and may be unforeseeable over the long term. In this article, we consider how expanded data sharing poses new challenges, illustrated by genomics and the transition to new models of consent. We draw on the experiences of participants in an open data platform-the Personal Genome Project-to allow study participants to contribute their voices to inform ethical consent

  10. Pentosan polysulfate regulates scavenger receptor-mediated, but not fluid-phase, endocytosis in immortalized cerebral endothelial cells.

    Science.gov (United States)

    Deli, M A; Abrahám, C S; Takahata, H; Katamine, S; Niwa, M

    2000-12-01

    1. Effects of pentosan polysulfate (PPS) and the structurally related sulfated polyanions dextran sulfate, fucoidan, and heparin on the scavenger receptor-mediated and fluidphase endocytosis in GP8 immortalized rat brain endothelial cells were investigated. 2. Using 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarboxyamine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL), we found a binding site with high affinity and low binding capacity, and another one with low affinity and high binding capacity. Increasing ligand concentrations could not saturate DiI-AcLDL uptake. DiI-AcLDL uptake, but not binding, was sensitive to pretreatment with filipin, an inhibitor of caveola formation. 3. PPS (20-200 microg/ml) significantly reduced the binding of DiI-AcLDL after coincubation for 3 hr, though this effect was less expressed after 18 hr. Among other polyanions, only fucoidan decreased the DiI-AcLDL binding after 3 hr, whereas dextran sulfate significantly increased it after 18 hr. PPS treatment induced an increase in DiI-AcLDL uptake, whereas other polysulfated compounds caused a significant reduction. 4. Fluid-phase endocytosis determined by the accumulation of Lucifer yellow was concentration and time dependent in GP8 cells. Coincubation with PPS or other sulfated polyanions could not significantly alter the rate of Lucifer yellow uptake. 5. In conclusion. PPS decreased the binding and increased the uptake of DiI-AcLDL in cerebral endothelial cells, an effect not mimicked by the other polyanions investigated.

  11. A high-affinity human monoclonal IgM antibody reacting with multiple strains of Mycoplasma hominis

    DEFF Research Database (Denmark)

    Moller, SA; Birkelund, Svend; Borrebaeck, CA

    1990-01-01

    Human monoclonal antibodies were produced against Mycoplasma hominis by in vitro immunization of peripheral blood lymphocytes from a healthy seropositive donor using low amounts of antigen (5 ng/ml). The immune B lymphocytes were subsequently immortalized by Epstein-Barr virus transformation...

  12. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J.; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E.; Macdougall, Mary; Chen, Shuo

    2016-01-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2ko/ko dp) cell line by introducing Cre fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2fx/fx dp) cells. iBmp2ko/ko dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2ko/ko dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmpko/ko cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. PMID:26037045

  13. Establishment of Immortalized Mouse Bmp2 Knock-Out Dental Papilla Mesenchymal Cells Necessary for Study of Odontoblastic Differentiation and Odontogenesis.

    Science.gov (United States)

    Wu, Lian; Wang, Feng; Donly, Kevin J; Wan, Chunyan; Luo, Daoshu; Harris, Stephen E; MacDougall, Mary; Chen, Shuo

    2015-11-01

    Bmp2 is essential for dentin formation. Bmp2 cKO mice exhibited similar phenotype to dentinogenesis imperfecta, showing dental pulp exposure, hypomineralized dentin, and delayed odontoblast differentiation. As it is relatively difficult to obtain lot of primary Bmp2 cKO dental papilla mesenchymal cells and to maintain a long-term culture of these primary cells, availability of immortalized deleted Bmp2 dental papilla mesenchymal cells is critical for studying the underlying mechanism of Bmp2 signal in odontogenesis. In this study, our goal was to generate an immortalized deleted Bmp2 dental papilla mesenchymal (iBmp2(ko/ko)dp) cell line by introducing Cre recombinase and green fluorescent protein (GFP) into the immortalized mouse floxed Bmp2 dental papilla mesenchymal (iBmp2(fx/fx)dp) cells. iBmp2(ko/ko)dp cells were confirmed by GFP and PCR. The deleted Bmp2 cells exhibited slow cell proliferation rate and cell growth was arrested in G2 phase. Expression of tooth-related marker genes and cell differentiation were decreased in the deleted cells. Importantly, extracellular matrix remodeling was impaired in the iBmp2(ko/ko)dp cells as reflected by the decreased Mmp-9 expression. In addition, with exogenous Bmp2 induction, these cell differentiation and mineralization were rescued as well as extracellular matrix remodeling was enhanced. Therefore, we for the first time described establishment of iBmp(ko/ko) cells that are useful for study of mechanisms in regulating dental papilla mesenchymal cell lineages. © 2015 Wiley Periodicals, Inc.

  14. [Development of human embryonic stem cell model for toxicity evaluation].

    Science.gov (United States)

    Yu, Guang-yan; Cao, Tong; Ouyang, Hong-wei; Peng, Shuang-qing; Deng, Xu-liang; Li, Sheng-lin; Liu, He; Zou, Xiao-hui; Fu, Xin; Peng, Hui; Wang, Xiao-ying; Zhan, Yuan

    2013-02-18

    The current international standard for toxicity screening of biomedical devices and materials recommend the use of immortalized cell lines because of their homogeneous morphologies and infinite proliferation which provide good reproducibility for in vitro cytotoxicity screening. However, most of the widely used immortalized cell lines are derived from animals and may not be representative of normal human cell behavior in vivo, in particular in terms of the cytotoxic and genotoxic response. Therefore, It is vital to develop a model for toxicity evaluation. In our studies, two Chinese human embryonic stem cell (hESC) lines as toxicity model were established. hESC derived tissue/organ cell model for tissue/organ specific toxicity evaluation were developed. The efficiency and accuracy of using hESC model for cytoxicity, embryotoxicity and genotoxicity evaluation were confirmed. The results indicated that hESCs might be good tools for toxicity testing and biosafety evaluation in vitro.

  15. Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types

    DEFF Research Database (Denmark)

    Dabelsteen, Sally; Hercule, Paula; Barron, Patricia

    2009-01-01

    Human embryonic stem (hES) cells can generate cells expressing p63, K14, and involucrin, which have been proposed to be keratinocytes. Although these hES-derived, keratinocyte-like (hESderK) cells form epithelioid colonies when cultured in a fibroblast feeder system optimal for normal tissue......(+)/K14(+) urothelial and tracheobronchial epithelial cells. Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase...

  16. «El inmortal» de Jorge Luis Borges: el yo, infinitos, absolutos y vocabularios finales Jorge Luis Borges's «The Immortal»: the Self, Infinites, Absolutes, and Final Vocabularies

    Directory of Open Access Journals (Sweden)

    Jorge Sagastume

    2011-07-01

    ilosofía de la lengua.Jorge Luis Borges often consulted Mathematics and Imagination by E. Kasner and J. Newman, where they address the set theory (branch of mathematics that studies the relationship between sets proposed by Georg Cantor (18451918, and in which transfinite arithmetic (beyond finite arithmetic is established, and an epistemic system created to represent different levels of the ininite. Thus, Cantor labels the different levels of the ininite by assigning to each the irst letter of the Hebrew alphabet, the Aleph, followed by a number, depending on the level of infinite he is referring to (Aleph-zero, Aleph-one, etc.. Following these ideas, Borges weaves several narratives, discussing the ininite and the absolute. An example of such narratives is the collection of stories compiled under the title The Aleph, which opens with «The Immortal» and closes with the story that gives the collection its title. The objective of this paper is to study «The Immortal» under the Cantorian lens, so as to discuss one particular absolute, the self, and to suggest that it is impossible to establish a inal vocabulary, or a deinite deinition, about this topic. This impossibility, Borges proposes, is in part due to the apparent initude of language, on the one hand, and on the other, the fallible attributes of human memory are also crucial when it comes to deining anything. However, being the ironist Borges is, he is capable of providing through «The Immortal» a re-description of these issues by means of a transinite language that resolves some paradoxes while at the same time reveals others. Due to this way of writing, I propose Borges fosters a continuation of the dialogue among different disciplines. Though I will center my analysis on «The Immortal», to develop these ideas I will also revisit other stories in The Aleph departing from a theoretical approach rooted in the philosophy of language.

  17. Bioethics in popular science: evaluating the media impact of The Immortal Life of Henrietta Lacks on the biobank debate.

    Science.gov (United States)

    Nisbet, Matthew C; Fahy, Declan

    2013-02-28

    The global expansion of biobanks has led to a range of bioethical concerns related to consent, privacy, control, ownership, and disclosure. As an opportunity to engage broader audiences on these concerns, bioethicists have welcomed the commercial success of Rebecca Skloot's 2010 bestselling book The Immortal Life of Henrietta Lacks. To assess the impact of the book on discussion within the media and popular culture more generally, we systematically analyzed the ethics-related themes emphasized in reviews and articles about the book, and in interviews and profiles of Skloot. We conducted a content analysis of a population of relevant English-language articles and transcripts (n = 125) produced by news organizations and publications in the U.S., Canada, Great Britain/Ireland, and Australia/New Zealand. We scored each article for the emphasis and appearance of 9 ethics-related themes. These were informed consent, welfare of the vulnerable, compensation, scientific progress, control/access, accountability/oversight, privacy, public education, and advocacy. The informed consent theme dominated media discussion, with almost 39.2 percent of articles/transcripts featuring the theme as a major focus and 44.8 percent emphasizing the theme as a minor focus. Other prominent themes and frames of reference focused on the welfare of the vulnerable (18.4 percent major emphasis; 36.0 percent minor emphasis), and donor compensation (19.2 percent major; 52.8 percent minor). Ethical themes that comprised a second tier of prominence included those of scientific progress, control/access, and accountability/oversight. The least prominent themes were privacy, public education, and advocacy. The book has been praised as an opportunity to elevate media discussion of bioethics, but such claims should be re-considered. The relatively narrow focus on informed consent in the media discussion generated by Skloot's book may limit the ability of ethicists and advocates to elevate attention to

  18. Immortalized mesenchymal stem cells: an alternative to primary mesenchymal stem cells in neuronal differentiation and neuroregeneration associated studies

    Directory of Open Access Journals (Sweden)

    Gong Min

    2011-11-01

    Full Text Available Abstract Background Mesenchymal stem cells (MSCs can be induced to differentiate into neuronal cells under appropriate cellular conditions and transplanted in brain injury and neurodegenerative diseases animal models for neuroregeneration studies. In contrast to the embryonic stem cells (ESCs, MSCs are easily subject to aging and senescence because of their finite ability of self-renewal. MSCs senescence seriously affected theirs application prospects as a promising tool for cell-based regenerative medicine and tissue engineering. In the present study, we established a reversible immortalized mesenchymal stem cells (IMSCs line by using SSR#69 retrovirus expressing simian virus 40 large T (SV40T antigen as an alternative to primary MSCs. Methods 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. Results 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

  19. Bioethics in popular science: evaluating the media impact of The Immortal Llife of Henrietta Lacks on the biobank debate

    Science.gov (United States)

    2013-01-01

    Background The global expansion of biobanks has led to a range of bioethical concerns related to consent, privacy, control, ownership, and disclosure. As an opportunity to engage broader audiences on these concerns, bioethicists have welcomed the commercial success of Rebecca Skloot’s 2010 bestselling book The Immortal Life of Henrietta Lacks. To assess the impact of the book on discussion within the media and popular culture more generally, we systematically analyzed the ethics-related themes emphasized in reviews and articles about the book, and in interviews and profiles of Skloot. Methods We conducted a content analysis of a population of relevant English-language articles and transcripts (n = 125) produced by news organizations and publications in the U.S., Canada, Great Britain/Ireland, and Australia/New Zealand. We scored each article for the emphasis and appearance of 9 ethics-related themes. These were informed consent, welfare of the vulnerable, compensation, scientific progress, control/access, accountability/oversight, privacy, public education, and advocacy. Results The informed consent theme dominated media discussion, with almost 39.2 percent of articles/transcripts featuring the theme as a major focus and 44.8 percent emphasizing the theme as a minor focus. Other prominent themes and frames of reference focused on the welfare of the vulnerable (18.4 percent major emphasis; 36.0 percent minor emphasis), and donor compensation (19.2 percent major; 52.8 percent minor). Ethical themes that comprised a second tier of prominence included those of scientific progress, control/access, and accountability/oversight. The least prominent themes were privacy, public education, and advocacy. Conclusions The book has been praised as an opportunity to elevate media discussion of bioethics, but such claims should be re-considered. The relatively narrow focus on informed consent in the media discussion generated by Skloot’s book may limit the ability of

  20. Natural selection and immortality.

    Science.gov (United States)

    Danchin, Antoine

    2009-08-01

    Genomes replicate while the host cells reproduce. I explore the reproduction/replication dialogue, based on a deep analysis of bacterial genomes, in relation to ageing. Making young structures from aged ones implies creating information. I revisit Information Theory, showing that the laws of physics permit de novo creation of information, provided an energy-dependent process preserving functional entities makes room for entities accumulating information. I identify explicit functions involved in the process and characterise some of their genes. I suggest that the energy source necessary to establish reproduction while replication is temporarily stopped could be the ubiquitous polyphosphates. Finally, I show that rather than maintain and repair the original individual, organisms tend to metamorphose into young ones, sometimes totally, sometimes progressively. This permits living systems to accumulate information over generations, but has the drawback, in multicellular organisms, to open the door for immortalisation, leading to cancer.

  1. Perennial roots to immortality.

    Science.gov (United States)

    Munné-Bosch, Sergi

    2014-10-01

    Maximum lifespan greatly varies among species, and it is not strictly determined; it can change with species evolution. Clonal growth is a major factor governing maximum lifespan. In the plant kingdom, the maximum lifespans described for clonal and nonclonal plants vary by an order of magnitude, with 43,600 and 5,062 years for Lomatia tasmanica and Pinus longaeva, respectively. Nonclonal perennial plants (those plants exclusively using sexual reproduction) also present a huge diversity in maximum lifespans (from a few to thousands of years) and even more interestingly, contrasting differences in aging patterns. Some plants show a clear physiological deterioration with aging, whereas others do not. Indeed, some plants can even improve their physiological performance as they age (a phenomenon called negative senescence). This diversity in aging patterns responds to species-specific life history traits and mechanisms evolved by each species to adapt to its habitat. Particularities of roots in perennial plants, such as meristem indeterminacy, modular growth, stress resistance, and patterns of senescence, are crucial in establishing perenniality and understanding adaptation of perennial plants to their habitats. Here, the key role of roots for perennial plant longevity will be discussed, taking into account current knowledge and highlighting additional aspects that still require investigation. © 2014 American Society of Plant Biologists. All Rights Reserved.

  2. [Senescence and cellular immortality].

    Science.gov (United States)

    Trentesaux, C; Riou, J-F

    2010-11-01

    Senescence was originally described from the observation of the limited ability of normal cells to grow in culture, and may be generated by telomere erosion, accumulation of DNA damages, oxidative stress and modulation of oncogenes or tumor suppressor genes. Senescence corresponds to a cellular response aiming to control tumor progression by limiting cell proliferation and thus constitutes an anticancer barrier. Senescence is observed in pre-malignant tumor stages and disappears from malignant tumors. Agents used in standard chemotherapy also have the potential to induce senescence, which may partly explain their therapeutic activities. It is possible to restore senescence in tumors using targeted therapies that triggers telomere dysfunction or reactivates suppressor genes functions, which are essential for the onset of senescence.

  3. Toxicity evaluation of chlorinated organic compounds using immortalized rat hepatocytes; Fushika rat kansaibo wo mochiita yuki enso kagobutsu no dokusei hyoka no kokoromi

    Energy Technology Data Exchange (ETDEWEB)

    Sone, H.; Nakajima, M.; Yonemoto, J. [National Institute for Environmental Studies, Tsukuba (Japan)

    1997-11-10

    Chlorinated organic compounds has high priority for toxicity screening among environmental hazardous chemicals. In the present study, we used immortalized rat hepatocytes as a liver model in vitro to evaluate the toxicity of nine chlorinated organic compounds. Toxicity of nine chlorinated organic compounds were evaluated to cellular viability of immortalized rat hapatocytes. The potency of the toxicity based on 50% inhibitory concentration (IC50) value was in the following order: triclocalban>triclosan>3,4-dichloroaniline>2,5-diclorophenol> 2,5-dichloroanisole>p-dichlorobenzene> p-chloroaniline>o-dichlorobenzene=tris (2-chloroethyl) phosphate. The rank order of cytotoxic potency of nine chemicals was compared with toxicity information using animals. The rank order of cytotoxic potency did not relative to the order referenced mean lethal dose (LD50) as an index of acute toxicity of rats or mice. However, the rank order of cytotoxic potency relatively correlated non-observed adverse effect level (NOAEL) under the exposure duration adjusted for chronic toxicity in vivo. These data suggests that the origin of testing cell had better to make match target organ of toxic chemicals for extrapolation from data of bioassay in vitro to in vivo. 16 refs., 2 figs., 3 tabs.

  4. Genetic synthetic lethality screen at the single gene level in cultured human cells

    OpenAIRE

    Simons, Arnold H.; Dafni, Naomi; Dotan, Iris; Oron, Yoram; Canaani, Dan

    2001-01-01

    Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elem...

  5. Lack of the p42 form of C/EBPα leads to spontaneous immortalization and lineage infidelity of committed myeloid progenitors

    DEFF Research Database (Denmark)

    Schuster, Mikkel B; Frank, Anne-Katrine; Bagger, Frederik O

    2013-01-01

    Acute myeloid leukemia (AML) develops via a multistep process involving several genetic and epigenetic events, which ultimately leads to the formation of a heterogeneous population of malignant cells, of which only a small subpopulation termed the leukemia initiating cell (LIC) is able to sustain...... the leukemia. The identity of the LIC is highly diverse and ranges from populations resembling hematopoietic stem cells or multipotent progenitors (MPPs) to more committed myeloid progenitors, and the question still remains whether this is a direct consequence of which cells are targets of the final...... transforming events. In this study, we use premalignant cells from a Cebpa mutant AML model, in which the LIC population resembles granulocyte-macrophage progenitors (GMPs), to show that premalignant GMPs undergo spontaneous immortalization with a high clonal frequency when cultured in vitro, suggesting...

  6. Differential RNA expression between schizophrenic patients and controls of the dystrobrevin binding protein 1 and neuregulin 1 genes in immortalized lymphocytes.

    Science.gov (United States)

    Chagnon, Y C; Roy, M-A; Bureau, A; Mérette, C; Maziade, M

    2008-03-01

    The dystrobrevin binding protein 1 (DTNBP1) and neuregulin 1 (NRG1) genes have been related to schizophrenia (SZ) and bipolar disorder (BP) by several whole-genome linkage and associations studies. Few expression studies in post-mortem brains have also reported a lower or a higher expression of DTNBP1 and NRG1, respectively, in SZ. Since the difficulty to access post-mortem brains, we evaluated RNA expression of DTNBP1 and NRG1 in immortalized lymphocytes of SZ patients and unrelated-family controls. An antipsychotic stimulation was also used to challenge the genetic background of the subjects and enhance differential expression. Immortalized lymphocytes of twelve SZ and twelve controls were grown individually in the presence or not of the antipsychotic olanzapine (Zyprexa; EliLilly). RNA was extracted and pooled in four groups of three SZ and four groups of three controls, and used to probe Agilent 18K microchips. Mean gene expression values were contrasted between SZ and control groups using a T-test. For DTNBP1, RNA expression was lower in SZ than in controls before (-28%; p=0.02) and after (-30%; p=0.01) olanzapine stimulation. Similarly, NRG1 GGF2 isoform showed a lower expression in SZ before (-29%; p=0.04) and after (-33%; p=0.02) olanzapine stimulation. In contrast, NRG1 GGF isoform showed no significant difference between SZ and controls (-7%; p=0.61, +3%; p=0.86, respectively), but was slightly repressed by olanzapine in controls (-8%; p=0.008) but not in SZ (+1%; p=0.91). These results are in agreement with those observed in post-mortem brain when the isoforms involved are considered.

  7. Expression of vitamin D receptor and cathelicidin in human corneal epithelium cells during fusarium solani infection.

    Science.gov (United States)

    Cong, Lin; Xia, Yi-Ping; Zhao, Gui-Qiu; Lin, Jing; Xu, Qiang; Hu, Li-Ting; Qu, Jian-Qiu; Peng, Xu-Dong

    2015-01-01

    To observe the expression of vitamin D receptor (VDR) in human specimen and immortalized human corneal epithelium cells (HCEC) when challenged with fusarium solani. Moreover, we decided to discover the pathway of VDR expression. Also, we would like to detect the expression of cathelicidin antimicrobial peptide (CAMP) in the downstream pathway of VDR. Immunohistochemistry was used to examine the VDR expression in HCEC from healthy and fungal keratitis patients. Real time quantitative polymerase chain reaction (qPCR) was performed to observe the messenger ribonucleic acid (mRNA) change of VDR when immortalized HCEC were challenged with fusarium solani for different hours. CAMP was detected at both mRNA and protein levels. We found out that the VDR expression in fusarium solani keratitis patients' specimen was much more than that in healthy people. The mRNA and protein expression of VDR increased when we stimulated HCEC with fusarium solani antigen (Pfusarium solani antigen stimulation (Pfusarium solani antigen.

  8. Efficient derivation and genetic modifications of human pluripotent stem cells on engineered human feeder cell lines.

    Science.gov (United States)

    Zou, Chunlin; Chou, Bin-Kuan; Dowey, Sarah N; Tsang, Kitman; Huang, Xiaosong; Liu, Cyndi F; Smith, Cory; Yen, Jonathan; Mali, Prashant; Zhang, Yu Alex; Cheng, Linzhao; Ye, Zhaohui

    2012-08-10

    Derivation of pluripotent stem cells (iPSCs) induced from somatic cell types and the subsequent genetic modifications of disease-specific or patient-specific iPSCs are crucial steps in their applications for disease modeling as well as future cell and gene therapies. Conventional procedures of these processes require co-culture with primary mouse embryonic fibroblasts (MEFs) to support self-renewal and clonal growth of human iPSCs as well as embryonic stem cells (ESCs). However, the variability of MEF quality affects the efficiencies of all these steps. Furthermore, animal sourced feeders may hinder the clinical applications of human stem cells. In order to overcome these hurdles, we established immortalized human feeder cell lines by stably expressing human telomerase reverse transcriptase, Wnt3a, and drug resistance genes in adult mesenchymal stem cells. Here, we show that these immortalized human feeders support efficient derivation of virus-free, integration-free human iPSCs and long-term expansion of human iPSCs and ESCs. Moreover, the drug-resistance feature of these feeders also supports nonviral gene transfer and expression at a high efficiency, mediated by piggyBac DNA transposition. Importantly, these human feeders exhibit superior ability over MEFs in supporting homologous recombination-mediated gene targeting in human iPSCs, allowing us to efficiently target a transgene into the AAVS1 safe harbor locus in recently derived integration-free iPSCs. Our results have great implications in disease modeling and translational applications of human iPSCs, as these engineered human cell lines provide a more efficient tool for genetic modifications and a safer alternative for supporting self-renewal of human iPSCs and ESCs.

  9. Integrin α3β1 signaling through MEK/ERK determines alternative polyadenylation of the MMP-9 mRNA transcript in immortalized mouse keratinocytes.

    Science.gov (United States)

    Missan, Dara S; Mitchell, Kara; Subbaram, Sita; DiPersio, C Michael

    2015-01-01

    Integrin α3β1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for α3β1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by α3β1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3β1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of α3β1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in α3β1-expressing keratinocytes compared with α3β1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3β1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3β1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in α3β1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1

  10. 永生化转基因成纤维细胞治疗帕金森病%Treatment of Parkinson's Disease by Genetically Modified Immortalized Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    许德华; 徐舲飞; 曹蕾; 刘新垣; 郑仲承; 陈先文; 陈生弟

    2001-01-01

    将SV40大T抗原的基因(LTAg)转染大鼠原代成纤维细胞并筛选到永生化成纤维细胞(RFLT), 将此细胞皮下植入裸鼠和大鼠脑内均无致瘤性, 植入脑内时LTAg基因即停止表达, 3个月后取出细胞培养时LTAg基因又会重新表达。 将RFLT细胞分别转入酪氨酸羟化酶(TH)、GTP环水解酶-1(GCH)的基因, 筛选出稳定表达株。 混合培养的这两种细胞经HPLC-ECD法测出多巴胺(DA), 将它们移植入帕金森病(PD)大鼠模型可明显改善动物的旋转行为(近4个月), 在脑切片中观察到TH和增强型绿色荧光蛋白(EGFP)基因的表达产物。 本细胞株的建立为人类PD的基因治疗提供了一种获得新的适用的效应细胞的方法。%Primary rat fibroblast cells were immortalized by genetic modification of SV40 large T antigen (LTAg) gene and called RFLT. This cell line was non-tumorigenic after grafting into nud-mouse and rat. The LTAg gene stopped expression when the cells were transplanted in rat striatum, but it resumed the expression ability after the transplanted cells were recovered from striatum and cultured in vitro. TH gene and GCH gene were transfected into RFLT, respectively, and two types of transfected cells, RFLT-TH and RFLT-GCH, were obtained. In mixed culture with these two cell lines, DA was detected with HPLC-ECD. Implanting mixture of those cells into the striatum of PD rats significantly decreased their rotational asymmetry for up to 12 weeks. The expression of TH gene was proved by TH immunohistochemical staining in the sections of rat brain. The establishment of the genetically modified immortalized cells may play role in the gene therapy of PD.

  11. [Towards an industrial control of the cloning of lymphocytes B human for the manufacturing of monoclonal antibodies stemming from the human repertoire].

    Science.gov (United States)

    Guillot-Chene, P; Lebecque, S; Rigal, D

    2009-05-01

    Monoclonal antibodies (mAbs) are efficient drugs for treating infectious, inflammatory and cancer diseases. Antibodies secreted by human lymphocytes that have been isolated from either peripheral blood or tissues present the definite interest of being part of the physiological or disease-related response to antigens present in the human body. However, attempts to generate hybridomas with human B cells have been largely unsuccessful, and cloning of human B cells has been achieved only via their inefficient immortalization with Epstein Barr Virus (EBV). However, recent progress in our understanding of the molecular mechanisms of polyclonal B cell activation has dramatically increased the capacity to clone human B cells. In particular, activation of human naïve and memory B cells through CD40 or memory B cells only through TLR9 was shown to greatly facilitate their immortalization by EBV. Industrial development based on these observations will soon provide large collections of high affinity human mAbs of every isotype directly selected by the human immune system directed to recognize epitopes relevant for individual patients. Moreover, after CD40 activation, these mAbs will cover the full human repertoire, including the natural auto-immune repertoire. Full characterization of the biological activity of these mAbs will in turn bring useful information for selecting vaccine epitopes. This breakthrough in human B cell cloning opens the way into new areas for therapeutic use of mAbs.

  12. Pseudo-immortalization of postnatal cochlear progenitor cells yields a scalable cell line capable of transcriptionally regulating mature hair cell genes.

    Science.gov (United States)

    Walters, Brandon J; Diao, Shiyong; Zheng, Fei; Walters, Bradley J; Layman, Wanda S; Zuo, Jian

    2015-12-07

    The mammalian cochlea is a highly specialized organ within the inner ear. Sensory hair cells (HC) in the cochlea detect and transduce sound waves into electrical impulses that are sent to the brain. Studies of the molecular pathways regulating HC formation are hindered by the very sparse nature of HCs, where only ~3300 are found within an entire mouse cochlea. Current cell lines mimic certain aspects of HCs but lack terminal HC marker expression. Here we successfully "pseudo-immortalized" cochlear progenitor cells using the "conditional reprogramming" technique. These cells, termed "Conditionally Reprogrammed Otic Stem Cells" (CR-OSC), are able to bypass the senescence inherent to cochlear progenitor cells without genetic alterations, allowing for the generation of over 15 million cells from a single cochlea. These cells can be differentiated and up-regulate both early and terminal differentiation genes associated with HCs, including the terminal HC differentiation marker prestin. CR-OSCs also respond to known HC cues, including upregulation of HC genes in response to Atoh1 overexpression, and upregulation of prestin expression after thyroid hormone application. Overall, we describe the creation of a HC line capable of regulated expression of HC genes that can easily be recreated in any laboratory from any mouse of interest.

  13. Mapping of QTL for tiller number at different stages of growth in wheat using double haploid and immortalized F2 populations

    Indian Academy of Sciences (India)

    Zhuokun Li; Tao Peng; Quangang Xie; Shuxiao Han; Jichun Tian

    2010-12-01

    Effective tiller number is one of the most important traits for wheat (Triticum aestivum L.) yield, but the inheritance of tillering is poorly understood. A set of 168 doubled haploid (DH) lines derivatives of a cross between two winter wheat cultivars (Huapei 3 and Yumai 57), and an immortalized F2 (IF2) population generated by randomly permutated intermating of these DHs were investigated, and QTLs of tillering related to the maximum tillering of pre-winter (MTW), maximum tillering in spring (MTS), and effective tillering in harvest (ETH) were mapped. Phenotypic data were collected for the two populations from two different environments. Using inclusive composite interval mapping (ICIM), a total of 9 and 18 significant QTL were detected across environments for tillering in the DH and IF2 populations, respectively. Four QTLs were common between two populations. A major QTL located on the 5D chromosome with the allele originating from Yumai 57 was detected and increased 1.92 and 3.55 tillers in MTW and MTS, respectively. QTLs (QMts6D, QEth6D) having a neighbouring marker interval at Xswes679.1 and Xcfa2129 on chromosome 6D was detected in MTS and ETH. These results provide a better understanding of the genetic factors for selectively expressing the control of tiller number in different growth stages and facilitate marker-assisted selection strategy in breeding.

  14. An In Vitro System Comprising Immortalized Hypothalamic Neuronal Cells (GT1–7 Cells for Evaluation of the Neuroendocrine Effects of Essential Oils

    Directory of Open Access Journals (Sweden)

    Dai Mizuno

    2015-01-01

    Full Text Available Aromatherapy and plant-based essential oils are widely used as complementary and alternative therapies for symptoms including anxiety. Furthermore, it was reportedly effective for the care of several diseases such as Alzheimer’s disease and depressive illness. To investigate the pharmacological effects of essential oils, we developed an in vitro assay system using immortalized hypothalamic neuronal cells (GT1–7 cells. In this study, we evaluated the effects of essential oils on neuronal death induced by hydrogen peroxide (H2O2, aluminum, zinc, or the antagonist of estrogen receptor (tamoxifen. Among tests of various essential oils, we found that H2O2-induced neuronal death was attenuated by the essential oils of damask rose, eucalyptus, fennel, geranium, ginger, kabosu, mandarin, myrrh, and neroli. Damask rose oil had protective effects against aluminum-induced neurotoxicity, while geranium and rosemary oil showed protective activity against zinc-induced neurotoxicity. In contrast, geranium oil and ginger oil enhanced the neurotoxicity of tamoxifen. Our in vitro assay system could be useful for the neuropharmacological and endocrine pharmacological studies of essential oils.

  15. Type I interferon reaction to viral infection in interferon-competent, immortalized cell lines from the African fruit bat Eidolon helvum.

    Directory of Open Access Journals (Sweden)

    Susanne E Biesold

    Full Text Available Bats harbor several highly pathogenic zoonotic viruses including Rabies, Marburg, and henipaviruses, without overt clinical symptoms in the animals. It has been suspected that bats might have evolved particularly effective mechanisms to suppress viral replication. Here, we investigated interferon (IFN response, -induction, -secretion and -signaling in epithelial-like cells of the relevant and abundant African fruit bat species, Eidolon helvum (E. helvum. Immortalized cell lines were generated; their potential to induce and react on IFN was confirmed, and biological assays were adapted to application in bat cell cultures, enabling comparison of landmark IFN properties with that of common mammalian cell lines. E. helvum cells were fully capable of reacting to viral and artificial IFN stimuli. E. helvum cells showed highest IFN mRNA induction, highly productive IFN protein secretion, and evidence of efficient IFN stimulated gene induction. In an Alphavirus infection model, O'nyong-nyong virus exhibited strong IFN induction but evaded the IFN response by translational rather than transcriptional shutoff, similar to other Alphavirus infections. These novel IFN-competent cell lines will allow comparative research on zoonotic, bat-borne viruses in order to model mechanisms of viral maintenance and emergence in bat reservoirs.

  16. Differential localization of cytoplasmic myosin II isoforms A and B in avian interphase and dividing embryonic and immortalized cardiomyocytes and other cell types in vitro

    Science.gov (United States)

    Conrad, A. H.; Jaffredo, T.; Conrad, G. W.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Two principal isoforms of cytoplasmic myosin II, A and B (CMIIA and CMIIB), are present in different proportions in different tissues. Isoform-specific monoclonal and polyclonal antibodies to avian CMIIA and CMIIB reveal the cellular distributions of these isoforms in interphase and dividing embryonic avian cardiac, intestinal epithelial, spleen, and dorsal root ganglia cells in primary cell culture. Embryonic cardiomyocytes react with antibodies to CMIIB but not to CMIIA, localize CMIIB in stress-fiber-like-structures during interphase, and markedly concentrate CMIIB in networks in the cleavage furrow during cytokinesis. In contrast, cardiac fibroblasts localize both CMIIA and CMIIB in stress fibers and networks during interphase, and demonstrate slight and independently regulated concentration of CMIIA and CMIIB in networks in their cleavage furrows. V-myc-immortalized cardiomyocytes, an established cell line, have regained the ability to express CMIIA, as well as CMIIB, and localize both CMIIA and CMIIB in stress fibers and networks in interphase cells and in cleavage furrows in dividing cells. Conversely, some intestinal epithelial, spleen, and dorsal root ganglia interphase cells express only CMIIA, organized primarily in networks. Of these, intestinal epithelial cells express both CMIIA and CMIIB when they divide, whereas some dividing cells from both spleen and dorsal root ganglia express only CMIIA and concentrate it in their cleavage furrows. These results suggest that within a given tissue, different cell types express different isoforms of CMII, and that cells expressing either CMIIA or CMIIB alone, or simultaneously, can form a cleavage furrow and divide.

  17. Derivation and characterization of hepatic progenitor cells from human embryonic stem cells.

    Science.gov (United States)

    Zhao, Dongxin; Chen, Song; Cai, Jun; Guo, Yushan; Song, Zhihua; Che, Jie; Liu, Chun; Wu, Chen; Ding, Mingxiao; Deng, Hongkui

    2009-07-31

    The derivation of hepatic progenitor cells from human embryonic stem (hES) cells is of value both in the study of early human liver organogenesis and in the creation of an unlimited source of donor cells for hepatocyte transplantation therapy. Here, we report for the first time the generation of hepatic progenitor cells derived from hES cells. Hepatic endoderm cells were generated by activating FGF and BMP pathways and were then purified by fluorescence activated cell sorting using a newly identified surface marker, N-cadherin. After co-culture with STO feeder cells, these purified hepatic endoderm cells yielded hepatic progenitor colonies, which possessed the proliferation potential to be cultured for an extended period of more than 100 days. With extensive expansion, they co-expressed the hepatic marker AFP and the biliary lineage marker KRT7 and maintained bipotential differentiation capacity. They were able to differentiate into hepatocyte-like cells, which expressed ALB and AAT, and into cholangiocyte-like cells, which formed duct-like cyst structures, expressed KRT19 and KRT7, and acquired epithelial polarity. In conclusion, this is the first report of the generation of proliferative and bipotential hepatic progenitor cells from hES cells. These hES cell-derived hepatic progenitor cells could be effectively used as an in vitro model for studying the mechanisms of hepatic stem/progenitor cell origin, self-renewal and differentiation.

  18. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Laura N Bonifacio

    Full Text Available Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

  19. Development of a high-throughput screening assay for chemical effects on proliferation and viability of immortalized human neural progenitor cells

    Science.gov (United States)

    There is considerable public concern that the majority of commercial chemicals have not been evaluated for their potential to cause developmental neurotoxicity. Although several chemicals are assessed annually under the current developmental neurotoxicity guidelines, time, resour...

  20. Multistep process of neoplastic transformation of normal human fibroblasts by 60Co gamma rays and Harvey sarcoma viruses

    Energy Technology Data Exchange (ETDEWEB)

    Namba, M.; Nishitani, K.; Fukushima, F.; Kimoto, T.; Nose, K.

    1986-03-15

    As reported previously (Namba et al., 1985), normal human fibroblasts were transformed by 60Co gamma-ray irradiation into immortal cells with abnormal karyotypes. These transformed cells (KMST-6), however, showed a low cloning efficiency in soft agar and no transplantability. However, upon treatment with Harvey murine sarcoma virus (Ha-MSV), the cells acquired elevated clonability in soft agar and transplantability in nude mice. Ha-MSV alone, however, did not convert normal human fibroblasts into either immortal or tumorigenic cells. The Ha-MSV-transformed KMST-6 cells showed an enhanced expression of the ras oncogene, but normal and 60Co gamma-ray-transformed cells did not. Our current data suggest that gamma rays worked against normal human cells as an initiator, giving rise to chromosome aberrations and immortality, and that Ha-MSV, probably through its ras oncogene, played a role in the progression of the malignant cell population to a more malignant one showing enhanced colony formation in soft agar and tumorigenicity in nude mice.

  1. Human mammary progenitor cell fate decisions are productsof interactions with combinatorial microenvironments

    DEFF Research Database (Denmark)

    LaBarge, Mark A.; Nelson, Celeste M.; Villadsen, René

    2009-01-01

    as constellations of signaling molecules; and these were used in conjunction with physiologically relevant 3 dimensional human breast cultures. Both immortalized and primary human breast progenitors were analyzed. We report on the functional ability of those proteins of the mammary gland that maintain quiescence...... combinations of cell-extrinsic mammary gland proteins and ECM molecules that imposed specific cell fates on bipotent human mammary progenitor cells.Micropatterned cell culture surfaces were fabricated to distinguish between the instructive effects of cell-cell versus cell-ECM interactions, as well...

  2. Isolation, growth, and characterization of human renal epithelial cells using traditional and 3D methods.

    Science.gov (United States)

    Gildea, John J; McGrath, Helen E; Van Sciver, Robert E; Wang, Dora Bigler; Felder, Robin A

    2013-01-01

    The kidney is a highly heterogeneous organ that is responsible for fluid and electrolyte balance. Much interest is focused on determining the function of specific renal epithelial cells in humans, which can only be accomplished through the isolation and growth of nephron segment-specific epithelial cells. However, human renal epithelial cells are notoriously difficult to maintain in culture. This chapter describes the isolation, growth, immortalization, and characterization of the human renal proximal tubule cell. In addition, we describe new paradigms in 3D cell culture which allow the cells to maintain more in vivo-like morphology and function.

  3. Generation of immortal cell lines from the adult pituitary: role of cAMP on differentiation of SOX2-expressing progenitor cells to mature gonadotropes.

    Directory of Open Access Journals (Sweden)

    Ginah L Kim

    Full Text Available The pituitary is a complex endocrine tissue composed of a number of unique cell types distinguished by the expression and secretion of specific hormones, which in turn control critical components of overall physiology. The basic function of these cells is understood; however, the molecular events involved in their hormonal regulation are not yet fully defined. While previously established cell lines have provided much insight into these regulatory mechanisms, the availability of representative cell lines from each cell lineage is limited, and currently none are derived from adult pituitary. We have therefore used retroviral transfer of SV40 T-antigen to mass immortalize primary pituitary cell culture from an adult mouse. We have generated 19 mixed cell cultures that contain cells from pituitary cell lineages, as determined by RT-PCR analysis and immunocytochemistry for specific hormones. Some lines expressed markers associated with multipotent adult progenitor cells or transit-amplifying cells, including SOX2, nestin, S100, and SOX9. The progenitor lines were exposed to an adenylate cyclase activator, forskolin, over 7 days and were induced to differentiate to a more mature gonadotrope cell, expressing significant levels of α-subunit, LHβ, and FSHβ mRNAs. Additionally, clonal populations of differentiated gonadotropes were exposed to 30 nM gonadotropin-releasing hormone and responded appropriately with a significant increase in α-subunit and LHβ transcription. Further, exposure of the lines to a pulse paradigm of GnRH, in combination with 17β-estradiol and dexamethasone, significantly increased GnRH receptor mRNA levels. This array of adult-derived pituitary cell models will be valuable for both studies of progenitor cell characteristics and modulation, and the molecular analysis of individual pituitary cell lineages.

  4. Orthotopic transplantation of immortalized mesencephalic progenitors (CSM14.1 cells) into the substantia nigra of hemiparkinsonian rats induces neuronal differentiation and motoric improvement.

    Science.gov (United States)

    Haas, Stefan Jean-Pierre; Petrov, Stanislav; Kronenberg, Golo; Schmitt, Oliver; Wree, Andreas

    2008-01-01

    Neural progenitor cell grafting is a promising therapeutic option in the treatment of Parkinson's disease. In previous experiments we grafted temperature-sensitive immortalized CSM14.1 cells, derived from the ventral mesencephalon of E14-rats, bilaterally in the caudate putamen of adult hemiparkinsonian rats. In these studies we were not able to demonstrate either a therapeutic improvement or neuronal differentiation of transplanted cells. Here we examined whether CSM14.1 cells grafted bilaterally orthotopically in the substantia nigra of hemiparkinsonian rats have the potential to differentiate into dopaminergic neurons. Adult male rats received 6-hydroxydopamine into the right medial forebrain bundle, and successful lesions were evaluated with apomorphine-induced rotations 12 days after surgery. Two weeks after a successful lesion the animals received bilateral intranigral grafts consisting of either about 50 000 PKH26-labelled undifferentiated CSM14.1 cells (n = 16) or a sham-graft (n = 9). Rotations were evaluated 3, 6, 9 and 12 weeks post-grafting. Animals were finally perfused with 4% paraformaldehyde. Cryoprotected brain slices were prepared for immunohistochemistry using the freeze-thaw technique to preserve PKH26-labelling. Slices were immunostained against neuronal epitopes (NeuN, tyrosine hydroxylase) or glial fibrillary acidic protein. The CSM14.1-cell grafts significantly reduced the apomorphine-induced rotations 12 weeks post-grafting compared to the sham-grafts (P < 0.05). There was an extensive mediolateral migration (400-700 microm) of the PKH26-labelled cells within the host substantia nigra. Colocalization with NeuN or glial fibrillary acidic protein in transplanted cells was confirmed with confocal microscopy. No tyrosine hydroxylase-immunoreactive grafted cells were detectable. The therapeutic effect of the CSM14.1 cells could be explained either by their glial cell-derived neurotrophic factor-expression or their neural differentiation with

  5. Progesterone receptor membrane component-1 (PGRMC1) and PGRMC-2 interact to suppress entry into the cell cycle in spontaneously immortalized rat granulosa cells.

    Science.gov (United States)

    Peluso, John J; Griffin, Daniel; Liu, Xiufang; Horne, Meghan

    2014-11-01

    Progesterone receptor membrane component 1 (PGRMC1) and PGRMC2 are expressed in rat granulosa cells and spontaneously immortalized granulosa cells (SIGCs) but their biological roles are not well defined. The present studies demonstrate that depleting either Pgrmc1 or Pgrmc2 in SIGCs increases entry into the cell cycle but does not increase cell proliferation. Rather, PGRMC1 and/or PGRMC2-deplete cells accumulate in metaphase and undergo apoptosis. Because both PGRMC1 and PGRMC2 localize to the mitotic spindle, their absence likely accounts for cells arresting in metaphase. Moreover, pull-down assays, colocalization studies and in situ proximity ligation assays (PLA) indicate that PGRMC1 binds PGRMC2. Disrupting the PGRMC1:PGRMC2 complex through the use of siRNA or the cytoplasmic delivery of a PGRMC2 antibody increases entry into the cell cycle. Conversely, overexpressing either PGRMC1-GFP or GFP-PGRMC2 fusion protein inhibits entry into the cell cycle. Subsequent studies reveal that depleting PGRMC1 and/or PGRMC2 reduces the percentage of cells in G0 and increases the percentage of cells in G1. These observations indicate that in addition to their role at metaphase, PGRMC1 and PGRMC2 are involved in regulating entry into the G1 stage of the cell cycle. Interestingly, both PGRMC1 and PGRMC2 bind GTPase-activating protein-binding protein 2 (G3BP2) as demonstrated by pull-down assays, colocalization assays, and PLAs. G3bp2 siRNA treatment also promotes entry into the G1 stage. This implies that dynamic changes in the interaction among PGRMC1, PGRMC2, and G3BP2 play an important protein regulating the rate at which SIGCs enter into the cell cycle.

  6. Genetic dissection of the maize kernel development process via conditional QTL mapping for three developing kernel-related traits in an immortalized F2 population.

    Science.gov (United States)

    Zhang, Zhanhui; Wu, Xiangyuan; Shi, Chaonan; Wang, Rongna; Li, Shengfei; Wang, Zhaohui; Liu, Zonghua; Xue, Yadong; Tang, Guiliang; Tang, Jihua

    2016-02-01

    Kernel development is an important dynamic trait that determines the final grain yield in maize. To dissect the genetic basis of maize kernel development process, a conditional quantitative trait locus (QTL) analysis was conducted using an immortalized F2 (IF2) population comprising 243 single crosses at two locations over 2 years. Volume (KV) and density (KD) of dried developing kernels, together with kernel weight (KW) at different developmental stages, were used to describe dynamic changes during kernel development. Phenotypic analysis revealed that final KW and KD were determined at DAP22 and KV at DAP29. Unconditional QTL mapping for KW, KV and KD uncovered 97 QTLs at different kernel development stages, of which qKW6b, qKW7a, qKW7b, qKW10b, qKW10c, qKV10a, qKV10b and qKV7 were identified under multiple kernel developmental stages and environments. Among the 26 QTLs detected by conditional QTL mapping, conqKW7a, conqKV7a, conqKV10a, conqKD2, conqKD7 and conqKD8a were conserved between the two mapping methodologies. Furthermore, most of these QTLs were consistent with QTLs and genes for kernel development/grain filling reported in previous studies. These QTLs probably contain major genes associated with the kernel development process, and can be used to improve grain yield and quality through marker-assisted selection.

  7. Nuclear DNA methylation and chromatin condensation phenotypes are distinct between normally proliferating/aging, rapidly growing/immortal, and senescent cells.

    Science.gov (United States)

    Oh, Jin Ho; Gertych, Arkadiusz; Tajbakhsh, Jian

    2013-03-01

    This study reports on probing the utility of in situ chromatin texture features such as nuclear DNA methylation and chromatin condensation patterns - visualized by fluorescent staining and evaluated by dedicated three-dimensional (3D) quantitative and high-throughput cell-by-cell image analysis - in assessing the proliferative capacity, i.e. growth behavior of cells: to provide a more dynamic picture of a cell population with potential implications in basic science, cancer diagnostics/prognostics and therapeutic drug development. Two types of primary cells and four different cancer cell lines were propagated and subjected to cell-counting, flow cytometry, confocal imaging, and 3D image analysis at various points in culture. Additionally a subset of primary and cancer cells was accelerated into senescence by oxidative stress. DNA methylation and chromatin condensation levels decreased with declining doubling times when primary cells aged in culture with the lowest levels reached at the stage of proliferative senescence. In comparison, immortal cancer cells with constant but higher doubling times mostly displayed lower and constant levels of the two in situ-derived features. However, stress-induced senescent primary and cancer cells showed similar levels of these features compared with primary cells that had reached natural growth arrest. With regards to global DNA methylation and chromatin condensation levels, aggressively growing cancer cells seem to take an intermediate level between normally proliferating and senescent cells. Thus, normal cells apparently reach cancer-cell equivalent stages of the two parameters at some point in aging, which might challenge phenotypic distinction between these two types of cells. Companion high-resolution molecular profiling could provide information on possible underlying differences that would explain benign versus malign cell growth behaviors.

  8. Expression of progesterone receptor membrane component-2 within the immature rat ovary and its role in regulating mitosis and apoptosis of spontaneously immortalized granulosa cells.

    Science.gov (United States)

    Griffin, Daniel; Liu, Xiufang; Pru, Cindy; Pru, James K; Peluso, John J

    2014-08-01

    Progesterone receptor membrane component 2 (Pgrmc2) mRNA was detected in the immature rat ovary. By 48 h after eCG, Pgrmc2 mRNA levels decreased by 40% and were maintained at 48 h post-hCG. Immunohistochemical studies detected PGRMC2 in oocytes and ovarian surface epithelial, interstitial, thecal, granulosa, and luteal cells. PGRMC2 was also present in spontaneously immortalized granulosa cells, localizing to the cytoplasm of interphase cells and apparently to the mitotic spindle of cells in metaphase. Interestingly, PGRMC2 levels appeared to decrease during the G1 stage of the cell cycle. Moreover, overexpression of PGRMC2 suppressed entry into the cell cycle, possibly by binding the p58 form of cyclin dependent kinase 11b. Conversely, Pgrmc2 small interfering RNA (siRNA) treatment increased the percentage of cells in G1 and M stage but did not increase the number of cells, which was likely due to an increase in apoptosis. Depleting PGRMC2 did not inhibit cellular (3)H-progesterone binding, but attenuated the ability of progesterone to suppress mitosis and apoptosis. Taken together these studies suggest that PGRMC2 affects granulosa cell mitosis by acting at two specific stages of the cell cycle. First, PGRMC2 regulates the progression from the G0 into the G1 stage of the cell cycle. Second, PGRMC2 appears to localize to the mitotic spindle, where it likely promotes the final stages of mitosis. Finally, siRNA knockdown studies indicate that PGRMC2 is required for progesterone to slow the rate of granulosa cell mitosis and apoptosis. These findings support a role for PGRMC2 in ovarian follicle development.

  9. Dioxin exerts anti-estrogenic actions in a novel dioxin-responsive telomerase-immortalized epithelial cell line of the porcine oviduct (TERT-OPEC).

    Science.gov (United States)

    Hombach-Klonisch, Sabine; Pocar, Paola; Kauffold, Johannes; Klonisch, Thomas

    2006-04-01

    Oviduct epithelial cells are important for the nourishment and survival of ovulated oocytes and early embryos, and they respond to the steroid hormones estrogen and progesterone. Endocrine-disrupting polyhalogenated aromatic hydrocarbons (PHAH) are environmental toxins that act in part through the ligand-activated transcription factor arylhydrocarbon receptor (AhR; dioxin receptor), and exposure to PHAH has been shown to decrease fertility. To investigate effects of PHAHs on the oviduct epithelium as a potential target tissue of dioxin-type endocrine disruptors, we have established a novel telomerase-immortalized oviduct porcine epithelial cell line (TERT-OPEC). TERT-OPEC exhibited active telomerase and the immunoreactive epithelial marker cytokeratin but lacked the stromal marker vimentin. TERT-OPEC contained functional estrogen receptor (ER)-alpha and AhR, as determined by the detection of ER-alpha- and AhR-specific target molecules. Treatment of TERT-OPEC with the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a significant increase in the production of the cytochrome P-450 microsomal enzyme CYP1A1. Activated AhR caused a downregulation of ER nuclear protein fraction and significantly decreased ER-signaling in TERT-OPEC as determined by ERE-luciferase transient transfection assays. In summary, the TCDD-induced and AhR-mediated anti-estrogenic responses by TERT-OPEC suggest that PHAH affect the predominantly estrogen-dependent differentiation of the oviduct epithelium within the fallopian tube. This action then alters the local endocrine milieu, potentially resulting in a largely unexplored cause of impaired embryonic development and female infertility.

  10. Characterization of Murine Thymic Stromal-Cell Lines Immortalized by Temperature-Sensitive Simian Virus 40 Large T or Adenovirus 5 E1a

    Science.gov (United States)

    Larsson, Lena; Timms, Emma; Blight, Kenneth; Restall, Deborah E.; Jat, Parmjit S.; Fisher, Amanda G.

    1991-01-01

    The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33°C and arrest growth when transferred to the nonpermissive temperature of 39°C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined. PMID:1668372

  11. Palmitate alters the rhythmic expression of molecular clock genes and orexigenic neuropeptide Y mRNA levels within immortalized, hypothalamic neurons.

    Science.gov (United States)

    Fick, Laura J; Fick, Gordon H; Belsham, Denise D

    2011-09-30

    The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.

  12. Culture models of human mammary epithelial cell transformation

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2000-11-10

    Human pre-malignant breast diseases, particularly ductal carcinoma in situ (DCIS)3 already display several of the aberrant phenotypes found in primary breast cancers, including chromosomal abnormalities, telomerase activity, inactivation of the p53 gene and overexpression of some oncogenes. Efforts to model early breast carcinogenesis in human cell cultures have largely involved studies in vitro transformation of normal finite lifespan human mammary epithelial cells (HMEC) to immortality and malignancy. We present a model of HMEC immortal transformation consistent with the know in vivo data. This model includes a recently described, presumably epigenetic process, termed conversion, which occurs in cells that have overcome stringent replicative senescence and are thus able to maintain proliferation with critically short telomeres. The conversion process involves reactivation of telomerase activity, and acquisition of good uniform growth in the absence and presence of TFGB. We propose th at overcoming the proliferative constraints set by senescence, and undergoing conversion, represent key rate-limiting steps in human breast carcinogenesis, and occur during early stage breast cancer progression.

  13. Generation and In Vitro Expansion of Hepatic Progenitor Cells from Human iPS Cells.

    Science.gov (United States)

    Yanagida, Ayaka; Nakauchi, Hiromitsu; Kamiya, Akihide

    2016-01-01

    Stem cells have the unique properties of self-renewal and multipotency (producing progeny belonging to two or more lineages). Induced pluripotent stem (iPS) cells can be generated from somatic cells by simultaneous expression of pluripotent factors (Oct3/4, Klf4, Sox2, and c-Myc). They share the same properties as embryonic stem (ES) cells and can differentiate into several tissue cells, i.e., neurons, hematopoietic cells, and liver cells. Therefore, iPS cells are suitable candidate cells for regenerative medicine and analyses of disease mechanisms.The liver is the major organ that regulates a multitude of metabolic functions. Hepatocytes are the major cell type populating the liver parenchyma and express several metabolic enzymes that are necessary for liver functions. Although hepatocytes are essential for maintaining homeostasis, it is difficult to alter artificial and transplanted cells because of their multifunctionality, donor shortage, and immunorejection risk. During liver development, hepatic progenitor cells in the fetal liver differentiate into both mature hepatocytes and cholangiocytes. As hepatic progenitor cells have bipotency and high proliferation ability, they could present a potential source for generating transplantable cells or as a liver study model. Here we describe the induction and purification of hepatic progenitor cells derived from human iPS cells. These cells can proliferate for a long term under suitable culture conditions.

  14. Generation of human antigen-specific monoclonal IgM antibodies using vaccinated "human immune system" mice.

    Directory of Open Access Journals (Sweden)

    Pablo D Becker

    Full Text Available BACKGROUND: Passive transfer of antibodies not only provides immediate short-term protection against disease, but also can be exploited as a therapeutic tool. However, the 'humanization' of murine monoclonal antibodies (mAbs is a time-consuming and expensive process that has the inherent drawback of potentially altering antigenic specificity and/or affinity. The immortalization of human B cells represents an alternative for obtaining human mAbs, but relies on the availability of biological samples from vaccinated individuals or convalescent patients. In this work we describe a novel approach to generate fully human mAbs by combining a humanized mouse model with a new B cell immortalization technique. METHODOLOGY/PRINCIPAL FINDINGS: After transplantation with CD34+CD38⁻ human hematopoietic progenitor cells, BALB/c Rag2⁻/⁻IL-2Rγc⁻/⁻ mice acquire a human immune system and harbor B cells with a diverse IgM repertoire. "Human Immune System" mice were then immunized with two commercial vaccine antigens, tetanus toxoid and hepatitis B surface antigen. Sorted human CD19+CD27+ B cells were retrovirally transduced with the human B cell lymphoma (BCL-6 and BCL-XL genes, and subsequently cultured in the presence of CD40-ligand and IL-21. This procedure allows generating stable B cell receptor-positive B cells that secrete immunoglobulins. We recovered stable B cell clones that produced IgM specific for tetanus toxoid and the hepatitis B surface antigen, respectively. CONCLUSION/SIGNIFICANCE: This work provides the proof-of-concept for the usefulness of this novel method based on the immunization of humanized mice for the rapid generation of human mAbs against a wide range of antigens.

  15. Productive infection of human peripheral blood mononuclear cells by feline immunodeficiency virus: implications for vector development.

    Science.gov (United States)

    Johnston, J; Power, C

    1999-03-01

    Feline immunodeficiency virus (FIV) is a lentivirus causing immune suppression and neurological disease in cats. Like primate lentiviruses, FIV utilizes the chemokine receptor CXCR4 for infection. In addition, FIV gene expression has been demonstrated in immortalized human cell lines. To investigate the extent and mechanism by which FIV infected primary and immortalized human cell lines, we compared the infectivity of two FIV strains, V1CSF and Petaluma, after cell-free infection. FIV genome was detected in infected human peripheral blood mononuclear cells (PBMC) and macrophages at 21 and 14 days postinfection, respectively. Flow cytometry analysis of FIV-infected human PBMC indicated that antibodies to FIV p24 recognized 12% of the cells. Antibodies binding the CCR3 chemokine receptor maximally inhibited infection of human PBMC by both FIV strains compared to antibodies to CXCR4 or CCR5. Reverse transcriptase levels increased in FIV-infected human PBMC, with detection of viral titers of 10(1.3) to 10(2.1) 50% tissue culture infective doses/10(6) cells depending on the FIV strain examined. Cell death in human PBMC infected with either FIV strain was significantly elevated relative to uninfected control cultures. These findings indicate that FIV can productively infect primary human cell lines and that viral strain specificity should be considered in the development of an FIV vector for gene therapy.

  16. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hanwen [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States); Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, University of South Carolina, Columbia, SC 29208 (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States)

    2015-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.

  17. Elevated connexin 43 expression in arsenite-and cadmium-transformed human bladder cancer cells, tumor transplants and selected high grade human bladder cancers.

    Science.gov (United States)

    Zhang, Ruowen; Wang, Liping; Garrett, Scott H; Sens, Donald A; Dunlevy, Jane R; Zhou, Xu Dong; Somji, Seema

    2016-10-01

    Connexin 43 has been shown to play a role in cell migration and invasion; however, its role in bladder cancer is not well defined. Previous studies from our laboratory have shown that the environmental pollutants arsenite and cadmium can cause malignant transformation of the immortalized urothelial cell line UROtsa. These transformed cells can form tumors in immune-compromised mice. The goal of the present study was to determine if connexin 43 is expressed in the normal human bladder, the arsenite and cadmiun-transformed UROtsa cells as well as human urothelial cancer. The results obtained showed that connexin 43 is not expressed in the epithelial cells of the human bladder but is expressed in immortalized cultures of human urothelial cells and the expression is variable in the arsenite and cadmium- transformed urothelial cell lines derived from these immortalized cells. Tumor heterotransplants generated from the transformed cells expressed connexin 43 and the expression was localized to areas of squamous differentiation. Immuno-histochemical analysis of human bladder cancers also showed that the expression of connexin 43 was localized to areas of the tumor that showed early features of squamous differentiation. Treatment of UROtsa cells with various concentrations of arsenite or cadmium did not significantly alter the expression level of connexin 43. In conclusion, our results show that the expression of connexin 43 is localized to the areas of the tumor that show squamous differentiation, which may be an indicator of poor prognosis. This suggests that connexin 43 has the potential to be developed as a biomarker for bladder cancer that may have the ability to invade and metastasize.

  18. Identification of a novel brain derived neurotrophic factor (BDNF)-inhibitory factor: regulation of BDNF by teneurin C-terminal associated peptide (TCAP)-1 in immortalized embryonic mouse hypothalamic cells.

    Science.gov (United States)

    Ng, Tiffany; Chand, Dhan; Song, Lifang; Al Chawaf, Arij; Watson, John D; Boutros, Paul C; Belsham, Denise D; Lovejoy, David A

    2012-02-10

    The teneurins are a family of four large transmembrane proteins that are highly expressed in the central nervous system (CNS) where they have been implicated in development and CNS function. At the tip of the carboxyl terminus of each teneurin lies a 43-amino acid sequence, that when processed, could liberate an amidated 41-residue peptide. We have called this region the teneurin C-terminal associated peptide (TCAP). Picomolar concentrations of the synthetic version of TCAP-1 inhibit stress-induced cocaine reinstatement in rats. Because cocaine-seeking is associated with increased brain derived neurotrophic factor (BDNF) in the brain, we examined whether synthetic mouse TCAP-1 has the potential to regulate BDNF expression in immortalized mouse neurons. Immortalized mouse neurons (N38; mHypoE38) show strong FITC-labeled [K(8)]-TCAP-1 uptake and BDNF labeling in the cytosol. Moreover, FITC-labeled [K(8)]-TCAP-1 bound competitively to membrane fractions. In culture, the labeled TCAP-1 peptide could be detected on cell membranes within 15 min and subsequently became internalized in the cytosol and trafficked toward the nucleus. Administration of 10(-8)M unlabeled TCAP-1 to cultures of the N38 cells resulted in a significant decrease of total cell BDNF immunoreactivity over 4h as determined by western blot and ELISA analyses. Real-time PCR, utilizing primers to the various BDNF transcripts showed a significant decline of promoter IIB- and VI-driven transcripts. Taken together, these studies indicated that in vitro, TCAP-1 induces a significant decline in BDNF transcription and protein labeling in embyronic mouse immortalized hypothalamic neurons. Thus, TCAP-1 may act as a novel BDNF inhibitory factor.

  19. Establishment of a Human Conjunctival Epithelial Cell Line Lacking the Functional Tacstd2 Gene (An American Ophthalmological Society Thesis)

    Science.gov (United States)

    Kinoshita, Shigeru; Kawasaki, Satoshi; Kitazawa, Koji; Shinomiya, Katsuhiko

    2012-01-01

    Purpose: To report the establishment of a human conjunctival epithelial cell line lacking the functional tumor-associated calcium signal transducer 2 (TACSTD2) gene to be used as an in vitro model of gelatinous drop-like corneal dystrophy (GDLD), a rare disease in which the corneal epithelial barrier function is significantly compromized by the loss of function mutation of the TACSTD2 gene. Methods: A small piece of conjunctival tissue was obtained from a GDLD patient. The conjunctival epithelial cells were enzymatically separated and dissociated from the tissue and immortalized by the lentiviral introduction of the SV40 large T antigen and human telomerase reverse transcriptase (hTERT) genes. Population doubling, protein expression, and transepithelial resistance (TER) analyses were performed to assess the appropriateness of the established cell line as an in vitro model for GDLD. Results: The life span of the established cell line was found to be significantly elongated compared to nontransfected conjunctival epithelial cells. The SV40 large T antigen and hTERT genes were stably expressed in the established cell line. The protein expression level of the tight junction–related proteins was significantly low compared to the immortalized normal conjunctival epithelial cell line. TER of the established cell line was found to be significantly low compared to the immortalized normal conjunctival epithelial cell line. Conclusions: Our conjunctival epithelial cell line was successfully immortalized and well mimicked several features of GDLD corneas. This cell line may be useful for the elucidation of the pathogenesis of GDLD and for the development of novel treatments for GDLD. PMID:23818740

  20. Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses.

    Directory of Open Access Journals (Sweden)

    Jong-Ho Kim

    Full Text Available Adipose-derived stem cells (ADSCs have the potential to differentiate into various cell lineages and they are easily obtainable from patients, which makes them a promising candidate for cell therapy. However, a drawback is their limited life span during in vitro culture. Therefore, hTERT-immortalized CD34+ and CD34- mouse ADSC lines (mADSCshTERT tagged with GFP were established. We evaluated the proliferation capacity, multi-differentiation potential, and secretory profiles of CD34+ and CD34- mADSCshTERT in vitro, as well as their effects on cardiac function and systemic inflammation following transplantation into a rat model of acute myocardial infarction (AMI to assess whether these cells could be used as a novel cell source for regeneration therapy in the cardiovascular field. CD34+ and CD34- mADSCshTERT demonstrated phenotypic characteristics and multi-differentiation potentials similar to those of primary mADSCs. CD34+ mADSCshTERT exhibited a higher proliferation ability compared to CD34- mADSCshTERT, whereas CD34- mADSCshTERT showed a higher osteogenic differentiation potential compared to CD34+ mADSCshTERT. Primary mADSCs, CD34+, and CD34- mADSCshTERT primarily secreted EGF, TGF-β1, IGF-1, IGF-2, MCP-1, and HGFR. CD34+ mADSCshTERT had higher secretion of VEGF and SDF-1 compared to CD34- mADSCshTERT. IL-6 secretion was severely reduced in both CD34+ and CD34- mADSCshTERT compared to primary mADSCs. Transplantation of CD34+ and CD34- mADSCshTERT significantly improved the left ventricular ejection fraction and reduced infarct size compared to AMI-induced rats after 28 days. At 28 days after transplantation, engraftment of CD34+ and CD34- mADSCshTERT was confirmed by positive Y chromosome staining, and differentiation of CD34+ and CD34- mADSCshTERT into endothelial cells was found in the infarcted myocardium. Significant decreases were observed in circulating IL-6 levels in CD34+ and CD34- mADSCshTERT groups compared to the AMI

  1. Short-term inhibition of TERT induces telomere length-independent cell cycle arrest and apoptotic response in EBV-immortalized and transformed B cells

    Science.gov (United States)

    Celeghin, Andrea; Giunco, Silvia; Freguja, Riccardo; Zangrossi, Manuela; Nalio, Silvia; Dolcetti, Riccardo; De Rossi, Anita

    2016-01-01

    Besides its canonical role in stabilizing telomeres, telomerase reverse transcriptase (TERT) may promote tumorigenesis through extra-telomeric functions. The possible therapeutic effects of BIBR1532 (BIBR), a powerful TERT inhibitor, have been evaluated in different cellular backgrounds, but no data are currently available regarding Epstein–Barr virus (EBV)-driven B-cell malignancies. Our aim was to characterize the biological effects of TERT inhibition by BIBR on EBV-immortalized lymphoblastoid cell lines (LCLs) and fully transformed Burkitt's lymphoma (BL) cell lines. We found that BIBR selectively inhibits telomerase activity in TERT-positive 4134/Late and 4134/TERT+ LCLs and EBV-negative BL41 and EBV-positive BL41/B95.8 BL cell lines. TERT inhibition led to decreased cell proliferation, accumulation of cells in the S-phase and ultimately to increased apoptosis, compared with mock-treated control cells. All these effects occurred within 72 h and were not observed in BIBR-treated TERT-negative 4134/TERT- and U2OS cells. The cell cycle arrest and apoptosis, consequent upon short-term TERT inhibition, were associated with and likely dependent on the activation of the DNA damage response (DDR), highlighted by the increased levels of γH2AX and activation of ATM and ATR pathways. Analyses of the mean and range of telomere lengths and telomere dysfunction-induced foci indicated that DDR after short-term TERT inhibition was not related to telomere dysfunction, thus suggesting that TERT, besides stabilizing telomere, may protect DNA via telomere-independent mechanisms. Notably, TERT-positive LCLs treated with BIBR in combination with fludarabine or cyclophosphamide showed a significant increase in the number of apoptotic cells with respect to those treated with chemotherapeutic agents alone. In conclusion, TERT inhibition impairs cell cycle progression and enhances the pro-apoptotic effects of chemotherapeutic agents in TERT-positive cells. These results support new

  2. An implantable vascularized protein gel construct that supports human fetal hepatoblast survival and infection by hepatitis C virus in mice.

    Directory of Open Access Journals (Sweden)

    Martha J Harding

    Full Text Available BACKGROUND: Widely accessible small animal models suitable for the study of hepatitis C virus (HCV in vivo are lacking, primarily because rodent hepatocytes cannot be productively infected and because human hepatocytes are not easily engrafted in immunodeficient mice. METHODOLOGY/PRINCIPAL FINDINGS: We report here on a novel approach for human hepatocyte engraftment that involves subcutaneous implantation of primary human fetal hepatoblasts (HFH within a vascularized rat collagen type I/human fibronectin (rCI/hFN gel containing Bcl-2-transduced human umbilical vein endothelial cells (Bcl-2-HUVEC in severe combined immunodeficient X beige (SCID/bg mice. Maturing hepatic epithelial cells in HFH/Bcl-2-HUVEC co-implants displayed endocytotic activity at the basolateral surface, canalicular microvilli and apical tight junctions between adjacent cells assessed by transmission electron microscopy. Some primary HFH, but not Huh-7.5 hepatoma cells, appeared to differentiate towards a cholangiocyte lineage within the gels, based on histological appearance and cytokeratin 7 (CK7 mRNA and protein expression. Levels of human albumin and hepatic nuclear factor 4alpha (HNF4alpha mRNA expression in gel implants and plasma human albumin levels in mice engrafted with HFH and Bcl-2-HUVEC were somewhat enhanced by including murine liver-like basement membrane (mLBM components and/or hepatocyte growth factor (HGF-HUVEC within the gel matrix. Following ex vivo viral adsorption, both HFH/Bcl-2-HUVEC and Huh-7.5/Bcl-2-HUVEC co-implants sustained HCV Jc1 infection for at least 2 weeks in vivo, based on qRT-PCR and immunoelectron microscopic (IEM analyses of gel tissue. CONCLUSION/SIGNIFICANCE: The system described here thus provides the basis for a simple and robust small animal model of HFH engraftment that is applicable to the study of HCV infections in vivo.

  3. Sequence and expression analysis of gaps in human chromosome 20

    DEFF Research Database (Denmark)

    Minocherhomji, Sheroy; Seemann, Stefan; Mang, Yuan;

    2012-01-01

    The finished human genome-assemblies comprise several hundred un-sequenced euchromatic gaps, which may be rich in long polypurine/polypyrimidine stretches. Human chromosome 20 (chr 20) currently has three unfinished gaps remaining on its q-arm. All three gaps are within gene-dense regions and....../or overlap disease-associated loci, including the DLGAP4 locus. In this study, we sequenced ~99% of all three unfinished gaps on human chr 20, determined their complete genomic sizes and assessed epigenetic profiles using a combination of Sanger sequencing, mate pair paired-end high-throughput sequencing...... and chromatin, methylation and expression analyses. We found histone 3 trimethylated at Lysine 27 to be distributed across all three gaps in immortalized B-lymphocytes. In one gap, five novel CpG islands were predominantly hypermethylated in genomic DNA from peripheral blood lymphocytes and human cerebellum...

  4. The immortality of Ms Jones.

    Science.gov (United States)

    Gallagher, Timothy

    2014-07-01

    When I began my medical student clinical rotations, I quickly became overwhelmed by feelings of inadequacy. While the doctors around me conjured appropriate diagnoses and treatment approaches, I fumbled with the only tools I possessed: my time and a smile. It was only when I met the patient Ms Jones that I came to understand the potential impact of these simple tools. My encouragement became part of her recovery process. She gave me the confidence to construct this ability of comforting patients into a small platform of confidence from which I could safely venture to educate patients or suggest treatments to residents. It could be something that I could reliably fall back on in times of doubt and something I could pass along to other people I met. © 2014 Annals of Family Medicine, Inc.

  5. Therapeutic targeting of replicative immortality

    OpenAIRE

    Yaswen, Paul; MacKenzie, Karen L.; Keith, W. Nicol; Hentosh, Patricia; Rodier, Francis; Zhu, Jiyue; Firestone, Gary L.; Matheu, Ander; Carnero, Amancio; Bilsland, Alan; Sundin, Tabetha; Honoki, Kanya; Fujii, Hiromasa; Georgakilas, Alexandros G.; Amedei, Amedeo

    2015-01-01

    One of the hallmarks of malignant cell populations is the ability to undergo continuous proliferation. This property allows clonal lineages to acquire sequential aberrations that can fuel increasingly autonomous growth, invasiveness, and therapeutic resistance. Innate cellular mechanisms have evolved to regulate replicative potential as a hedge against malignant progression. When activated in the absence of normal terminal differentiation cues, these mechanisms can result in a state of persis...

  6. Aloe vera: Plant of Immortality

    Directory of Open Access Journals (Sweden)

    Sikarwar Mukesh. S.

    2010-02-01

    Full Text Available The Egyptians called Aloe the “Plant of Immortality” because it can live and even bloom without soil. Aloe has been used medicinally since at least the first century C.E. and continues to be used extensively worldwide.

  7. Comparative proteomic analysis of primary schwann cells and a spontaneously immortalized schwann cell line RSC 96: a comprehensive overview with a focus on cell adhesion and migration related proteins.

    Science.gov (United States)

    Ji, Yuhua; Shen, Mi; Wang, Xin; Zhang, Shuqiang; Yu, Shu; Chen, Gang; Gu, Xiaosong; Ding, Fei

    2012-06-01

    Schwann cells (SCs) are the principal glial cells of the peripheral nervous system (PNS). As a result of tissue heterogeneity and difficulties in the isolation and culture of primary SCs, a considerable understanding of SC biology is obtained from SC lines. However, the differences between the primary SCs and SC lines remain uncertain. In the present study, quantitative proteomic analysis based on isobaric tags for relative and absolute quantitation (iTRAQ) labeling was conducted to obtain an unbiased view of the proteomic profiles of primary rat SCs and RSC96, a spontaneously immortalized rat SC line. Out of 1757 identified proteins (FDR RSC96. Bioinformatics analysis indicated the unique features of spontaneous immortalization, illustrated the dedifferentiated state of RSC96, and highlighted a panel of novel proteins associated with cell adhesion and migration including CADM4, FERMT2, and MCAM. Selected proteomic data and the requirement of these novel proteins in SC adhesion and migration were properly validated. Taken together, our data collectively revealed proteome differences between primary SCs and RSC96, validated several differentially expressed proteins with potential biological significance, and generated a database that may serve as a useful resource for studies of SC biology and pathology.

  8. Proteasome inhibition-induces endoplasmic reticulum dysfunction and cell death of human cholangiocarcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Yucel Ustundag; Steven F Bronk; Gregory J Gores

    2007-01-01

    AIM: To determine if proteasome inhibition induces apoptosis in human cholangiocarcinoma cells, and if so, to elucidate the cellular mechanisms.METHODS: Studies were performed in the human KMCH, KMBC, and Mz-ChA-1 cholangiocarcinoma, and normal rat cell lines. MG132, a peptide aldehyde, which inhibits the chymotrypsin-like activity of the proteaosome was employed for this study. Apoptosis was assessed morphologically by 4'-6-Diamidino-2-phenylindole (DAPI) nuclear staining and fluorescence microscopy. Mitochondrial membrane potential was examined using a fluorescent unquenching assay. Ultrastructural changes during cell death were examined using transmission electron microscopy (TEM). Caspase 3/7 activity was assessed using an enzymatic-based fluorescent assay. Cytosolic-free calcium concentrations were measured using Fura-2 and digitized fluorescent microscopy.RESULTS: MG132, a proteasome inhibitor, induced apoptosis in all the cholangiocarcinoma cell lines examined. In contrast, minimal cytotoxicity was observed in normal rat cholangiocytes. Apoptosis was time- and -concentration-dependent. There was no change in the mitochondrial membrane potential between treated and untreated cells. Ultrastructural examination by transmission electron microscopy displayed the classic features of apoptosis, but in addition, there was also dramatic vacuolization of the endoplasmic reticulum (ER). Unexpectedly, no increase in caspase 3/7 activity was observed in MG132 treated cells, nor did the pancaspase inhibitor, Q-VD-OPh prevent cell death. The protein synthesis inhibitor, cycloheximide, blocked apoptosis induced by proteosome inhibitor indicating that ER dysfunction was dependent upon the formation of new proteins.CONCLUSION:Proteosome inhibition induces ER dysfunction and caspase-independent cell death selectively in human cholangiocarcinoma cells. Proteasome inhibitors warrant evaluation as anticancer agents for the treatment of human cholangiocarcinoma.

  9. L-MYC Expression Maintains Self-Renewal and Prolongs Multipotency of Primary Human Neural Stem Cells

    Directory of Open Access Journals (Sweden)

    Zhongqi Li

    2016-09-01

    Full Text Available Pre-clinical studies indicate that neural stem cells (NSCs can limit or reverse CNS damage through direct cell replacement, promotion of regeneration, or delivery of therapeutic agents. Immortalized NSC lines are in growing demand due to the inherent limitations of adult patient-derived NSCs, including availability, expandability, potential for genetic modifications, and costs. Here, we describe the generation and characterization of a new human fetal NSC line, immortalized by transduction with L-MYC (LM-NSC008 that in vitro displays both self-renewal and multipotent differentiation into neurons, oligodendrocytes, and astrocytes. These LM-NSC008 cells were non-tumorigenic in vivo, and migrated to orthotopic glioma xenografts in immunodeficient mice. When administered intranasally, LM-NSC008 distributed specifically to sites of traumatic brain injury (TBI. These data support the therapeutic development of immortalized LM-NSC008 cells for allogeneic use in TBI and other CNS diseases.

  10. In vivo Brain Delivery of v-myc Overproduced Human Neural Stem Cells via the Intranasal Pathway: Tumor Characteristics in the Lung of a Nude Mouse

    Directory of Open Access Journals (Sweden)

    Eun Seong Lee

    2015-01-01

    Full Text Available We aimed to monitor the successful brain delivery of stem cells via the intranasal route and to observe the long-term consequence of the immortalized human neural stem cells in the lungs of a nude mouse model. Stably immortalized HB1.F3 human neural stem cells with firefly luciferase gene (F3-effluc were intranasally delivered to BALB/c nude mice. Bioluminescence images were serially acquired until 41 days in vivo and at 4 hours and 41 days ex vivo after intranasal delivery. Lungs were evaluated by histopathology. After intranasal delivery of F3-effluc cells, the intense in vivo signals were detected in the nasal area, migrated toward the brain areas at 4 hours (4 of 13, 30.8%, and gradually decreased for 2 days. The brain signals were confirmed by ex vivo imaging (2 of 4, 50%. In the mice with initial lung signals (4 of 9, 44.4%, the lung signals disappeared for 5 days but reappeared 2 weeks later. The intense lung signals were confirmed to originate from the tumors in the lungs formed by F3-effluc cells by ex vivo imaging and histopathology. We propose that intranasal delivery of immortalized stem cells should be monitored for their successful delivery to the brain and their tumorigenicity longitudinally.

  11. 陆地棉永久 F2群体产量、杂种优势及相关关系分析%Analysis of yield, heterosis and their correlation of cotton immortalized F2 population

    Institute of Scientific and Technical Information of China (English)

    王保勤; 李宾; 李俊文; 刘书梅; 贾新合

    2016-01-01

    利用重组自交系RIL群体两两随机组配构建1套含98个组合的永久F2群体。通过对永久群体7个性状的杂种优势表现及其性状之间的相关性进行分析,探讨永久F2群体经济性状关系。结果表明,永久F2群体的7个性状在2年3点3个不同环境下均具有明显的杂种优势,中亲优势和优势组合率较大;产量构成因子的相关系数中铃重最大,其次是单株结铃数和衣分;永久群体的竞争优势为负向,理论上此永久F2群体不能在生产上直接利用来作为杂交亲本,但可以作为遗传群体的研究材料。%In order to analyse the heterosis of yield and traits of cotton , a set of recombinant inbred line ( RIL) de-rived from a hybrid combination ( sGK9708 × 0-153 ) was used to constructed an immortalized F 2 ( IF2 ) popula-tion.This IF2 population included 98 single crosses.The heterosis analysis of seven yield traits and their correlation of immortalized F2 population was studied .The results showed that this seven traits had obvious heterosis in different environments , middle parents advantage and advantage combination rate were both larger .In the correlation factors of production , boll weight was the greatest , followed by the boll number per plant and lint percentage .Permanent group competitive advantage was negative , so this immortalized F2 population could not be directly used theoretically in pro-duction, but it could be used as genetic group research materials .

  12. Integration sites of Epstein-Barr virus genome on chromosomes of human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Wuu, K.D.; Chen, Y.J.; Wang-Wuu, S. [Institute of Genetics, Taipei (Taiwan, Province of China)

    1994-09-01

    Epstein-Barr virus (EBV) is the pathogen of infectious mononucleosis. The viral genome is present in more than 95% of the African cases of Burkitt lymphoma and it is usually maintained in episomal form in the tumor cells. Viral integration has been described only for Nanalwa which is a Burkitt lymphoma cell line lacking episomes. In order to examine the role of EBV in the immortalization of human Blymphocytes, we investigated whether the EBV integration into the human genome is essential. If the integration does occur, we would like to know whether the integration is randomly distributed or whether the viral DNA integrates preferentially at certain sites. Fourteen in vitro immortalized human lymphoblastoid cell lines (LCLs) were examined by fluorescence in situ hybridization (FISH) with a biotinylated EBV BamHI w DNA fragment as probe. The episomal form of EBV DNA was found in all cells of these cell lines, while only about 65% of the cells have the integrated viral DNA. This might suggest that integration is not a pre-requisite for cell immortalization. Although all chromosomes, except Y, have been found with integrated viral genome, chromsomes 1 and 5 are the most frequent EBV DNA carrier (p<0.05). Nine chromosome bands, namely, 1p31, 1q31, 2q32, 3q13, 3q26, 5q14, 6q24, 7q31 and 12q21, are preferential targets for EBV integration (p<0.001). Eighty percent of the total 938 EBV hybridization signals were found to be at G-band-positive area. This suggests that the mechanism of EBV integration might be different from that of the retroviruses, which specifically integrate to G-band-negative areas. Thus, we conclude that the integration of EBV to host genome is non-random and it may have something to do with the structure of chromosome and DNA sequences.

  13. KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Objective: To establish two stably KGF-transfected, immortalized cell lines. Methods: HaCaT-keratinocytes and KMST-6-fibroblasts were transfected by liposome mediated gene transfer. Transfection effectivity, gene integration and configuration of the transgenic protein were investigated by ELISA, DANN-PCR and β-Gal-staining. Results: Most effective GF producing clones were tested by a colorimetric XTT-test. Conclusion: This is a significant acceleration of cell proliferation and mitosis of human keratinocytes in an Air Liquid Interface (ALI) test system.

  14. 从《搜神记》看魏晋时期的个体意识%On the Individual Consciousness of Wei and Jin Dynasty in Stories of Immortals

    Institute of Scientific and Technical Information of China (English)

    李小成; 唐海宁

    2012-01-01

    the mystery novel ,Stories of Immortals included the mystery stories and reflected the chaotic life and exposed the general cognition and the individual consciousness. The declination of Confucianism can be explored in the analysis.%《搜神记》作为魏晋志怪小说的代表,记载了魏晋及魏晋以前的怪异之事,反映了社会的动乱,体现了魏晋时期人们的普遍认识和个体意识的觉醒。从《搜神记》文本出发考察其中所展现的魏晋时期人们的思想状况,可以挖掘出儒学衰微,关注个体生命价值的真正原因。

  15. On Inheritance of the Dynasty Han and Wei Style in Poetry about Immortals by Guo Pu%试论郭璞《游仙诗》对汉魏风骨的继承

    Institute of Scientific and Technical Information of China (English)

    陈祥伟; 陈亮; 臧守刚

    2016-01-01

    初唐陈子昂在《修竹篇序》中说:“汉魏风骨,晋宋莫传。”这一说法不能成立。郭璞是东晋人,其《游仙诗》产生的时代背景与汉魏诗歌相近,其诗歌精神实质是坎壈自伤,运用的表现手法是比兴寄托,抒情风格表现为激烈悲愤,这几个方面是对汉魏风骨继承与发展。《游仙诗》具备汉魏风骨特征。%Chen Ziang, the poet in early Tang Dynasty , once remarked in his Preface to Long Bamboos,“ The Vigorous Style of the Han and Wei Dynasty hasn ’ t been inherited in the poems of Jin an Song Dynas-ty.” However, the statement isn’t proved true.The background of The Poetry about Immortals by Guo Pu who was born in the Eastern Jin Dynasty is similar to the historical background of poems composed during the Han and Wei Dynasty .The spirit of the poem is essentially sentimental .The analogy is applied as the? ex-pression technique? in the poem .Furthermore , the lyric style in this poem is presented as fierce grief .Ac-cording to these mentioned aspects , it is indisputable to draw a conclusion that the poetic features of Han and Wei Dynasty are inherited and improved in Poetry about Immortals.

  16. A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells.

    Science.gov (United States)

    Guo, Xuan; Wang, Shu; Dou, Ya-ling; Guo, Xiang-fei; Chen, Zhao-li; Wang, Xin-wei; Shen, Zhi-qiang; Qiu, Zhi-gang; Jin, Min; Li, Jun-wen

    2015-06-01

    Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.

  17. Early embryonic chromosome instability results in stable mosaic pattern in human tissues.

    Directory of Open Access Journals (Sweden)

    Hasmik Mkrtchyan

    Full Text Available The discovery of copy number variations (CNV in the human genome opened new perspectives on the study of the genetic causes of inherited disorders and the aetiology of common diseases. Here, a single-cell-level investigation of CNV in different human tissues led us to uncover the phenomenon of mitotically derived genomic mosaicism, which is stable in different cell types of one individual. The CNV mosaic ratios were different between the 10 individuals studied. However, they were stable in the T lymphocytes, immortalized B lymphoblastoid cells, and skin fibroblasts analyzed in each individual. Because these cell types have a common origin in the connective tissues, we suggest that mitotic changes in CNV regions may happen early during embryonic development and occur only once, after which the stable mosaic ratio is maintained throughout the differentiated tissues. This concept is further supported by a unique study of immortalized B lymphoblastoid cell lines obtained with 20 year difference from two subjects. We provide the first evidence of somatic mosaicism for CNV, with stable variation ratios in different cell types of one individual leading to the hypothesis of early embryonic chromosome instability resulting in stable mosaic pattern in human tissues. This concept has the potential to open new perspectives in personalized genetic diagnostics and can explain genetic phenomena like diminished penetrance in autosomal dominant diseases. We propose that further genomic studies should focus on the single-cell level, to better understand the aetiology of aging and diseases mediated by somatic mutations.

  18. Long-term cultivation of in vitro Apis mellifera cells by gene transfer of human c-myc proto-oncogene.

    Science.gov (United States)

    Kitagishi, Yasuko; Okumura, Naoko; Yoshida, Hitomi; Nishimura, Yuri; Takahashi, Jun-ichi; Matsuda, Satoru

    2011-08-01

    Establishment of cell lines representative of honeybee character would greatly assist in their analysis. Here, we show that immortalized cell line, designated as MYN9, has been generated from honeybee embryo by the gene transfer of human c-myc proto-oncogene. The morphology of the cell is characteristic of embryonic stem cell, although the cell is stable and does not spontaneously differentiate. Polymerase chain reaction analyses show that the cell is originated from authentic honeybee cell. It is proposed that the integration of human c-myc gene into honeybee precursor populations results in the establishment of stable cell line suitable for cellular and molecular studies.

  19. Changes in expression of imprinted genes following treatment of human cancer cell lines with non-mutagenic or mutagenic carcinogens.

    Science.gov (United States)

    Shibui, Takeo; Higo, Yukari; Tsutsui, Takeo W; Uchida, Minoru; Oshimura, Mitsuo; Barrett, J Carl; Tsutsui, Takeki

    2008-08-01

    It remains possible that chemicals that act by mutagenic mechanisms as well as chemicals that do not induce gene mutations may affect epigenetic gene expression. To test the possibility, we investigated the ability of both types of chemicals to alter the expression of five imprinted genes, PEG3, SNRPN, NDN, ZAC and H19, using two human colon cancer cell lines and a human breast cancer cell line. The expression of imprinted genes was changed by some non-mutagenic and mutagenic carcinogens independent of their mutagenic activity. The genes most commonly exhibiting the changes in expression were SNRPN and PEG3. Alterations of the expression of NDN and ZAC were also observed in some conditions. Methylation-specific PCR and chromatin immunoprecipitation assays suggest the possibility that changes in the expression of SNRPN may be associated with DNA hypomethylation and histone acetylation of the promoters and euchromatinization of the heterochromatic domains of the promoters. Changes in expression of the imprinted genes, PEG3 and NDN, were also observed in cells immortalized by treatment of normal human fibroblasts with 4-nitroquinoline 1-oxide or aflatoxin B1. We previously demonstrated that expression of the cancer-related gene, INK4a, in these immortal cells was lost via epigenetic mechanisms. The results prove that, in cancer cells, some mutagenic or non-mutagenic carcinogens can epigenetically influence the transcription levels of imprinted genes and also suggest the possibility that some chemical carcinogens may have epigenetic carcinogenic effects in human cells.

  20. BAD-mediated apoptotic pathway is associated with human cancer development.

    Science.gov (United States)

    Stickles, Xiaomang B; Marchion, Douglas C; Bicaku, Elona; Al Sawah, Entidhar; Abbasi, Forough; Xiong, Yin; Bou Zgheib, Nadim; Boac, Bernadette M; Orr, Brian C; Judson, Patricia L; Berry, Amy; Hakam, Ardeshir; Wenham, Robert M; Apte, Sachin M; Berglund, Anders E; Lancaster, Johnathan M

    2015-04-01

    The malignant transformation of normal cells is caused in part by aberrant gene expression disrupting the regulation of cell proliferation, apoptosis, senescence and DNA repair. Evidence suggests that the Bcl-2 antagonist of cell death (BAD)-mediated apoptotic pathway influences cancer chemoresistance. In the present study, we explored the role of the BAD-mediated apoptotic pathway in the development and progression of cancer. Using principal component analysis to derive a numeric score representing pathway expression, we evaluated clinico-genomic datasets (n=427) from corresponding normal, pre-invasive and invasive cancers of different types, such as ovarian, endometrial, breast and colon cancers in order to determine the associations between the BAD-mediated apoptotic pathway and cancer development. Immunofluorescence was used to compare the expression levels of phosphorylated BAD [pBAD (serine-112, -136 and -155)] in immortalized normal and invasive ovarian, colon and breast cancer cells. The expression of the BAD-mediated apoptotic pathway phosphatase, PP2C, was evaluated by RT-qPCR in the normal and ovarian cancer tissue samples. The growth-promoting effects of pBAD protein levels in the immortalized normal and cancer cells were assessed using siRNA depletion experiments with MTS assays. The expression of the BAD-mediated apoptotic pathway was associated with the development and/or progression of ovarian (n=106, pcancers, as well as with ovarian endometriosis (n=20, pcancer cells compared to the immortalized normal cells, whereas PP2C gene expression was lower in the cancer compared to the ovarian tumor tissue samples (n=76, pcancer cells. The BAD-mediated apoptotic pathway is thus associated with the development of human cancers likely influenced by the protein levels of pBAD.

  1. 一种永生化的大鼠颈动脉血管球细胞系的生化特性%The neurochemical properties of an immortalized glomus cell line from the carotid body of rat

    Institute of Scientific and Technical Information of China (English)

    周光斗

    2006-01-01

    目的 神经干细胞在帕金森病的治疗中有十分巨大的潜在价值,然而,神经干细胞含有多种神经细胞成分,目前选择性分离纯化其中的多巴胺能神经元尚不完全成熟.在这里我们报告一种来自颈动脉体的克隆细胞系.方法 我们用逆转录病毒转导SV40 T抗原和Cre/lox P到大鼠颈动脉血管球细胞使其永生化,然后这些原癌基因被腺病毒转导的Cre重组酶切除.结果 克隆的血管球细胞表达高水平的酪氨酸羟化酶免疫反应性物质并且选择性地释放多巴胺.这些克隆细胞几乎不表达多巴胺转运体(DAT)而且抵抗多巴胺和MPTP介导的神经毒性.结论 克隆的血管球细胞可能为帕金森病的治疗提供一种新的方法.%Objective Embryonic stem cells (ES) are potentially useful for treatment of Parkinson's disease. However, ES cells contain a mixed population of neurons, and selective enrichment of dopaminergic components remains difficult. Here, we report that a dopaminergic clonal cell line developed from carotid body. Methods Glomus cells of the rat carotid body were conditionally immortalized by retroviral transfer of the SV40 T antigen and Cre/lox P elements. The oncogene was subsequently removed adenoviral delivery of the Cre recombinase. Results Cloned glomus cells expressed high levels of tyrosine hydroxylase immunoreactivity and selectively released dopamine in culture. Remarkably, they expressed little dopamine transporter DAT, and were resistant to dopamine- and MPTP-mediated neurotoxicity. Conclusion Immortalized glomus cell line might provide an alternative way to the treatment of Parkinson's disease.

  2. BRCA1 controls the cell division axis and governs ploidy and phenotype in human mammary cells.

    Science.gov (United States)

    He, Zhengcheng; Kannan, Nagarajan; Nemirovsky, Oksana; Chen, Helen; Connell, Marisa; Taylor, Brian; Jiang, Jihong; Pilarski, Linda M; Fleisch, Markus C; Niederacher, Dieter; Pujana, Miguel Angel; Eaves, Connie J; Maxwell, Christopher A

    2017-05-16

    BRCA1 deficiency may perturb the differentiation hierarchy present in the normal mammary gland and is associated with the genesis of breast cancers that are genomically unstable and typically display a basal-like transcriptome. Oriented cell division is a mechanism known to regulate cell fates and to restrict tumor formation. We now show that the cell division axis is altered following shRNA-mediated BRCA1 depletion in immortalized but non-tumorigenic, or freshly isolated normal human mammary cells with graded consequences in progeny cells that include aneuploidy, perturbation of cell polarity in spheroid cultures, and a selective loss of cells with luminal features. BRCA1 depletion stabilizes HMMR abundance and disrupts cortical asymmetry of NUMA-dynein complexes in dividing cells such that polarity cues provided by cell-matrix adhesions were not able to orient division. We also show that immortalized mammary cells carrying a mutant BRCA1 allele (BRCA1 185delAG/+) reproduce many of these effects but in this model, oriented divisions were maintained through cues provided by CDH1+ cell-cell junctions. These findings reveal a previously unknown effect of BRCA1 suppression on mechanisms that regulate the cell division axis in proliferating, non-transformed human mammary epithelial cells and consequent downstream effects on the mitotic integrity and phenotype control of their progeny.

  3. Biocompatibility Evaluation of Dental Luting Cements Using Cytokine Released from Human Oral Fibroblasts and Keratinocytes

    Directory of Open Access Journals (Sweden)

    Jae-Sung Kwon

    2015-10-01

    Full Text Available Dental luting cements are commonly used in dentistry for cementation of prosthetic restoration. Many previous studies focused on the measurement of the cell viability as the method of cytotoxicity evaluation during biocompatibility study for the material. In this study, the biocompatibility of various dental luting cements were evaluated using the new method of cytokine release measurement in order to better simulate inflammatory reactions in animal or clinical model using two different oral cells; immortalized human gingival fibroblast and immortalized human oral keratinocytes. Cells were exposed to extractions of various commercially available dental luting cements for different durations. Cytokines of IL-1α and IL-8 were measured from the supernatants of the cells and the results were then compared to the conventional MTT viability test. The result from the conventional cell viability study showed a relatively simple and straight forward indication that only one of the dental luting cements tested in this study was cytotoxic with increasing duration of exposure for both cells. Meanwhile, the result from the cytokine measurement study was much more complex at the time point they were measured, type of cells used for the study and the type of cytokines measured, all of which influenced the interpretation of the results. Hence, the better understanding of the cytokine release would be required for the application in biocompatibility evaluation.

  4. Lipopolysaccharide (LPS-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR.

    Directory of Open Access Journals (Sweden)

    Christy E Trussoni

    Full Text Available Cholangiocytes (biliary epithelial cells actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells, or low passage normal human cholangiocytes (NHC, were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05 and proliferation (p<0.01. Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC livers exhibited increased phospho-EGFR (p<0.01. Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  5. Human papillomavirus: E6 and E7 oncogenes.

    Science.gov (United States)

    Boulet, Gaëlle; Horvath, Caroline; Vanden Broeck, Davy; Sahebali, Shaira; Bogers, Johannes

    2007-01-01

    The recognition of a causal relationship between human papillomaviruses and cancer almost 30 years ago led to a rapid expansion of knowledge in the field, resulting in the description of the main mediators of HPV-induced carcinogenesis, the viral proteins E6 and E7. These oncoproteins show a remarkable pleiotropism in binding host-cell proteins, with the tumour suppressor genes p53 and pRb as their major targets. These interactions induce proliferation, immortalization and malignant transformation of infected cells. The link between HPV and cervical cancer led to the development of molecular methods, often based on the detection of E6 and E7, for screening and diagnosis. Therapeutic vaccines and gene therapy are primarily directed at E6 and E7. Although prophylactic vaccines are available, further understanding of the viral life cycle and the mechanisms underlying HPV-induced oncogenesis is necessary to face the many challenges in the field of HPV and cancer.

  6. Neoplastic transformation of a human prostate epithelial cell line by the v-Ki-ras oncogene.

    Science.gov (United States)

    Parda, D S; Thraves, P J; Kuettel, M R; Lee, M S; Arnstein, P; Kaighn, M E; Rhim, J S; Dritschilo, A

    1993-01-01

    Investigations of mechanisms of human prostate carcinogenesis are limited by the unavailability of a suitable in vitro model system. We have demonstrated that an immortal, but nontumorigenic, human epithelial cell line (267B1) established from fetal prostate tissue can be malignantly transformed by a biological carcinogen, and can serve as a useful model for investigations of the progression steps of carcinogenesis. Activated Ki-ras was introduced into 267B1 cells by infection with the Kirsten murine sarcoma virus. Morphological alterations and anchorage-independent growth were observed; when cells were injected into nude mice, poorly differentiated adenocarcinomas developed. These findings represent the first evidence of malignant transformation of human prostate epithelial cells in culture, and support a role for Ki-ras activation in a multistep process for prostate neoplastic transformation.

  7. MIO-M1 cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics.

    Science.gov (United States)

    Lawrence, Jean M; Singhal, Shweta; Bhatia, Bhairavi; Keegan, David J; Reh, Thomas A; Luthert, Philip J; Khaw, Peng T; Limb, Gloria Astrid

    2007-08-01

    Growing evidence suggests that glial cells may have a role as neural precursors in the adult central nervous system. Although it has been shown that Müller cells exhibit progenitor characteristics in the postnatal chick and rat retinae, their progenitor-like role in developed human retina is unknown. We first reported the Müller glial characteristics of the spontaneously immortalized human cell line MIO-M1, but recently we have derived similar cell lines from the neural retina of several adult eye donors. Since immortalization is one of the main properties of stem cells, we investigated whether these cells expressed stem cell markers. Cells were grown as adherent monolayers, responded to epidermal growth factor, and could be expanded indefinitely without growth factors under normal culture conditions. They could be frozen and thawed without losing their characteristics. In the presence of extracellular matrix and fibroblast growth factor-2 or retinoic acid, they acquired neural morphology, formed neurospheres, and expressed neural stem cell markers including betaIII tubulin, Sox2, Pax6, Chx10, and Notch 1. They also expressed markers of postmitotic retinal neurons, including peripherin, recoverin, calretinin, S-opsin, and Brn3. When grafted into the subretinal space of dystrophic Royal College of Surgeons rats or neonatal Lister hooded rats, immortalized cells migrated into the retina, where they expressed various markers of retinal neurons. These observations indicate that adult human neural retina harbors a population of cells that express both Müller glial and stem cell markers and suggest that these cells may have potential use for cell-based therapies to restore retinal function. Disclosure of potential conflicts of interest is found at the end of this article.

  8. Three-Dimensionally Engineered Normal Human Lung Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    Science.gov (United States)

    Goodwin, Thomas J.; McCarthy, M.; Lin, Y-H.; Deatly, A. M.

    2008-01-01

    In vitro three-dimensional (3D) human lung epithelio-mesenchymal tissue-like assemblies (3D hLEM TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and the detection of membrane bound glycoproteins over time confirm productive infection with the virus. Therefore, we assert TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host s immune system.

  9. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    Science.gov (United States)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  10. Three-Dimensionally Engineered Normal Human Broncho-epithelial Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    Science.gov (United States)

    Goodwin, T. J.; McCarthy, M.; Lin, Y-H

    2006-01-01

    In vitro three-dimensional (3D) human broncho-epithelial (HBE) tissue-like assemblies (3D HBE TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and parainfluenza virus type 3 (wtPIV3 JS) and the detection of membrane bound glycoproteins over time confirm productive infections with both viruses. Therefore, TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host's immune system.

  11. Is cancer a metabolic rebellion against host aging? In the quest for immortality, tumor cells try to save themselves by boosting mitochondrial metabolism.

    Science.gov (United States)

    Ertel, Adam; Tsirigos, Aristotelis; Whitaker-Menezes, Diana; Birbe, Ruth C; Pavlides, Stephanos; Martinez-Outschoorn, Ubaldo E; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

    2012-01-15

    Aging drives large systemic reductions in oxidative mitochondrial function, shifting the entire body metabolically towards aerobic glycolysis, a.k.a, the Warburg effect. Aging is also one of the most significant risk factors for the development of human cancers, including breast tumors. How are these two findings connected? One simplistic idea is that cancer cells rebel against the aging process by increasing their capacity for oxidative mitochondrial metabolism (OXPHOS). Then, local and systemic aerobic glycolysis in the aging host would provide energy-rich mitochondrial fuels (such as L-lactate and ketones) to directly "fuel" tumor cell growth and metastasis. This would establish a type of parasite-host relationship or "two-compartment tumor metabolism", with glycolytic/oxidative metabolic-coupling. The cancer cells ("the seeds") would flourish in this nutrient-rich microenvironment ("the soil"), which has been fertilized by host aging. In this scenario, cancer cells are only trying to save themselves from the consequences of aging, by engineering a metabolic mutiny, through the amplification of mitochondrial metabolism. We discuss the recent findings of Drs. Ron DePinho (MD Anderson) and Craig Thomspson (Sloan-Kettering) that are also consistent with this new hypothesis, linking cancer progression with metabolic aging. Using data mining and bioinformatics approaches, we also provide key evidence of a role for PGC1a/NRF1 signaling in the pathogenesis of (1) two-compartment tumor metabolism, and (2) mitochondrial biogenesis in human breast cancer cells.

  12. Precancerous model of human breast epithelial cells induced by NNK for prevention.

    Science.gov (United States)

    Siriwardhana, Nalin; Choudhary, Shambhunath; Wang, Hwa-Chain Robert

    2008-06-01

    Epidemiological investigations have suggested that exposure to tobacco and environmental carcinogens increase the risk of developing human breast cancer. In light of the chronic exposure of human breast tissues to tobacco and environmental carcinogens, we have taken an approach of analyzing cellular changes of immortalized non-cancerous human breast epithelial MCF10A cells during the acquisition of cancerous properties induced by repeated exposure to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at a low concentration of 100 pM. We found that accumulated exposures of MCF10A cells to NNK result in progressive development of cellular carcinogenesis from a stage of immortalization to precancerous sub-stages of acquiring a reduced dependence on growth factors and acquiring anchorage-independent growth. Using Matrigel for MCF10A cells to form size-restricted acini, we detected that exposures to NNK resulted in altered acinar conformation. Analysis of gene expression profiles by cDNA microarrays revealed up- and down-regulated genes associated with NNK-induced carcinogenesis. Using this cellular carcinogenesis model as a target system to identify anticancer agents, we detected that grape seed proanthocyanadin extract significantly suppressed NNK-induced carcinogenesis of MCF10A cells. Our studies provide a carcinogenesis-cellular model mimicking the accumulative exposure to carcinogens in the progression of human breast epithelial cells to increasingly acquire cancerous properties, as likely occurs in the development of precancerous human breast cells. Our cellular model also serves as a cost-efficient, in vitro system to identify preventive agents that inhibit human breast cell carcinogenesis induced by chronic exposures to carcinogens.

  13. Relocalization of cell adhesion molecules during neoplastic transformation of human fibroblasts.

    Science.gov (United States)

    Belgiovine, Cristina; Chiodi, Ilaria; Mondello, Chiara

    2011-11-01

    Studying neoplastic transformation of telomerase immortalized human fibroblasts (cen3tel), we found that the transition from normal to tumorigenic cells was associated with the loss of growth contact inhibition, the acquisition of an epithelial-like morphology and a change in actin organization, from stress fibers to cortical bundles. We show here that these variations were paralleled by an increase in N-cadherin expression and relocalization of different adhesion molecules, such as N-cadherin, α-catenin, p-120 and β-catenin. These proteins presented a clear membrane localization in tumorigenic cells compared to a more diffuse, cytoplasmic distribution in primary fibroblasts and non-tumorigenic immortalized cells, suggesting that tumorigenic cells could form strong cell-cell contacts and cell contacts did not induce growth inhibition. The epithelial-like appearance of tumorigenic cells did not reflect a mesenchymal-epithelial transition; in fact, cen3tel cells expressed vimentin and did not express cytokeratins at all transformation stages. Moreover, they did not express epithelial proteins such as occluding and claudin-1. In contrast, ZO-1 showed higher levels and a more defined membrane localization in tumorigenic cells compared to non-tumorigenic cells; this confirms its role in adherens junction formation in mesenchymal cells and is in agreement with the strong cell-cell contact formation by neoplastically transformed cells. Finally, we found α-catenin and ZO-1 nuclear localization in non-transformed cells, suggestive of possible additional roles of these proteins besides cell junction formation.

  14. Effect of exogenous surfactants on viability and DNA synthesis in A549, immortalized mouse type II and isolated rat alveolar type II cells

    Directory of Open Access Journals (Sweden)

    Haller Thomas

    2011-02-01

    Full Text Available Abstract Background In mechanically ventilated preterm infants with respiratory distress syndrome (RDS, exogenous surfactant application has been demonstrated both to decrease DNA-synthesis but also and paradoxically to increase epithelial cell proliferation. However, the effect of exogenous surfactant has not been studied directly on alveolar type II cells (ATII cells, a key cell type responsible for alveolar function and repair. Objective The aim of this study was to investigate the effects of two commercially available surfactant preparations on ATII cell viability and DNA synthesis. Methods Curosurf® and Alveofact® were applied to two ATII cell lines (human A549 and mouse iMATII cells and to primary rat ATII cells for periods of up to 24 h. Cell viability was measured using the redox indicator resazurin and DNA synthesis was measured using BrdU incorporation. Results Curosurf® resulted in slightly decreased cell viability in all cell culture models. However, DNA synthesis was increased in A549 and rat ATII cells but decreased in iMATII cells. Alveofact® exhibited the opposite effects on A549 cells and had very mild effects on the other two cell models. Conclusion This study showed that commercially available exogenous surfactants used to treat preterm infants with RDS can have profound effects on cell viability and DNA synthesis.

  15. Generation and properties of a new human ventral mesencephalic neural stem cell line

    DEFF Research Database (Denmark)

    Villa, Ana; Liste, Isabel; Courtois, Elise T

    2009-01-01

    Neural stem cells (NSCs) are powerful research tools for the design and discovery of new approaches to cell therapy in neurodegenerative diseases like Parkinson's disease. Several epigenetic and genetic strategies have been tested for long-term maintenance and expansion of these cells in vitro....... Here we report the generation of a new stable cell line of human neural stem cells derived from ventral mesencephalon (hVM1) based on v-myc immortalization. The cells expressed neural stem cell and radial glia markers like nestin, vimentin and 3CB2 under proliferation conditions. After withdrawal...... derivatives may constitute good candidates for the study of development and physiology of human dopaminergic neurons in vitro, and to develop tools for Parkinson's disease cell replacement preclinical research and drug testing....

  16. ABOUT THE BASICS OF HUMAN THINKING APPARATUS

    Directory of Open Access Journals (Sweden)

    Yuriy Mikhailovich AMIRBEGOV

    2015-01-01

    Full Text Available The main subject of our research is the integrity of the thinking apparatus of humans with its extremes, one of which is known to us as cause-effect materiality, denoted by the term ―brain‖, and the other extreme is unknown but is supposed as an immaterial cause of the motion needed to ―revitalize‖ the material extreme in response to revitalizing effects, reacting with changes the order and direction of which are realized by light streams (oscillation spectrum in the range of light waves and sounds of different tones (oscillation spectrum in the range of sound waves. But for realization of light streams and sound streams into images and words is needed not only information streams regulator (dominant factor, changing their vectors in the integrity of the human thinking apparatus, but also verifier of oscillations is needed which requires philosophical comprehension.While modeling the thinking apparatus of humans by dialectical method of reasoning, we had to abandon the conventional opinions about reality, matter, motion, and take on trust the opinions which have not been confirmed but are logically essential – a divine spark of Aristotle and permanent impacts taking the material world out of a cold death…, what led to the solution of the problem: as a result was found the second, immaterial extreme of the integrity of human thinking apparatus and the dominant factor focusing the flow of information in the CNS and having the significance of the status of the monarch. Actually, the proposed model of human thinking apparatus, which has no analogue, presents the novelty of the worldview which answers the question: what is thinking, why and how, and what is realized by us, our mortal body and immortal soul identifying our ―I‖.

  17. Determination of acute lethal and chronic lethal dose thresholds of valproic acid using 3D spheroids constructed from the immortal human hepatocyte cell line HepG2/C3A

    DEFF Research Database (Denmark)

    Fey, S. J.; Wrzesinski, K.

    2013-01-01

    Valproic acid (VPA) is a broad-spectrum antiepileptic drug that is now used commonly for several other neurological and psychiatric indications. While VPA is usually well tolerated, on rare occasions, it has been associated with severe, and sometimes, fatal liver injuries. These complications may...

  18. Functional human artificial chromosomes are generated and stably maintained in human embryonic stem cells

    Science.gov (United States)

    Mandegar, Mohammad A.; Moralli, Daniela; Khoja, Suhail; Cowley, Sally; Chan, David Y.L.; Yusuf, Mohammed; Mukherjee, Sayandip; Blundell, Michael P.; Volpi, Emanuela V.; Thrasher, Adrian J.; James, William; Monaco, Zoia L.

    2011-01-01

    We present a novel and efficient non-integrating gene expression system in human embryonic stem cells (hESc) utilizing human artificial chromosomes (HAC), which behave as autonomous endogenous host chromosomes and segregate correctly during cell division. HAC are important vectors for investigating the organization and structure of the kinetochore, and gene complementation. HAC have so far been obtained in immortalized or tumour-derived cell lines, but never in stem cells, thus limiting their potential therapeutic application. In this work, we modified the herpes simplex virus type 1 amplicon system for efficient transfer of HAC DNA into two hESc. The deriving stable clones generated green fluorescent protein gene-expressing HAC at high frequency, which were stably maintained without selection for 3 months. Importantly, no integration of the HAC DNA was observed in the hESc lines, compared with the fibrosarcoma-derived control cells, where the exogenous DNA frequently integrated in the host genome. The hESc retained pluripotency, differentiation and teratoma formation capabilities. This is the first report of successfully generating gene expressing de novo HAC in hESc, and is a significant step towards the genetic manipulation of stem cells and potential therapeutic applications. PMID:21593218

  19. YThe BigH3 Tumor Suppressor Gene in Radiation-Induced Malignant Transformation of Human Bronchial Epithelial Cells

    Science.gov (United States)

    Zhao, Y.; Shao, G.; Piao, C.; Hei, T.

    Carcinogenesis is a multi-stage process with sequences of genetic events governing the phenotypic expression of a series of transformation steps leading to the development of metastatic cancer Previous studies from this laboratory have identified a 7 fold down- regulation of the novel tumor suppressor Big-h3 among radiation induced tumorigenic BEP2D cells Furthermore ectopic re-expression of this gene suppresses tumorigenic phenotype and promotes the sensitivity of these tumor cells to etoposide-induced apoptosis To extend these studies using a genomically more stable bronchial cell line we ectopically expresses the catalytic subunit of telomerase hTERT in primary human small airway epithelial SAE cells and generated several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice These cells show no alteration in the p53 gene but a decrease in p16 expression Exponentially growing SAEh cells were exposed to graded doses of 1 GeV nucleon of 56 Fe ions accelerated at the Brookhaven National Laboratory Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium These findings indicate

  20. On Research of the Era of Kechang Chen Became Immortal in the Dragon Boat Festival%《陈可常端阳仙化》为明代作品考

    Institute of Scientific and Technical Information of China (English)

    田冬梅; 张颖夫

    2013-01-01

    "San Yan"are the best novels' collections of vernacular short stories in ancient China. There are still lots of controversies and research space about the novels' creative era. Through investigation, the author believes that the novel of Kechang Chen Became Immortal in the Dragon Boat Festival was created in the Ming Dynasty. First of all, the author questioned the previous researching point of view that they thought the novel was written in the Song Dynasty;secondly, according to the differences of the criminal justice system in the Song, the Yuan, the Ming Dynasty, the change of "Samen Island" territorial right, proved that the novel couldn't be written in the Southern Song Dynasty;finally, according to the administrative divisions' history of the "Wenzhou government" and "Lin'an government", the author proved that the novel was written in the Ming Dynasty.%  “三言”是中国古代成就最高的白话短篇小说集,关于其中作品的成书时代依然存在着很大的争议和研究空间。《警世通言·陈可常端阳仙化》一文,胡士莹和郑振铎两位先生认为是宋人话本,实则应为明代作品。首先,对前人提出的“宋人话本”说的研究观点提出质疑;其次,根据宋元明时期刑法制度的差异,“沙门岛”领土所属权的变化,证明这篇作品绝非创作于南宋时期;最后,根据“温州府”和“临安府”行政区划的历史沿革,进一步证明这篇小说为明代作品。

  1. Radiogenic transformation of human mammary epithelial cells in vitro

    Science.gov (United States)

    Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

    1996-01-01

    Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

  2. A human-human hybridoma producing cytotoxic antibody to HLA-B15, cross-reacting with B17, B5, B35 and B18.

    Science.gov (United States)

    Hansen, T; Kolstad, A; Thorsby, E; Hannestad, K

    1987-05-01

    Mononuclear blood cells from a multiparous woman were transformed with Epstein Barr virus, and a cell line (Tr2D8) producing anti-HLA antibody was obtained. This cell line was immortalized by hybridization to the human fusion partners KR4 and KR12. While the EBV line died after 7 months, the hybridomas have remained stable for 13 months. The EBV line supernatant (40 micrograms IgM/ml) lysed peripheral blood mononuclear cells (PBMC) bearing B15, B17, B5 and B35. Consistent lysis of B18 bearing cells was only observed with lymphoblastoid cell lines. The supernatant from the Tr2D8 (EBV line X KR4) hybridoma (2.7 micrograms IgM/ml) only lysed B15 bearing PBMC. At a concentration of 13.5 micrograms IgM/ml, the hybridoma antibody lysed lymphoblastoid cell lines bearing B15, B17, B5, B35 and B18.

  3. Effects of insulin on human pancreatic cancer progression modeled in vitro.

    Science.gov (United States)

    Chan, Michelle T; Lim, Gareth E; Skovsø, Søs; Yang, Yu Hsuan Carol; Albrecht, Tobias; Alejandro, Emilyn U; Hoesli, Corinne A; Piret, James M; Warnock, Garth L; Johnson, James D

    2014-11-06

    Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways

  4. An Alternate Splicing Variant of the Human Telomerase Catalytic Subunit Inhibits Telomerase Activity

    Directory of Open Access Journals (Sweden)

    Xiaoming Yi

    2000-09-01

    Full Text Available Telomerase, a cellular reverse transcriptase, adds telomeric repeats to chromosome ends. In normal human somatic cells, telomerase is repressed and telomeres progressively shorten, leading to proliferative senescence. Introduction of the telomerase (hTERT cDNA is sufficient to produce telomerase activity and immortalize normal human cells, suggesting that the repression of telomerase activity is transcriptional. The telomerase transcript has been shown to have at least six alternate splicing sites (four insertion sites and two deletion sites, and variants containing both or either of the deletion sites are present during development and in a panel of cancer cell lines we surveyed. One deletion (β site and all four insertions cause premature translation terminations, whereas the other deletion (α site is 36 by and lies within reverse transcriptase (RT motif A, suggesting that this deletion variant may be a candidate as a dominant-negative inhibitor of telomerase. We have cloned three alternately spliced hTERT variants that contain the α,β or both α and,β deletion sites. These alternate splicing variants along with empty vector and wild-type hTERT were introduced into normal human fibroblasts and several telomerase-positive immortal and tumor cell lines. Expression of the α site deletion variant (hTERT α− construct was confirmed by Western blotting. We found that none of the three alternate splicing variants reconstitutes telomerase activity in fibroblasts. However, hTERT α− inhibits telomerase activities in telomerase-positive cells, causes telomere shortening and eventually cell death. This alternately spliced dominant-negative variant may be important in understanding telomerase regulation during development, differentiation and in cancer progression.

  5. Growth and characterization of different human rhinovirus C types in three-dimensional human airway epithelia reconstituted in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Tapparel, Caroline, E-mail: Caroline.Tapparel@hcuge.ch [Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14 (Switzerland); Sobo, Komla [Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14 (Switzerland); Constant, Samuel; Huang, Song [Epithelix sárl, 14 Chemin des Aulx, 1228 Plan les Ouates, Geneva (Switzerland); Van Belle, Sandra; Kaiser, Laurent [Laboratory of Virology, Division of Infectious Diseases and Division of Laboratory Medicine, University of Geneva Hospitals, 4 Rue Gabrielle-Perret-Gentil, 1211 Geneva 14 (Switzerland)

    2013-11-15

    New molecular diagnostic tools have recently allowed the discovery of human rhinovirus species C (HRV-C) that may be overrepresented in children with lower respiratory tract complications. Unlike HRV-A and HRV-B, HRV-C cannot be propagated in conventional immortalized cell lines and their biological properties have been difficult to study. Recent studies have described the successful amplification of HRV-C15, HRV-C11, and HRV-C41 in sinus mucosal organ cultures and in fully differentiated human airway epithelial cells. Consistent with these studies, we report that a panel of clinical HRV-C specimens including HRV-C2, HRV-C7, HRV-C12, HRV-C15, and HRV-C29 types were all capable of mediating productive infection in reconstituted 3D human primary upper airway epithelial tissues and that the virions enter and exit preferentially through the apical surface. Similar to HRV-A and HRV-B, our data support the acid sensitivity of HRV-C. We observed also that the optimum temperature requirement during HRV-C growth may be type-dependent. - Highlights: • A 3D human upper airway epithelia reconstituted in vitro supports HRV-C growth. • HRV-Cs enter and exit preferentially at the apical side of this ALI culture system. • HRV-Cs are acid sensitive. • Temperature sensitivity may be type-dependent for HRV-Cs.

  6. Cell adhesion markers are expressed by a stable human endothelial cell line transformed by the SV40 large T antigen under vimentin promoter control.

    Science.gov (United States)

    Vicart, P; Testut, P; Schwartz, B; Llorens-Cortes, C; Perdomo, J J; Paulin, D

    1993-10-01

    Markers of endothelium have been studied in a new endothelial cell line derived from human umbilical cord vein cells by microinjection of a recombinant gene that includes a deletion mutant of the human vimentin gene regulatory region controlling the large T and small t antigen coding region of the SV40 virus. In culture, this immortalized venous endothelial cell line (IVEC) demonstrated morphological characteristics of endothelium; uptake of acetylated low density lipoprotein and presence of the Factor VIII-related antigen. Treatment of IVEC cells with Interleukin-1 beta (IL-1 beta) at 10 U.ml-1 activates the expression of cell adhesion molecules such as endothelial leucocyte adhesion molecule (ELAM-1), intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1), as observed in primary culture. Prostacyclin secretion was induced in the IVEC cells by 100 nM PMA treatment and thrombin at 0.5 U/ml. Angiotensin converting enzyme (ACE) activity detected in IVEC cells was present but lower than ACE activity in primary endothelial cells and was completely blocked by enalaprilat (1 microM), a specific ACE inhibitor. The presence of ACE mRNA was also demonstrated in IVEC cells by RT-PCR amplification. Our data demonstrate that endothelial cells immortalized by use of this recombinant gene retain the morphological organization and numerous differentiated properties of endothelium.

  7. DNA replication timing is maintained genome-wide in primary human myoblasts independent of D4Z4 contraction in FSH muscular dystrophy.

    Directory of Open Access Journals (Sweden)

    Benjamin D Pope

    Full Text Available Facioscapulohumeral muscular dystrophy (FSHD is linked to contraction of an array of tandem 3.3-kb repeats (D4Z4 at 4q35.2 from 11-100 copies to 1-10 copies. The extent to which D4Z4 contraction at 4q35.2 affects overall 4q35.2 chromatin organization remains unclear. Because DNA replication timing is highly predictive of long-range chromatin interactions, we generated genome-wide replication-timing profiles for FSHD and control myogenic precursor cells. We compared non-immortalized myoblasts from four FSHD patients and three control individuals to each other and to a variety of other human cell types. This study also represents the first genome-wide comparison of replication timing profiles in non-immortalized human cell cultures. Myoblasts from both control and FSHD individuals all shared a myoblast-specific replication profile. In contrast, male and female individuals were readily distinguished by monoallelic differences in replication timing at DXZ4 and other regions across the X chromosome affected by X inactivation. We conclude that replication timing is a robust cell-type specific feature that is unaffected by FSHD-related D4Z4 contraction.

  8. Prevalence of Telomerase Activity in Human Cancer

    Directory of Open Access Journals (Sweden)

    Chi-Hau Chen

    2011-05-01

    Full Text Available Telomerase activity has been measured in a wide variety of cancerous and non-cancerous tissue types, and the vast majority of clinical studies have shown a direct correlation between it and the presence of cancerous cells. Telomerase plays a key role in cellular immortality and tumorigenesis. Telomerase is activated in 80–90% of human carcinomas, but not in normal somatic cells, therefore, its detection holds promise as a diagnostic marker for cancer. Measurable levels of telomerase have been detected in malignant cells from various samples: tissue from gestational trophoblastic neoplasms; squamous carcinoma cells from oral rinses; lung carcinoma cells from bronchial washings; colorectal carcinoma cells from colonic luminal washings; bladder carcinoma cells from urine or bladder washings; and breast carcinoma or thyroid cancer cells from fine needle aspirations. Such clinical tests for telomerase can be useful as non-invasive and cost-effective methods for early detection and monitoring of cancer. In addition, telomerase activity has been shown to correlate with poor clinical outcome in late-stage diseases such as non-small cell lung cancer, colorectal cancer, and soft tissue sarcomas. In such cases, testing for telomerase activity can be used to identify patients with a poor prognosis and to select those who might benefit from adjuvant treatment. Our review of the latest medical advances in this field reveals that telomerase holds great promise as a biomarker for early cancer detection and monitoring, and has considerable potential as the basis for developing new anticancer therapies.

  9. Telomere--the twilight to immortality.

    Science.gov (United States)

    Shukla, Samarth; Acharya, Sourya; Rajput, Devendra; Vagha, S; Grover, Shobha

    2010-09-01

    Besides forming a very important component of the chromosome, the telomeres have extremely significant modes of action and functions, right from maintaining a basic infrastructure and integrity of the chromosome vis a vis the other chromosomes, telomeres are responsible for the cell divisions and replicative senescence of the cell. The number of mitotic divisions which a cell will go through in its life span while passing through the cell cycle is governed inturn by these telomeres, the crux of the entire functioning of these chromosomal components suggests that they are the ticking clocks of the cell and when they diminish or are worn out so does the cell reach it's senility at the fag end of it's replicative life--resulting fate being--the cell is sent to it's grave yard (the final destination). Clinical implications include--regulation of cell life spans, regulating the cell's replicative behavior and it's utility in forming cells which usually are impossible to divide or replicate, telomeres regulate the cloning process,the telomeres play a major role in predicting the fate of a neoplastic cell and finally enhancing the life span of a single cell, the organ, the body as a whole by enzymes which expand the telomeres--the telomerase.

  10. Diffused Knowledge Immortalizes Itself The LOCKSS Program

    CERN Document Server

    Reich, Victoria A

    2003-01-01

    The LOCKSS model, 'Lots of Copies Keeps Stuff Safe,' creates low-cost, persistent digital 'caches' of authoritative versions of http-delivered content. The LOCKSS software enables institutions to locally collect, store, preserve and archive authorized content, thus safeguarding their community's access to that content. The current version of LOCKSS software is restricted to electronic journals. Accuracy and completeness of LOCKSS caches are assured through a peer-to-peer polling and reputation system (operated through LCAP, LOCKSS' communication protocol), which is both robust and secure. LOCKSS replicas cooperate to detect and repair preservation failures. LOCKSS is designed to run on inexpensive hardware and to require almost no technical administration. The software has been under development since 1999 and is distributed as open source.

  11. Cellular reprogramming in pursuit of immortality.

    Science.gov (United States)

    Surani, M Azim

    2012-12-07

    The discovery that phenotypic diversity among differentiated cells results from epigenetic and not genetic differences, and can be reset to restore pluripotency, promises revolutionary advances in medicine. I discuss how this and related seminal discoveries have brought us to an exciting future. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Stem cells' exodus: a journey to immortality.

    Science.gov (United States)

    Zhou, Yi; Lewallen, Michelle; Xie, Ting

    2013-01-28

    Stem cell niches provide a regulatory microenvironment that retains stem cells and promotes self-renewal. Recently in Developmental Cell, Rinkevich et al. (2013) showed that cell islands (CIs) of Botryllus schlosseri, a colonial chordate, provide niches for maintaining cycling stem cells that migrate from degenerated CIs to newly formed buds. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Suicide, Immortality, and the Method of Embarrassments

    Science.gov (United States)

    Bakan, David

    1976-01-01

    This paper, presented at Self-Destruction and Self-Creation, at Duquesne University (April, 1969) explains that the suicide, reacting to his experience of "being in question" (Heidegger), kills himself to arrogate death to his will. He allows the possibility that if he wished it, he could live forever and usurp death. (Author)

  14. Aging and immortality in unicellular species.

    Science.gov (United States)

    Florea, Michael

    2017-08-24

    It has been historically thought that in conditions that permit growth, most unicellular species do not to age. This was particularly thought to be the case for symmetrically dividing species, as such species lack a clear distinction between the soma and the germline. Despite this, studies of the symmetrically dividing species Escherichia coli and Schizosaccharomyces pombe have recently started to challenge this notion. They indicate that E. coli and S. pombe do age, but only when subjected to environmental stress. If true, this suggests that aging may be widespread among microbial species in general, and that studying aging in microbes may inform other long-standing questions in aging. This review examines the recent evidence for and against replicative aging in symmetrically dividing unicellular organisms, the mechanisms that underlie aging, why aging evolved in these species, and how microbial aging fits into the context of other questions in aging. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Cell culture from sponges: pluripotency and immortality.

    Science.gov (United States)

    de Caralt, Sònia; Uriz, María J; Wijffels, René H

    2007-10-01

    Sponges are a source of compounds with potential pharmaceutical applications. In this article, methods of sponge cell culture for production of these bioactive compounds are reviewed, and new approaches for overcoming the problem of metabolite supply are examined. The use of embryos is proposed as a new source of sponge material for cell culture. Stem cells are present in high amounts in embryos and are more versatile and resistant to infections than adult cells. Additionally, genetic engineering and cellular research on apoptotic mechanisms are promising new fields that might help to improve cell survival in sponge-cell lines. We propose that one topic for future research should be how to reduce apoptosis, which appears to be very high in sponge cell cultures.

  16. Microteaching: From Infant Death to Immortality?

    Science.gov (United States)

    Davis, Brian K.

    A general introduction to the concept of microteaching and its development is presented, and the generally accepted format and the skills practiced for microteaching are described. Aspects of microteaching commonly perceived as favorable and unfavorable are addressed, and a review of current research is provided and followed by a discussion of the…

  17. The search of a digital immortality?

    Directory of Open Access Journals (Sweden)

    Carla Landuzzi

    2015-12-01

    Full Text Available The text intends to examine how technological innovations allow the construction of web spaces for shared funeral rituals and their partecipation by those same communities that move across the network. It can be assumed a new resacralization process of rituals referring to an individual system of meanings. The web may be capable of reappropriating the experience of pain through new ways that allow flexible access and fruition without spatial or temporal limits. They are interactive rituals that build an elective sociality, as practiced by the inclusion decisions under emotionally meaningful groups. The search for a digital alter ego that will survive the earthly physicality threatens to tarnish the very identity of the dead.

  18. Taiji Ruler: Legacy of the Sleeping Immortal

    Directory of Open Access Journals (Sweden)

    Kenneth S. Cohen

    2012-07-01

    Full Text Available This article describes the history, development, and principles of the two major Taiji Ruler lineages: one associated with the Song Dynasty (960-1279 CE imperial family, and the other lesser known, Daoist lineage, transmitted by Hu Yaozhen and his successors, including the well known Chen Style Taijiquan teacher, Feng Zhiqiang. The Taiji Ruler is a traditional system of health-enhancing qigong that is attributed to the Song Dynasty Daoist recluse Chen Xiyi and was first taught publicly in the 1950s. The exercises may be practiced while holding a foot-long wooden object, the Ruler, or with a variety of training devices, such as a wooden or stone ball. Mr. Cohen began studying Taji Ruler with various teachers more than 30 years ago, but also bases his research on works in Chinese and English. Both the teachers and literature agree that the ultimate goal of the Ruler is to blend with the original qi of the universe and, in the process, to achieve vitality and longevity.    

  19. Proteome alteration induced by hTERT transfection of human fibroblast cells

    Directory of Open Access Journals (Sweden)

    Riou Jean-François

    2008-04-01

    Full Text Available Abstract Background Telomerase confers cellular immortality by elongating telomeres, thereby circumventing the Hayflick limit. Extended-life-span cells have been generated by transfection with the human telomerase reverse transcriptase (hTERT gene. hTERT transfected cell lines may be of outstanding interest to monitor the effect of drugs targeting the telomerase activity. The incidence of hTERT gene transfection at the proteome level is a prerequisite to that purpose. The effect of the transfection has been studied on the proteome of human fibroblast (WI38. Cytosolic and nuclear fractions of WI38 cells, empty vector transfected WI38 (WI38-HPV and hTERT WI38 cells were submitted to a 2D-DIGE (Two-Dimensional Differential In-Gel Electrophoresis analysis. Only spots that had a similar abundance in WI38 and WI38-HPV, but were differentially expressed in WI38 hTERT were selected for MS identification. This method directly points to the proteins linked with the hTERT expression. Number of false positive differentially expressed proteins has been excluded by using control WI38-HPV cells. The proteome alteration induced by hTERT WI38 transfection should be taken into account in subsequent use of the cell line for anti-telomerase drugs evaluation. Results 2D-DIGE experiment shows that 57 spots out of 2246 are significantly differentially expressed in the cytosolic fraction due to hTERT transfection, and 38 were confidently identified. In the nuclear fraction, 44 spots out of 2172 were selected in the differential proteome analysis, and 14 were identified. The results show that, in addition to elongating telomeres, hTERT gene transfection has other physiological roles, among which an enhanced ER capacity and a potent cell protection against apoptosis. Conclusion We show that the methodology reduces the complexity of the proteome analysis and highlights proteins implicated in other processes than telomere elongation. hTERT induced proteome changes suggest

  20. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    Science.gov (United States)

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (pphospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes.

  1. Human microvascular endothelial cell toxicity caused by Brazilian purpuric fever-associated strains of Haemophilus influenzae biogroup aegyptius.

    Science.gov (United States)

    Weyant, R S; Quinn, F D; Utt, E A; Worley, M; George, V G; Candal, F J; Ades, E W

    1994-02-01

    An in vitro cytotoxicity model that uses an immortalized human microvascular endothelial cell line (HMEC-1) differentiates Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius (HAE) strains from non-BPF-associated HAE strains. Toxic strains produced a characteristic HMEC-1 phenotype at an MOI of 1000 bacteria/TCC to produce an observable effect. The cytotoxic phenotype was characterized by the presence of large clumps of HMEC-1 cells, which detached from the monolayer within 48 h of inoculation by HAE cells. The cytotoxic phenotype was observed with 100% of BPF-associated HAE (40/40) and 14% of non-BPF-associated HAE (8/57; P < .001). The ability to study a BPF-associated phenotype in vitro using human microvascular cells should enhance our knowledge of BPF pathogenesis.

  2. Adaptive regulation of taurine and beta-alanine uptake in a human kidney cell line from the proximal tubule

    DEFF Research Database (Denmark)

    Jessen, H; Jacobsen, Christian

    1997-01-01

    homeostasis occurs predominantly via changes in the activity of the high-affinity taurine transport system by alterations in the uptake capacity and with an unaffected half-saturation constant. An adaptive response was not observed for the structurally related beta-alanine. 3. Only colchicine, which......), mimicking the effects of diacylglycerol, induced inhibition of both beta-alanine and taurine uptake. By contrast, the Ca2(+)-ionophore A23187, mimicking the effects of IP3, only stimulated the uptake of taurine but not the influx of beta-alanine. However, the effect of PMA down-regulation and A23187 up......1. The underlying mechanisms involved in the adaptive regulation of beta-amino acid uptake in the human proximal tubule were examined by use of an immortalized human embryonic kidney epithelial cell line (IHKE). 2. The results indicated that the adaptive response to maintain whole-body taurine...

  3. A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.

    Directory of Open Access Journals (Sweden)

    Hadassah Sade

    Full Text Available We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3, to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.

  4. Uptake of neutral alpha- and beta-amino acids by human proximal tubular cells

    DEFF Research Database (Denmark)

    Jessen, H; Røigaard, H; Jacobsen, Christian

    1996-01-01

    The transport characteristics of amino acids in primary cell cultures from the proximal tubule of human adults (AHKE cells) were examined, using alpha-aminoisobutyric acid (AIB) and beta-alanine as representatives of alpha- and beta-amino acids, respectively. The Na(+)-gradient dependent influx...... experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, L-aspartic acid, and L-arginine. By contrast, the influx of beta-alanine was only drastically reduced by beta-amino acids, whereas the inhibition by neutral alpha-amino acids...... was relatively low. Nor did L-arginine and L-aspartic acid affect the uptake of beta-alanine into AHKE cells. Comparison with the results obtained for normal (NHKE) and immortalized (IHKE) embryonic cells suggested an unaltered expression of the types of transport carriers for neutral alpha- and beta-amino acids...

  5. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    Science.gov (United States)

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses.

  6. Detection of telomerase activity in malignant neoplasms and nonmalignantepithelial tissues of human esophagus

    Institute of Scientific and Technical Information of China (English)

    Shah Min Yang; Tian Jiao Wang; Bao Yu Li; Yuan Huan Wu

    2000-01-01

    AIM To study the expression of telomerase activity in malignant esophageal neoplasms and normal humanesophageal epithelia.METHODS Telomerase activity was assayed by the telomere repeat amplification protocol (TRAP)method. All the neoplasms and epithelia of esophagus were confirmed by routine pathological diagnosis.RESULTS Telomerase activity was assayed in 18 normal esophageal epithelial tissues and in 35 malignantneoplasms of esophagus, including 27 cases of esophageal carcinoma and 8 cases of cardiac carcinoma.Telomerase activity was detected in most of malignant neoplasms of esophagus (91.4%, 32/35) and in allthe normal esophageal epithelial tissues except one (18/19).CONCLUSION The results suggest that in addition to contributing to proliferation of immortal blast cellsand neoplastic cells, telomerase activity may also play a similar role in regeneration of normal epithelia ofhuman esophagus. The potential use of telomerase activity as a diagnostic marker in human esophagealneoplasm might not be suitable.

  7. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity Using Human Keratinocyte Cells

    Science.gov (United States)

    Shvedova, Anna A.; Castranova, Vincent; Kisin, Elena R.; Schwegler-Berry, Diane; Murray, Ashley R.; Gandelsman, Vadim Z.; Maynard, Andrew; Baron, Paul

    2003-01-01

    Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

  8. Tissuelike 3D Assemblies of Human Broncho-Epithelial Cells

    Science.gov (United States)

    Goodwin, Thomas J.

    2010-01-01

    Three-dimensional (3D) tissuelike assemblies (TLAs) of human broncho-epithelial (HBE) cells have been developed for use in in vitro research on infection of humans by respiratory viruses. The 2D monolayer HBE cell cultures heretofore used in such research lack the complex cell structures and interactions characteristic of in vivo tissues and, consequently, do not adequately emulate the infection dynamics of in-vivo microbial adhesion and invasion. In contrast, the 3D HBE TLAs are characterized by more-realistic reproductions of the geometrical and functional complexity, differentiation of cells, cell-to-cell interactions, and cell-to-matrix interactions characteristic of human respiratory epithelia. Hence, the 3D HBE TLAs are expected to make it possible to perform at least some of the research in vitro under more-realistic conditions, without need to infect human subjects. The TLAs are grown on collagen-coated cyclodextran microbeads under controlled conditions in a nutrient liquid in the simulated microgravitational environment of a bioreactor of the rotating- wall-vessel type. Primary human mesenchymal bronchial-tracheal cells are used as a foundation matrix, while adult human bronchial epithelial immortalized cells are used as the overlying component. The beads become coated with cells, and cells on adjacent beads coalesce into 3D masses. The resulting TLAs have been found to share significant characteristics with in vivo human respiratory epithelia including polarization, tight junctions, desmosomes, and microvilli. The differentiation of the cells in these TLAs into tissues functionally similar to in vivo tissues is confirmed by the presence of compounds, including villin, keratins, and specific lung epithelium marker compounds, and by the production of tissue mucin. In a series of initial infection tests, TLA cultures were inoculated with human respiratory syncytial viruses and parainfluenza type 3 viruses. Infection was confirmed by photomicrographs that

  9. Transcriptional profiling of human brain endothelial cells reveals key properties crucial for predictive in vitro blood-brain barrier models.

    Directory of Open Access Journals (Sweden)

    Eduard Urich

    Full Text Available Brain microvascular endothelial cells (BEC constitute the blood-brain barrier (BBB which forms a dynamic interface between the blood and the central nervous system (CNS. This highly specialized interface restricts paracellular diffusion of fluids and solutes including chemicals, toxins and drugs from entering the brain. In this study we compared the transcriptome profiles of the human immortalized brain endothelial cell line hCMEC/D3 and human primary BEC. We identified transcriptional differences in immune response genes which are directly related to the immortalization procedure of the hCMEC/D3 cells. Interestingly, astrocytic co-culturing reduced cell adhesion and migration molecules in both BECs, which possibly could be related to regulation of immune surveillance of the CNS controlled by astrocytic cells within the neurovascular unit. By matching the transcriptome data from these two cell lines with published transcriptional data from freshly isolated mouse BECs, we discovered striking differences that could explain some of the limitations of using cultured BECs to study BBB properties. Key protein classes such as tight junction proteins, transporters and cell surface receptors show differing expression profiles. For example, the claudin-5, occludin and JAM2 expression is dramatically reduced in the two human BEC lines, which likely explains their low transcellular electric resistance and paracellular leakiness. In addition, the human BEC lines express low levels of unique brain endothelial transporters such as Glut1 and Pgp. Cell surface receptors such as LRP1, RAGE and the insulin receptor that are involved in receptor-mediated transport are also expressed at very low levels. Taken together, these data illustrate that BECs lose their unique protein expression pattern outside of their native environment and display a more generic endothelial cell phenotype. A collection of key genes that seems to be highly regulated by the local

  10. Differential transforming activity of the retroviral Tax oncoproteins in human T lymphocytes

    Directory of Open Access Journals (Sweden)

    Tong eRen

    2013-09-01

    Full Text Available 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.

  11. Establishment and characterization of human engineered cells stably expressing large extracellular matrix proteins.

    Science.gov (United States)

    Kwon, Daekee; Kang, Gwang-Sik; Han, Dong Keun; Park, Kwideok; Kim, Jae-Hwan; Lee, Soo-Hong

    2014-01-01

    Commercially available extracellular matrix (ECM) hydrogel-coated culture plates have been used to study the relationship between the ECM microenvironment and stem cell behavior. However, it is unclear whether ECM-coated dishes mimic the natural ECM microenvironment because the architecture of the ECM is constructed of randomly distributed fibers. The purpose of this study was the production and confirmation of human engineered cell lines stably expressing large ECM proteins such as collagen I/II and fibronectin. First, large (over 10 kb) ECM vectors encoding human collagen I/II and fibronectin were constructed and the circular vectors were linearized. Second, the linear ECM vectors were introduced into immortalized human embryonic kidney cells using various transfection methods. The polyethylenimine and liposome methods showed higher efficiencies than electroporation for transfection of these large vectors. Third, human ECM engineered cells were established by stable integration of the vector into the genomic DNA and resulted in stable overexpression of mRNA and proteins. In summary, human engineered cell lines stably expressing large ECM proteins such as human collagen I/II and fibronectin were successfully prepared, and secretion of the ECM components into the surrounding environment was confirmed by immunocytochemistry. Thus, human ECM engineered cells naturally secreting ECM components could be valuable for studying the relationship between the native ECM microenvironment and stem cell behavior.

  12. Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Yira Bermudez

    Full Text Available BACKGROUND: Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. RESULTS: Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibrobl