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Sample records for h9c2 cell line

  1. Cardiomyocyte H9c2 cells present a valuable alternative to fish lethal testing for azoxystrobin.

    PubMed

    Rodrigues, Elsa T; Pardal, Miguel Â; Laizé, Vincent; Cancela, M Leonor; Oliveira, Paulo J; Serafim, Teresa L

    2015-11-01

    The present study aims at identifying, among six mammalian and fish cell lines, a sensitive cell line whose in vitro median inhibitory concentration (IC50) better matches the in vivo short-term Sparus aurata median lethal concentration (LC50). IC50s and LC50 were assessed after exposure to the widely used fungicide azoxystrobin (AZX). Statistical results were relevant for most cell lines after 48 h of AZX exposure, being H9c2 the most sensitive cells, as well as the ones which provided the best prediction of fish toxicity, with a LC50,96h/IC50,48h = 0.581. H9c2 cell proliferation upon 72 h of AZX exposure revealed a LC50,96h/IC50,72h = 0.998. Therefore, identical absolute sensitivities were attained for both in vitro and in vivo assays. To conclude, the H9c2 cell-based assay is reliable and represents a suitable ethical alternative to conventional fish assays for AZX, and could be used to get valuable insights into the toxic effects of other pesticides. PMID:26319055

  2. PPARd signaling mediates the cytotoxicity of DHA in H9c2 cells Victor Samokhvalov a,1

    E-print Network

    Hammock, Bruce D.

    PPARd signaling mediates the cytotoxicity of DHA in H9c2 cells Victor Samokhvalov a,1 , Igor T S Investigated the mechanisms of DHA mediated cytotoxicity in H9c2 cardiac cells. DHA treatment decreased cell of this study was to examine the involvement of PPARd in DHA-mediated cytotoxicity toward H9c2 cells. Treatment

  3. Overexpression of MIP2, a novel WD-repeat protein, promotes proliferation of H9c2 cells

    SciTech Connect

    Wei, Xing; Institute of Cardiovascular Disease, Department of Pathophysiology, School of Medicine, University of South China, 28 Changsheng Xi Road, Hengyang, Hunan 421001 ; Song, Lan; Jiang, Lei; Wang, Guiliang; Luo, Xinjing; Zhang, Bin; Xiao, Xianzhong

    2010-03-19

    WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the role of MIP2 in regulating cellular proliferation. Transfection and constitutive expression of MIP2 in the rat cardiomyoblast cell line H9c2 results in enhanced growth of those cells as measured by cell number and is proportional to the amount of MIP2 expressed. Overexpression of MIP2 results in a shorter cell cycle, as measured by flow cytometry. Collectively, these data suggest that MIP2 may participate in the progression of cell proliferation in H9c2 cells.

  4. Cytoprotective effect of rhamnetin on miconazole-induced H9c2 cell damage

    PubMed Central

    Lee, Kang Pa; Kim, Jai-Eun

    2015-01-01

    BACKGROUND/OBJECTIVES Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis. MATERIALS/METHODS Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount™ cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate (H2DCF-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction. RESULTS Miconazole (3 and 10 µM) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole (3 µM)-treated cells in a dose-dependent manner. Rhamnetin (1 and 3 µM) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation. CONCLUSIONS Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels. PMID:26634046

  5. H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation

    PubMed Central

    Kuznetsov, Andrey V.; Javadov, Sabzali; Sickinger, Stephan; Frotschnig, Sandra; Grimm, Michael

    2015-01-01

    Dysfunction of cardiac energy metabolism plays a critical role in many cardiac diseases, including heart failure, myocardial infarction and ischemia–reperfusion injury and organ transplantation. The characteristics of these diseases can be elucidated in vivo, though animal-free in vitro experiments, with primary adult or neonatal cardiomyocytes, the rat ventricular H9c2 cell line or the mouse atrial HL-1 cells, providing intriguing experimental alternatives. Currently, it is not clear how H9c2 and HL-1 cells mimic the responses of primary cardiomyocytes to hypoxia and oxidative stress. In the present study, we show that H9c2 cells are more similar to primary cardiomyocytes than HL-1 cells with regard to energy metabolism patterns, such as cellular ATP levels, bioenergetics, metabolism, function and morphology of mitochondria. In contrast to HL-1, H9c2 cells possess beta-tubulin II, a mitochondrial isoform of tubulin that plays an important role in mitochondrial function and regulation. We demonstrate that H9c2 cells are significantly more sensitive to hypoxia-reoxygenation injury in terms of loss of cell viability and mitochondrial respiration, whereas HL-1 cells were more resistant to hypoxia as evidenced by their relative stability. In comparison to HL-1 cells, H9c2 cells exhibit a higher phosphorylation (activation) state of AMP-activated protein kinase, but lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha levels, suggesting that each cell type is characterized by distinct regulation of mitochondrial biogenesis. Our results provide evidence that H9c2 cardiomyoblasts are more energetically similar to primary cardiomyocytes than are atrial HL-1 cells. H9c2 cells can be successfully used as an in vitro model to simulate cardiac ischemia–reperfusion injury. PMID:25450968

  6. H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation.

    PubMed

    Kuznetsov, Andrey V; Javadov, Sabzali; Sickinger, Stephan; Frotschnig, Sandra; Grimm, Michael

    2015-02-01

    Dysfunction of cardiac energy metabolism plays a critical role in many cardiac diseases, including heart failure, myocardial infarction and ischemia-reperfusion injury and organ transplantation. The characteristics of these diseases can be elucidated in vivo, though animal-free in vitro experiments, with primary adult or neonatal cardiomyocytes, the rat ventricular H9c2 cell line or the mouse atrial HL-1 cells, providing intriguing experimental alternatives. Currently, it is not clear how H9c2 and HL-1 cells mimic the responses of primary cardiomyocytes to hypoxia and oxidative stress. In the present study, we show that H9c2 cells are more similar to primary cardiomyocytes than HL-1 cells with regard to energy metabolism patterns, such as cellular ATP levels, bioenergetics, metabolism, function and morphology of mitochondria. In contrast to HL-1, H9c2 cells possess beta-tubulin II, a mitochondrial isoform of tubulin that plays an important role in mitochondrial function and regulation. We demonstrate that H9c2 cells are significantly more sensitive to hypoxia-reoxygenation injury in terms of loss of cell viability and mitochondrial respiration, whereas HL-1 cells were more resistant to hypoxia as evidenced by their relative stability. In comparison to HL-1 cells, H9c2 cells exhibit a higher phosphorylation (activation) state of AMP-activated protein kinase, but lower peroxisome proliferator-activated receptor gamma coactivator 1-alpha levels, suggesting that each cell type is characterized by distinct regulation of mitochondrial biogenesis. Our results provide evidence that H9c2 cardiomyoblasts are more energetically similar to primary cardiomyocytes than are atrial HL-1 cells. H9c2 cells can be successfully used as an in vitro model to simulate cardiac ischemia-reperfusion injury. PMID:25450968

  7. Three pentacyclic triterpenes protect H9c2 cardiomyoblast cells against high-glucose-induced injury.

    PubMed

    Chan, C Y; Mong, M C; Liu, W H; Huang, C Y; Yin, M C

    2014-04-01

    H9c2 cardiomyoblast cell line was used to examine the protection of three triterpenes, asiatic acid, boswellic acid, and oleanolic acid, at 5 or 10 ?M against high-glucose-induced injury. High glucose stimulated reactive oxygen species (ROS), oxidized glutathione (GSSG), interleukin-6, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1 production, as well as decreased glutathione peroxidase (GPX), glutathione reductase (GR) and catalase activities, and protein expression. However, pre-treatments of three triterpenes reserved glutathione, maintained activity and expression of GPX, GR, and catalase, as well as lowered ROS, GSSG, and inflammatory cytokines generation. High glucose reduced Na(+)-K(+)-ATPase activity, raised nuclear factor kappa (NF-?) B and caspase-3 activities, up-regulated protein expression of NF-?B, mitogen-activated protein kinase, Bax, and cleaved caspase-3, as well as down-regulated Bcl-2 expression. Pre-treatments of three triterpenes retained Na(+)-K(+)-ATPase activity, declined NF-?B and caspase-3 activities, reserved Bcl-2 expression, as well as suppressed protein expression of NF-?B, p-p38, Bax, and cleaved caspase-3. These findings suggest that these triterpenes are potent cardiac-protective agents. PMID:24393047

  8. Effect of Alcohol Administration on Mg2+ Homeostasis in H9C2 Cells

    PubMed Central

    Nguyen, Huy; Romani, Andrea

    2015-01-01

    Alcoholic cardiomyopathy represents one of the main clinical complications in chronic alcoholics. This pathology contrasts the seemingly beneficial effect of small doses of alcohol on the cardiovascular system. Studies carried out in liver cells exposed acutely or chronically to varying doses of EtOH indicate that intrahepatic alcohol metabolism results in a major loss of cellular Mg2+. To investigate whether EtOH administration also induced Mg2+ extrusion in cardiac cells, H9C2 cells were exposed to varying doses of EtOH for short- or ling-term periods of time. The results indicate that H9C2 cells exposed to EtOH doses higher than 0.1% (v/v, or 15 mM) extruded Mg2+ into the extracellular medium on a time- and dose-dependent manner. Consistent with the involvement of cyP4502E1 in metabolizing EtOH, administration of chloro-methiazole (CMZ) as an inhibitor of the cytochrome prevented EtOH-induced Mg2+ loss to a large extent. EtOH-induced Mg2+ extrusion was also prevented by the administration of di-thio-treitol (DTT) and n-acetyl-cysteine (NAC), two agents that prevent the negative effects of ROS formation and free radicals generation associated with EtOH metabolism by cyP4502E1. Taken together, our data indicate that Mg2+ extrusion also occur in cardiac cells exposed to EtOH as a result of alcohol metabolism by cyP4502E1 and associated free radical formation. Interestingly, Mg2+ extrusion only occurs at doses of EtOH higher than 0.1% administered for an extended period of time. The significance of Mg2+ extrusion for the onset of alcoholic cardiomyopathy remains to be elucidated. PMID:25793216

  9. Autophagy plays an important role in sunitinib-mediated cell death in H9c2 cardiac muscle cells.

    PubMed

    Zhao, Yuqin; Xue, Tao; Yang, Xiaochun; Zhu, Hong; Ding, Xiaofei; Lou, Liming; Lu, Wei; Yang, Bo; He, Qiaojun

    2010-10-01

    Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity. PMID:20637791

  10. Autophagy plays an important role in Sunitinib-mediated cell death in H9c2 cardiac muscle cells

    SciTech Connect

    Zhao Yuqin; Xue Tao; Yang Xiaochun; Zhu Hong; Ding Xiaofei; Lou Liming; Lu Wei; Yang Bo; He Qiaojun

    2010-10-01

    Sunitinib, which is a multitargeted tyrosine-kinase inhibitor, exhibits antiangiogenic and antitumor activity, and extends survival of patients with metastatic renal-cell carcinoma (mRCC) and gastrointestinal stromal tumors (GIST). This molecule has also been reported to be associated with cardiotoxicity at a high frequency, but the mechanism is still unknown. In the present study, we observed that Sunitinib showed high anti-proliferative effect on H9c2 cardiac muscle cells measured by PI staining and the MTT assay. But apoptotic markers (PARP cleavage, caspase 3 cleavage and chromatin condensation) were uniformly negative in H9c2 cells after Sunitinib treatment for 48 h, indicating that another cell death pathway may be involved in Sunitinib-induced cardiotoxicity. Here we found Sunitinib dramatically increased autophagic flux in H9c2 cells. Acidic vesicle fluorescence and high expression of LC3-II in H9c2 cells identified autophagy as a Sunitinib-induced process that might be associated with cytotoxicity. Furthermore, knocking down Beclin 1 by RNA-interference to block autophagy in H9c2 cells revealed that the death rate was decreased when treated with Sunitinib in comparison to control cells. These results confirmed that autophagy plays an important role in Sunitinib-mediated H9c2 cells cytotoxicity. Taken together, the data presented here strongly suggest that autophagy is associated with Sunitinib-induced cardiotoxicity, and that inhibition of autophagy constitutes a viable strategy for reducing Sunitinib-induced cardiomyocyte death thereby alleviating Sunitinib cardiotoxicity.

  11. FGF2 Prevents Sunitinib-Induced Cardiotoxicity in Zebrafish and Cardiomyoblast H9c2 Cells.

    PubMed

    Cui, Guozhen; Chen, Huanxian; Cui, Wei; Guo, Xiaogang; Fang, Jiansong; Liu, Ailin; Chen, Yonglong; Lee, Simon Ming Yuen

    2016-01-01

    Sunitinib is used extensively in the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. However, the undesirable cardiotoxic effects of sunitinib, such as congestive heart failure and hypertension, limit its use in the clinical setting. As multiple receptor tyrosine kinases are inhibited by sunitinib, it raises a question as to which target mediates sunitinib-induced cardiotoxicity. Here, we reported that the injection of fibroblast growth factor 2 (FGF2) mRNA into one- to two-cell stage embryos protected against sunitinib-induced cardiotoxicity in zebrafish. In addition, FGF2 significantly prevented sunitinib-induced cardiotoxicity in cardiomyoblast H9c2 cells, possibly via activating the PLC-?/c-Raf/CREB pathway. Importantly, FGF2 did not compromise the antitumor activity of sunitinib in Caki-1 and OS-RC-2 renal cell carcinoma cells. Molecular docking simulations further revealed an interaction between the tyrosine kinase domain of FGF receptor 1 (FGFR1) and sunitinib. Taken together, our results clearly demonstrated that FGF2 inhibition plays an important role in sunitinib-induced cardiotoxicity both in vitro and in vivo. This study also provided a basis for further research on sunitinib-induced cardiotoxicity and may allow rational design of new sunitinib derivatives with fewer or weak cardiotoxic effects. PMID:25701259

  12. Curcumin potentiates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells through generation of reactive oxygen species.

    PubMed

    Hosseinzadeh, Leila; Behravan, Javad; Mosaffa, Fatemeh; Bahrami, Gholamreza; Bahrami, Ahmadreza; Karimi, Gholamreza

    2011-05-01

    Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin, a natural compound extracted from Curcuma Longa L., is known for its anti-oxidant properties. It has been identified as increased apoptosis in several cancer cell lines in combination with doxorubicin, but there are few studies about the effect of curcumin and doxorubicin on normal cardiac cells. Therefore, we evaluated the effects of curcumin on apoptosis induced by DOX in cardiac muscle cells. Pretreatment with curcumin significantly increased DOX-induced apoptosis of cardiac muscle cells through down regulation of Bcl-2, up-regulation of caspase-8 and caspase-9. The Bax/Bcl-2 ratio increased significantly after 1h pretreatment with curcumin. As well, curcumin increases ROS generation by DOX. In response to DOX, NF-?B was activated. However, curcumin was able to inhibit NF-?B activation. In conclusion, our results indicated that pretreatment with nontoxic concentrations of curcumin sensitized H9c2 cells to DOX-mediated apoptosis by generation of ROS. PMID:21295102

  13. Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells.

    PubMed

    Hahn, Nynke E; Musters, René J P; Fritz, Jan M; Pagano, Patrick J; Vonk, Alexander B A; Paulus, Walter J; van Rossum, Albert C; Meischl, Christof; Niessen, Hans W M; Krijnen, Paul A J

    2014-09-01

    Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 ?M PE to induce hypertrophy after 24 and 48h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells. PMID:24794531

  14. Early NADPH oxidase-2 activation is crucial in phenylephrine-induced hypertrophy of H9c2 cells

    PubMed Central

    Hahn, Nynke E.; Musters, René J.P.; Fritz, Jan M.; Pagano, Patrick J.; Vonk, Alexander B.A.; Paulus, Walter J.; van Rossum, Albert C.; Meischl, Christof; Niessen, Hans W.M.; Krijnen, Paul A.J.

    2015-01-01

    Reactive oxygen species (ROS) produced by different NADPH oxidases (NOX) play a role in cardiomyocyte hypertrophy induced by different stimuli, such as angiotensin II and pressure overload. However, the role of the specific NOX isoforms in phenylephrine (PE)-induced cardiomyocyte hypertrophy is unknown. Therefore we aimed to determine the involvement of the NOX isoforms NOX1, NOX2 and NOX4 in PE-induced cardiomyocyte hypertrophy. Hereto rat neonatal cardiomyoblasts (H9c2 cells) were incubated with 100 ?M PE to induce hypertrophy after 24 and 48 h as determined via cell and nuclear size measurements using digital imaging microscopy, electron microscopy and an automated cell counter. Digital-imaging microscopy further revealed that in contrast to NOX1 and NOX4, NOX2 expression increased significantly up to 4 h after PE stimulation, coinciding and co-localizing with ROS production in the cytoplasm as well as the nucleus. Furthermore, inhibition of NOX-mediated ROS production with apocynin, diphenylene iodonium (DPI) or NOX2 docking sequence (Nox2ds)-tat peptide during these first 4 h of PE stimulation significantly inhibited PE-induced hypertrophy of H9c2 cells, both after 24 and 48 h of PE stimulation. These data show that early NOX2-mediated ROS production is crucial in PE-induced hypertrophy of H9c2 cells. PMID:24794531

  15. Tribulus terrestris (Linn.) Attenuates Cellular Alterations Induced by Ischemia in H9c2 Cells Via Antioxidant Potential.

    PubMed

    Reshma, P L; Lekshmi, V S; Sankar, Vandana; Raghu, K G

    2015-06-01

    Tribulus terrestris L. was evaluated for its cardioprotective property against myocardial ischemia in a cell line model. Initially, methanolic extract was prepared and subjected to sequential extraction with various solvents. The extract with high phenolic content (T. terrestris L. ethyl acetate extract-TTME) was further characterized for its chemical constituents and taken forward for evaluation against cardiac ischemia. HPLC analysis revealed the presence of phenolic compounds like caffeic acid (12.41?±?0.22?mg?g(-1)), chlorogenic acid (0.52?±?0.06?mg?g(-1)) and 4-hydroxybenzoic acid (0.60?±?0.08?mg?g(-1)). H9c2 cells were pretreated with TTME (10, 25, 50 and 100?µg/ml) for 24?h before the induction of ischemia. Then ischemia was induced by exposing cells to ischemia buffer, in a hypoxic chamber, maintained at 0.1% O2, 95% N2 and 5% CO2, for 1?h. A significant (p???0.05) increase in reactive oxygen species generation (56%), superoxide production (18%), loss of plasma membrane integrity, dissipation of transmembrane potential, permeability transition pore opening and apoptosis had been observed during ischemia. However, pretreatment with TTME was found to significantly (p???0.05) attenuate the alterations caused by ischemia. The overall results of this study partially reveal the scientific basis of the use of T. terrestris L. in the traditional system of medicine for heart diseases. PMID:25858861

  16. 3,3'-Diindolylmethane attenuates cardiac H9c2 cell hypertrophy through 5'-adenosine monophosphate-activated protein kinase-?.

    PubMed

    Zong, Jing; Wu, Qing-Qing; Zhou, Heng; Zhang, Jie-Yu; Yuan, Yuan; Bian, Zhou-Yan; Deng, Wei; Dai, Jia; Li, Fang-Fang; Xu, Man; Fang, Yi; Tang, Qi-Zhu

    2015-07-01

    3,3'-Diindolylmethane (DIM) is the major product of the acid-catalyzed condensation of indole-3-carbinol (I3C), a component of extracts of Brassica food plants. Numerous studies have suggested that DIM has several beneficial biological activities, including elimination of free radicals, antioxidant and anti-angiogenic effects and activation of apoptosis of various tumor cells. In the present study, an in vitro model was established, using 1 µM angiotensin II (Ang II) in cultured rat cardiac H9c2 cells, to observe the effects of DIM on cardiac hypertrophy. Following 24 h stimulation with DIM (1, 5, and 10 µM) with or without Ang II, cells were characterized by immuno?uorescence to analyze cardiac ?-actinin expression. Cardiomyocyte hypertrophy and molecular markers of cardiac hypertrophy were assessed by quantitative polymerase chain reaction. Atrial natriuretic peptide, brain natriuretic peptide and myosin heavy chain ? mRNA expression were induced by Ang II in H9c2 cells treated with the optimal concentration of DIM for 6, 12, and 24 h. The levels of phosphorylated and total proteins of the 5' AMP-activated protein kinase ? (AMPK?)/mitogen-activated protein kinase (MAPK)/mechanistic target of rapamycin (mTOR) signaling pathways in H9c2 cells treated with DIM for 0, 15, 30, and 60 min induced by Ang II were determined by western blot analysis. The results showed that DIM attenuated cellular hypertrophy in vitro, enhanced the phosphorylation of AMPK? and inhibited the MAPK?mTOR signaling pathway in response to hypertrophic stimuli. PMID:25816057

  17. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

    PubMed

    Jin, Hyou-Ju; Xie, Xiao-Liang; Ye, Ji-Ming; Li, Chun-Guang

    2013-01-01

    Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA) and cryptotanshinone (CT) on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 ??) 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1? expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak). TIIA and CT (0.3 and 3 ??), in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins. PMID:23341883

  18. ZNF667/Mipu1 Is a Novel Anti-Apoptotic Factor That Directly Regulates the Expression of the Rat Bax Gene in H9c2 Cells

    PubMed Central

    Jiang, Lei; Wang, Hao; Shi, Chunli; Liu, Ke; Liu, Meidong; Wang, Nian; Wang, Kangkai; Zhang, Huali; Wang, Guiliang; Xiao, Xianzhong

    2014-01-01

    ZNF667/Mipu1, a C2H2-type zinc finger transcription factor, was suggested to play an important role in oxidative stress. However, none of the target genes or potential roles of ZNF667 in cardiomyocytes have been elucidated. Here, we investigated the functional role of ZNF667 in H9c2 cell lines focusing on its molecular mechanism by which it protects the cells from apoptosis. We found that ZNF667 inhibited the expression and the promoter activity of the rat proapoptotic gene Bax gene, and at the same time prevented apoptosis of H9c2 cells, induced by H2O2 and Dox. Western immunoblotting analysis revealed that ZNF667 also inhibited Bax protein expression, accompanied by attenuation of the mitochondrial translocation of Bax protein, induced by H2O2. EMSA and target detection assay showed that the purified ZNF667 fusion proteins could interact with the Bax promoter sequence in vitro, and this interaction was dependent upon the ZNF667 DNA binding sequences or its core sequence in the promoter. Furthermore, ChIP assay demonstrated that a stimulus H2O2 could enhance the ability of ZNF667 protein binding to the promoter. Finally, a reporter gene assay showed that ZNF667 could repress the activity of the Bax gene promoter, and the repression was dependent upon its binding to the specific DNA sequence in the promoter. Our work demonstrates that ZNF667 that confers cytoprotection is a novel regulator of the rat Bax gene, mediating the inhibition of the Bax mRNA and protein expression in H9c2 cardiomyocytes in response to H2O2 treatment. PMID:25397408

  19. Alcohol exposure increases the expression of cardiac transcription factors through ERK1/2-mediated histone3 hyperacetylation in H9c2 cells.

    PubMed

    Gao, Wenqun; Pan, Bo; Liu, Lingjuan; Huang, Xupei; Liu, Zhenguo; Tian, Jie

    2015-10-30

    Alcohol abuse during pregnancy may cause fetal cardiac developmental abnormalities. Our previous studies showed that alcohol could induce histone hyperacetylation and over-expression of cardiac transcription factors both in vivo and in vitro. The objective of the present study was to investigate the role of ERK1/2 signaling pathway in alcohol-induced histone hyperacetylation and up-regulation of cardiac transcription factors in H9c2 cells. The Cardiac cell line H9c2 was cultured with alcohol. U0126, a specific inhibitor of ERK1/2 pathway was employed to block the ERK1/2 signaling pathway. Western blotting analysis showed that alcohol significantly enhanced the levels of phosphorylated ERK1/2 and induced hyperacetylation of histone3, which were both effectively prevented with U0126. Real-time PCR showed that U0126 treatment significantly decreased alcohol-induced over-expression of GATA4 and MEF2c, and the basal expression level of GATA4, but did not affect MEF2c. ChIP assay showed that U0126 treatment significantly decreased alcohol-induced hyperacetylation of histone3 near the promoter regions of GATA4 and MEF2c. The basal acetylation level of histone3 near the promoter region of GATA4 was affected by U0126 as well, but not that near the promoter region of MEF2c. These data indicated that ERK1/2 signaling played an important role in mediating alcohol induced over-expression of GATA4 and MEF2c, which is possibly through the up-regulation of acetylation of histone3 near the gene promoters that affects the expression of GATA4 and MEF2c in H9c2 cells. ERK1/2 pathway might be a potential target for the intervention of alcohol induced congenital heart diseases. PMID:26392309

  20. Mechanism of endothelin-1-induced cytosolic Ca(2+) mobility in cultured H9c2 myocardiac ventricular cells.

    PubMed

    Hong, Show-Jen

    2002-10-01

    The effect of endothelin-1 (ET-1) on the intracellular free Ca(2+) ([Ca(2+)](i)) mobility in cultured H9c2 myocardiac ventricular cells was studied after loading with fura-2-AM. In Ca(2+)-containing buffer, ET-1 induced [Ca(2+)](i) rise from 10(-7) to 10(-9) M. ET-1 induced [Ca(2+)](i), which was composed of a first small peak and a secondary persistent plateau. In Ca(2+)-free buffer, pretreatment with 10(-7) M ET-1 inhibited the thapsigargin and carbonylcyanide m-chlorophenylhydrazone (CCCP)-induced [Ca(2+)](i) increase. Meanwhile, pretreatment with thapsigargin and CCCP also inhibited ET-1-induced [Ca(2+)](i) rise. In Ca(2+)-containing buffer, the ET(A) receptor antagonist (BQ123) completely abolished the secondary rising peak and plateau. Conversely, the ET(B) receptor antagonist (BQ788) completely inhibited the first small peak and secondary peak plateau. Nifedipine and La(3+) also abolished the 10(-7) M ET-1-induced [Ca(2+)](i) in the first rising peak. The internal Ca(2+) release induced by ET-1 was inhibited by U73122 (phospholipase C inhibitor), propranolol (phospholipase D inhibitor) and aristolochic acid (phospholipase A2 inhibitor). After incubation of 10(-7) M ET-1 in Ca(2+)-free buffer, the addition of 5 mM CaCl(2) increased Ca(2+) influx, implying that release of Ca(2+) from internal stores further induces capacitative Ca(2+) entry. Taken together, these results suggest that both ET(A) and ET(B) receptors are involved in ET-1-induced [Ca(2+)](i) rise in H9c2 myocardiac ventricular cells. Whereas ET(B) receptor seems to mediate the initial Ca(2+) influx via L-type Ca(2+) channel, ET(A) receptor appears to be involved in the subsequent Ca(2+) release from endoplasmic reticulum and mitochondria Ca(2+) stores. PMID:12135702

  1. Allicin protects rat cardiomyoblasts (H9c2 cells) from hydrogen peroxide-induced oxidative injury through inhibiting the generation of intracellular reactive oxygen species.

    PubMed

    Chan, Jackie Yan-Yan; Tsui, Hei-Tung; Chung, Ivan Ying-Ming; Chan, Robbie Yat-Kan; Kwan, Yiu-Wa; Chan, Shun-Wan

    2014-11-01

    Oxidative stress is considered an important factor that promotes cell death in response to a variety of pathophysiological conditions. This study investigated the antioxidant properties of allicin, the principle ingredient of garlic, on preventing oxidative stress-induced injury. The antioxidant capacities of allicin were measured by using 1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and hydrogen peroxide (H(2)O(2))-induced cell damage on H9c2 cardiomyoblasts. Allicin (0.3-10??M) pre-incubation could concentration-dependently attenuate the intracellular reactive oxygen species (ROS) increase induced by H(2)O(2) on H9c2 cells. It could also protect H9c2 cells against H(2)O(2)-induced cell damage. However, the DPPH free radical scavenging activity of allicin was shown to be low. Therefore, it is believed that the protective effect of allicin on H9c2 cells could inhibit intracellular ROS production instead of scavenging extracellular H(2)O(2) or free radicals. For the observed protective effect on H9c2 cells, allicin might also be effective in reducing free radical-induced myocardial cell death in ischemic condition. PMID:24945597

  2. Upregulation of peroxiredoxin III in doxorubicin-induced cytotoxicity and the FoxO3a-dependent expression in H9c2 cardiac cells

    PubMed Central

    LIU, MI-HUA; ZHANG, YUAN; HE, JUN; TAN, TIAN-PING; WU, SHAO-JIAN; FU, HONG-YUN; CHEN, YU-DAN; LIU, JUN; LE, QUN-FANG; HU, HENG-JING; YUAN, CONG; LIN, XIAO-LONG

    2015-01-01

    Doxorubicin (DOX) is an efficient drug used in cancer therapy; however, it produces reactive oxygen species (ROS) that induce severe cytotoxicity, limiting its clinical application. The aim of the present study was to investigate the role of peroxiredoxin III (Prx III) in DOX-induced H9c2 cell injuries. Following DOX treatment, the expression of phosphorylated-FoxO3a (p-FoxO3a) was decreased and Prx III expression was increased in H9c2 cells. In order to detect whether oxidative stress was involved in the induction of Prx III expression by FoxO3a, exogenous H2O2 was used to induce oxidative stress in the H9c2 cells. Apoptosis of H9c2 cardiomyocytes was assessed using methyl thiazolyl tetrazolium assay and Hoechst staining. The levels of Prx III and p-FoxO3a were evaluated using western blot analysis. As expected, H2O2 was found to mimic the effect of DOX, decreasing the expression of p-FoxO3a and increasing the expression of Prx III. In addition, the study evaluated whether the transcription factor FoxO3a was essential for the expression of Prx III. Pretreatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of ROS, prior to exposure to DOX dramatically increased the phosphorylation of FoxO3a and led to a marked reduction in Prx III expression in the H9c2 cells. In conclusion, the results of the current study suggest that FoxO3a mediates the expression of Prx III in DOX-induced injuries.

  3. Role of calreticulin in the sensitivity of myocardiac H9c2 cells to oxidative stress caused by hydrogen peroxide.

    PubMed

    Ihara, Yoshito; Urata, Yoshishige; Goto, Shinji; Kondo, Takahito

    2006-01-01

    Calreticulin (CRT), a Ca2+-binding molecular chaperone in the endoplasmic reticulum, plays a vital role in cardiac physiology and pathology. Oxidative stress is a main cause of myocardiac apoptosis in the ischemic heart, but the function of CRT under oxidative stress is not fully understood. In the present study, the effect of overexpression of CRT on susceptibility to apoptosis under oxidative stress was examined using myocardiac H9c2 cells transfected with the CRT gene. Under oxidative stress due to H2O2, the CRT-overexpressing cells were highly susceptible to apoptosis compared with controls. In the overexpressing cells, the levels of cytoplasmic free Ca2+ ([Ca2+]i) were significantly increased by H2O2, whereas in controls, only a slight increase was observed. The H2O2-induced apoptosis was enhanced by the increase in [Ca2+]i caused by thapsigargin in control cells but was suppressed by BAPTA-AM, a cell-permeable Ca2+ chelator in the CRT-overexpressing cells, indicating the importance of the level of [Ca2+]i in the sensitivity to H2O2-induced apoptosis. Suppression of CRT by the introduction of the antisense cDNA of CRT enhanced cytoprotection against oxidative stress compared with controls. Furthermore, we found that the levels of activity of calpain and caspase-12 were elevated through the regulation of [Ca2+]i in the CRT-overexpressing cells treated with H2O2 compared with controls. Thus we conclude that the level of CRT regulates the sensitivity to apoptosis under oxidative stress due to H2O2 through a change in Ca2+ homeostasis and the regulation of the Ca2+-calpain-caspase-12 pathway in myocardiac cells. PMID:16135540

  4. Palmitate induces ER stress and autophagy in H9c2 cells: implications for apoptosis and adiponectin resistance.

    PubMed

    Park, Min; Sabetski, Anna; Kwan Chan, Yee; Turdi, Subat; Sweeney, Gary

    2015-03-01

    The association between obesity and heart failure is well documented and recent studies have indicated that understanding the physiological role of autophagy will be of great significance. Cardiomyocyte apoptosis is one component of cardiac remodeling which leads to heart failure and in this study we used palmitate-treated H9c2 cells as an in vitro model of lipotoxicity to investigate the role of autophagy in cell death. Temporal analysis revealed that palmitate (100??M) treatment induced a gradual increase of intracellular lipid accumulation as well as apoptotic cell death. Palmitate induced autophagic flux, determined via increased LC3-II formation and p62 degradation as well as by detecting reduced colocalization of GFP with RFP in cells overexpressing tandem fluorescent GFP/RFP-LC3. The increased level of autophagy indicated by these measures were confirmed using transmission electron microscopy (TEM). Upon inhibiting autophagy using bafilomycin we observed an increased level of palmitate-induced cell death assessed by Annexin V/PI staining, detection of active caspase-3 and MTT cell viability assay. Interestingly, using TEM and p-PERK or p-eIF2? detection we observed increased endoplasmic reticulum (ER) stress in response to palmitate. Autophagy was induced as an adaptive response against ER stress since it was sensitive to ER stress inhibition. Palmitate-induced ER stress also induced adiponectin resistance, assessed via AMPK phosphorylation, via reducing APPL1 expression. This effect was independent of palmitate-induced autophagy. In summary, our data indicate that palmitate induces autophagy subsequent to ER stress and that this confers a prosurvival effect against lipotoxicity-induced cell death. Palmitate-induced ER stress also led to adiponecin resistance. PMID:25164368

  5. CREB Negatively Regulates IGF2R Gene Expression and Downstream Pathways to Inhibit Hypoxia-Induced H9c2 Cardiomyoblast Cell Death

    PubMed Central

    Chen, Wei-Kung; Kuo, Wei-Wen; Hsieh, Dennis Jine-Yuan; Chang, Hsin-Nung; Pai, Pei-Ying; Lin, Kuan-Ho; Pan, Lung-Fa; Ho, Tsung-Jung; Viswanadha, Vijaya Padma; Huang, Chih-Yang

    2015-01-01

    During hypoxia, gene expression is altered by various transcription factors. Insulin-like growth factor-II (IGF2) is known to be induced by hypoxia, which binds to IGF2 receptor IGF2R that acts like a G protein-coupled receptor, might cause pathological hypertrophy or activation of the mitochondria-mediated apoptosis pathway. Cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) is central to second messenger-regulated transcription and plays a critical role in the cardiomyocyte survival pathway. In this study, we found that IGF2R level was enhanced in H9c2 cardiomyoblasts exposed to hypoxia in a time-dependent manner but was down-regulated by CREB expression. The over-expression of CREB in H9c2 cardiomyoblasts suppressed the induction of hypoxia-induced IGF2R expression levels and reduced cell apoptosis. Gel shift assay results further indicated that CREB binds to the promoter sequence of IGF2R. With a luciferase assay method, we further observed that CREB represses IGF2R promoter activity. These results suggest that CREB plays an important role in the inhibition of IGF2R expression by binding to the IGF2R promoter and further suppresses H9c2 cardiomyoblast cell apoptosis induced by IGF2R signaling under hypoxic conditions. PMID:26610485

  6. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-?-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells

    SciTech Connect

    Yang, Chuen-Mao; Lee, I-Ta; Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der

    2013-10-15

    TNF-? plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-? in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-?-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-? induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-?-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-L-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-?B (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47{sup phox}, p42, p38, JNK1, p65, or PYK2. Moreover, TNF-? markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-?-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-?B (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-?-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-?-stimulated MAPKs and NF-?B activation. Thus, in H9c2 cells, we are the first to show that TNF-?-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-?B cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-?-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-? on H9c2 cells may provide potential therapeutic targets of chronic heart failure. - Highlights: • TNF-? induces MMP-9 secretion and expression via a TNFR1-dependent pathway. • TNF-? induces ROS/PYK2-dependent MMP-9 expression in H9c2 cells. • TNF-? induces MMP-9 expression via a NADPH oxidase/ROS-dependent NF-?B signaling. • TNF-? activates MAPK phosphorylation through NADPH oxidase/ROS generation.

  7. TRPV1 Activation Exacerbates Hypoxia/Reoxygenation-Induced Apoptosis in H9C2 Cells via Calcium Overload and Mitochondrial Dysfunction

    PubMed Central

    Sun, Zewei; Han, Jie; Zhao, Wenting; Zhang, Yuanyuan; Wang, Shuai; Ye, Lifang; Liu, Tingting; Zheng, Liangrong

    2014-01-01

    Transient potential receptor vanilloid 1 (TRPV1) channels, which are expressed on sensory neurons, elicit cardioprotective effects during ischemia reperfusion injury by stimulating the release of neuropeptides, namely calcitonin gene-related peptide (CGRP) and substance P (SP). Recent studies show that TRPV1 channels are also expressed on cardiomyocytes and can exacerbate air pollutant-induced apoptosis. However, whether these channels present on cardiomyocytes directly modulate cell death and survival pathways during hypoxia/reoxygenation (H/R) injury remains unclear. In the present study, we investigated the role of TRPV1 in H/R induced apoptosis of H9C2 cardiomyocytes. We demonstrated that TRPV1 was indeed expressed in H9C2 cells, and activated by H/R injury. Although neuropeptide release caused by TRPV1 activation on sensory neurons elicits a cardioprotective effect, we found that capsaicin (CAP; a TRPV1 agonist) treatment of H9C2 cells paradoxically enhanced the level of apoptosis by increasing intracellular calcium and mitochondrial superoxide levels, attenuating mitochondrial membrane potential, and inhibiting mitochondrial biogenesis (measured by the expression of ATP synthase ?). In contrast, treatment of cells with capsazepine (CPZ; a TRPV1 antagonist) or TRPV1 siRNA attenuated H/R induced-apoptosis. Furthermore, CAP and CPZ treatment revealed a similar effect on cell viability and mitochondrial superoxide production in primary cardiomyocytes. Finally, using both CGRP8–37 (a CGRP receptor antagonist) and RP67580 (a SP receptor antagonist) to exclude the confounding effects of neuropeptides, we confirmed aforementioned detrimental effects as TRPV1?/? mouse hearts exhibited improved cardiac function during ischemia/reperfusion. In summary, direct activation of TRPV1 in myocytes exacerbates H/R-induced apoptosis, likely through calcium overload and associated mitochondrial dysfunction. Our study provides a novel understanding of the role of myocyte TRPV1 channels in ischemia/reperfusion injury that sharply contrasts with its known extracardiac neuronal effects. PMID:25314299

  8. Ultrasound-Targeted Microbubble Destruction (UTMD) Assisted Delivery of shRNA against PHD2 into H9C2 Cells

    PubMed Central

    Ren, Pingping; Lee, Robert J.; Xiang, Guangya; Lv, Qing; Han, Wei; Wang, Jing; Ge, Shuping; Xie, Mingxing

    2015-01-01

    Gene therapy has great potential for human diseases. Development of efficient delivery systems is critical to its clinical translation. Recent studies have shown that microbubbles in combination with ultrasound (US) can be used to facilitate gene delivery. An aim of this study is to investigate whether the combination of US-targeted microbubble destruction (UTMD) and polyethylenimine (PEI) (UTMD/PEI) can mediate even greater gene transfection efficiency than UTMD alone and to optimize ultrasonic irradiation parameters. Another aim of this study is to investigate the biological effects of PHD2-shRNA after its transfection into H9C2 cells. pEGFP-N1 or eukaryotic shPHD2-EGFP plasmid was mixed with albumin-coated microbubbles and PEI to form complexes for transfection. After these were added into H9C2 cells, the cells were exposed to US with various sets of parameters. The cells were then harvested and analyzed for gene expression. UTMD/PEI was shown to be highly efficient in gene transfection. An US intensity of 1.5 W/cm2, a microbubble concentration of 300?l/ml, an exposure time of 45s, and a plasmid concentration of 15?g/ml were found to be optimal for transfection. UTMD/PEI-mediated PHD2-shRNA transfection in H9C2 cells significantly down regulated the expression of PHD2 and increased expression of HIF-1? and downstream angiogenesis factors VEGF, TGF-? and bFGF. UTMD/PEI, combined with albumin-coated microbubbles, warrants further investigation for therapeutic gene delivery. PMID:26267649

  9. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-?B signaling pathway

    PubMed Central

    SUN, Zhan; MA, Yi-Tong; CHEN, Bang-Dang; LIU, Fen

    2014-01-01

    Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-?B (NF-?B). The NF-?B transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-?B p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-?B p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the underlying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease. PMID:25593580

  10. Sanguinarine inhibits angiotensin II-induced apoptosis in H9c2 cardiac cells via restoring reactive oxygen species-mediated decreases in the mitochondrial membrane potential

    PubMed Central

    LIU, YUAN; JIAO, RONG; MA, ZHEN-GUO; LIU, WEI; WU, QING-QING; YANG, ZHENG; LI, FANG-FANG; YUAN, YUAN; BIAN, ZHOU-YAN; TANG, QI-ZHU

    2015-01-01

    Cell apoptosis induced by Angiotensin II (Ang II) has a critical role in the development of cardiovascular diseases. The aim of the present study was to investigate whether sanguinarine (SAN), a drug which was proved to have anti-oxidant, anti-proliferative and immune enhancing effects, can abolish cell apoptosis induced by Ang II. In the present study, H9c2 cardiac cells were stimulated with 10 µM Ang II with or without SAN. The level of intracellular reactive oxygen species (ROS) generation was assessed using dichlorodihydrofluorescein diacetate, and changes of the mitochondrial membrane potential (MMP) were assessed using JC-1 staining. Furthermore, mRNA expression of NOX2 was determined by reverse transcription quantitative polymerase chain reaction, and apoptosis was detected by Annexin V/propidium iodide staining and flow cytometry. The expression of B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) as well as cleaved (c)-caspase 3 and -9 were detected by western blot analysis, and the activity of caspase 3 and -9 was detected using an ELISA. The results of the present study showed that NOX2 expression and ROS generation induced by Ang II were inhibited by SAN, and the Ang 2-induced MMP loss was also ameliorated. Furthermore, Ang II-induced H9c2 cardiac cell apoptosis as well as c-caspase 3 and -9 levels were significantly reduced by SAN. Investigation of the possible pathway involved in the anti-apoptotic effect of SAN showed that the expression of Bcl-2 was decreased, while that of Bax was increased following stimulation with Ang II, which was reversed following treatment with SAN. In addition, Ang II enhanced the activity of caspase 9 and cleaved downstream caspases such as caspase-3, initiating the caspase cascade, while pre-treatment of H9c2 cardiac cells with SAN blocked these effects. In conclusion, the findings of the present study indicated that SAN inhibits the apoptosis of H9c2 cardiac cells induced by Ang II, most likely via restoring ROS-mediated decreases of the MMP. PMID:26017473

  11. Gene Expression Profiling of H9c2 Myoblast Differentiation towards a Cardiac-Like Phenotype

    PubMed Central

    Branco, Ana F.; Pereira, Susana P.; Gonzalez, Susana; Gusev, Oleg; Rizvanov, Albert A.; Oliveira, Paulo J.

    2015-01-01

    H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays. PMID:26121149

  12. Tanshinone IIA and Cryptotanshinone Prevent Mitochondrial Dysfunction in Hypoxia-Induced H9c2 Cells: Association to Mitochondrial ROS, Intracellular Nitric Oxide, and Calcium Levels.

    PubMed

    Jin, Hyou-Ju; Li, Chun-Guang

    2013-01-01

    The protective actions of tanshinones on hypoxia-induced cell damages have been reported, although the mechanisms have not been fully elucidated. Given the importance of nitric oxide (NO) and reactive oxygen species (ROS) in regulation of cell functions, the present study investigated the effects of two major tanshinones, Tanshinone IIA (TIIA) and cryptotanshinone (CT), on hypoxia-induced myocardial cell injury and its relationships with intracellular NO and ROS, calcium, and ATP levels in H9c2 cells. Chronic hypoxia significantly reduced cell viability which accompanied with LDH release, increase in mitochondrial ROS, intracellular NO and calcium levels, decrease in superoxide dismutase (SOD) activity, and cellular ATP contents. TIIA and CT significantly prevented cell injury by increasing cell viability and decreasing LDH release. The protective effects of tanshinones were associated with reduced mitochondrial superoxide production and enhanced mitochondrial SOD activity. Tanshinones significantly reduced intracellular NO and Ca(2+) levels. ATP levels were also restored by TIIA. These findings suggest that the cytoprotective actions of tanshinones may involve regulation of intracellular NO, Ca(2+), ATP productions, mitochondrial superoxide production, and SOD activity, which contribute to their actions against hypoxia injuries. PMID:23533503

  13. S-propranolol protected H9C2 cells from ischemia/reperfusion-induced apoptosis via downregultion of RACK1 Gene

    PubMed Central

    Jia, Xiongfei; Zhang, Li; Mao, Xiaoqin

    2015-01-01

    Ischemia-reperfusion (I/R) injury can lead to apoptotic death of heart cells and subsequently heart failure. Propranolol is widely used in the management of cardiovascular disorders, but the mechanism is still unclear. Our previous studies showed that activated protein kinase C1 (RACK1) was significantly down-regulated in human umbilical vein endothelial cells by S-propranolol. RACK1 may be a target protein of S-propranolol during I/R. At present, we constructed a lentiviral expression vector for RNA interference (RNAi) of RACK1. The interference efficiency of the lentivirus was confirmed by RT-PCR and western blot. H9C2 cells infected with Lv-RACK1-shRNA or control were subjected to simulate I/R in the presence and absence of S-propranolol. The release of cytokines and chemokines was determined by ELISA assay. Flow cytometry was employed to determine mitochondrial membrane potential (MMP), Ca2+ concentration, reactive oxygen species (ROS) production, and cell apoptosis. We found that RACK1 RNAi and S-propranolol treatment remarkably protected I/R injured cells from apoptosis via attenuating the release of cytokines and chemokines, Ca2+ overload, ROS concentration, and MMP. Furthermore, RACK1 RNAi and S-propranolol, separately and in combination, significantly reduced caspase-3 activity, cytochrome c release and JNK activation. RACK 1 can be considered as a target for drug development. PMID:26617741

  14. Combination of Nigella sativa with Glycyrrhiza glabra and Zingiber officinale augments their protective effects on doxorubicin-induced toxicity in h9c2 cells

    PubMed Central

    Hosseini, Azar; Shafiee-Nick, Reza; Mousavi, Seyed Hadi

    2014-01-01

    Objective(s): The use of doxorubicin (DOX) is limited by its dose-dependent cardio toxicity in which reactive Oxygen Species (ROS) play an important role in the pathological process. The aim of this study was to evaluate the protective effect of three medicinal plants, Nigella sativa (N), Glycyrrhiza glabra (G) and Zingiber officinale (Z), and their combination (NGZ), against DOX-induced apoptosis and death in H9c2 cells. Materials and Methods: The cells were incubated with different concentrations of each extract or NGZ for 4 hr which continued in the presence or absence of 5µM doxorubicin for 24 hr. Cell viability and the apoptotic rate were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) and propidium iodide (PI) staining assays, respectively. The level of ROS and lipid peroxidation were measured by fluorimetric methods. Results: Treatment with doxorubicin increased ROS generation, enhanced malondialdehyde (MDA) formation, and induced apoptosis. Co-treatment of the cells with each herb extract increased viability of cells dose-dependently with a maximum protection effect of about 30%, and their potencies were N>G>Z. The combination of the threshold dose of each extract (NGZ) produced a similar effect, which was increased dose-dependently to a maximum protection of 70%. These effects were correlated with the effects of NGZ on ROS and MDA. Conclusion: All of the extracts have some protective effects against DOX-induced toxicity in cardiomyocytes with similar efficacies, but with different potencies. However, NGZ produced much higher protective effect via reducing oxidative stress and inhibiting of apoptotic induction processes. Further investigations are needed to determine the effects of NGZ on DOX chemotherapy. PMID:25859303

  15. Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism

    PubMed Central

    Zhang, Hong; Liu, Xiao-Peng; Zhang, Dong; Wu, Ai-Li; Li, Jian-Jun; Tang, Yue

    2015-01-01

    Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway. PMID:26146991

  16. Danshensu protects against ischemia/reperfusion injury and inhibits the apoptosis of H9c2 cells by reducing the calcium overload through the p-JNK-NF-?B-TRPC6 pathway.

    PubMed

    Meng, Ying; Li, Wei-Zhu; Shi, You-Wei; Zhou, Bing-Feng; Ma, Rong; Li, Wei-Ping

    2016-01-01

    Ischemia-reperfusion (I/R) plays an important role in myocardial injury. In the present study, we aimed to examine the protective effects of Danshensu (DSS) against I/R injury and to elucidate the underlying mechanisms. For this purpose, H9c2 cells were cultured in hypoxic solution in a hypoxic incubator for 2 h, and then cultured in a high oxygen incubator for various periods of time and pre-treated with or without DSS, ammonium pyrrolidine dithiocarbamate (PDTC) or SP600125 [a c-Jun N-terminal kinase (JNK) inhibitor]. Cell apoptosis and cytosolic free Ca2+ ([Ca2+]i) levels were analyzed by flow cytometry. The protein expression levels of JNK, phosphorylated (p-)JNK, nuclear factor-?B (NF-?B) and transient receptor potential cation channel, subfamily C, member 6 (TRPC6) were measured by western blot analysis. The mRNA expression levels of JNK were measured by RT-qPCR. The results revealed that TRPC6 protein expression, the cell apoptotic rate and the [Ca2+]i levels increased in a time-dependent manner in the H9c2 cells following the induction of I/R injury. The apoptotic rate and TRPC6 protein expression decreased when the cells were treated with DSS prior to the induction of I/R injury. The knockdown of JNK expression by siRNA decreased the p-JNK and TRPC6 protein expression levels in the H9c2 cells subjected to I/R injury. The protein expression levels of p-JNK and NF-?B in the nucleus increased significantly when the H9c2 cells were subjected to I/R injury, whereas NF-?B expression in the cytoplasm decreased in a time?dependent manner. However, p-JNK, NF-?B and TRPC6 protein expression, the [Ca2+]i level and cell apoptosis decreased when the H9c2 cells were pre-treated with DSS or SP600125. Therefore, our data suggest that DSS prevents myocardial I/R injury by inhibiting p-JNK activation and NF-?B translocation, which potentially upregulate TRPC6 expression, increase the [Ca2+]i level, and result in the apoptosis of H9c2 cells. PMID:26718129

  17. Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

    PubMed

    Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

    2016-02-01

    Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75?M 4HNE and its vehicle, ethanol as well as 25, 50 and 75?M disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. PMID:26577527

  18. Berberine treatment prevents cardiac dysfunction and remodeling through activation of 5'-adenosine monophosphate-activated protein kinase in type 2 diabetic rats and in palmitate-induced hypertrophic H9c2 cells.

    PubMed

    Chang, Wenguang; Zhang, Ming; Meng, Zhaojie; Yu, Yang; Yao, Fan; Hatch, Grant M; Chen, Li

    2015-12-15

    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3?) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (?-MHC) and increased expression of beta-myosin heavy chain (?-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased ?-MHC expression and decreased ?-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3? activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. PMID:26522928

  19. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

    PubMed

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells. PMID:24866243

  20. Smad4 mediated BMP2 signal is essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells

    SciTech Connect

    Si, Lina; Shi, Jin; Gao, Wenqun; Zheng, Min; Liu, Lingjuan; Zhu, Jing; Tian, Jie

    2014-07-18

    Highlights: • BMP2 can upregulated cardiac related gene GATA4, Nkx2.5, MEF2c and Tbx5. • Inhibition of Smad4 decreased BMP2-induced hyperacetylation of histone H3. • Inhibition of Smad4 diminished BMP2-induced overexpression of GATA4 and Nkx2.5. • Inhibition of Smad4 decreased hyperacetylated H3 in the promoter of GATA4 and Nkx2.5. • Smad4 is essential for BMP2 induced hyperacetylated histone H3. - Abstract: BMP2 signaling pathway plays critical roles during heart development, Smad4 encodes the only common Smad protein in mammals, which is a pivotal nuclear mediator. Our previous studies showed that BMP2 enhanced the expression of cardiac transcription factors in part by increasing histone H3 acetylation. In the present study, we tested the hypothesis that Smad4 mediated BMP2 signaling pathway is essential for the expression of cardiac core transcription factors by affecting the histone H3 acetylation. We successfully constructed a lentivirus-mediated short hairpin RNA interference vector targeting Smad4 (Lv-Smad4) in rat H9c2 embryonic cardiac myocytes (H9c2 cells) and demonstrated that it suppressed the expression of the Smad4 gene. Cultured H9c2 cells were transfected with recombinant adenoviruses expressing human BMP2 (AdBMP2) with or without Lv-Smad4. Quantitative real-time RT-PCR analysis showed that knocking down of Smad4 substantially inhibited both AdBMP2-induced and basal expression levels of cardiac transcription factors GATA4 and Nkx2.5, but not MEF2c and Tbx5. Similarly, chromatin immunoprecipitation (ChIP) analysis showed that knocking down of Smad4 inhibited both AdBMP2-induced and basal histone H3 acetylation levels in the promoter regions of GATA4 and Nkx2.5, but not of Tbx5 and MEF2c. In addition, Lv-Smad4 selectively suppressed AdBMP2-induced expression of HAT p300, but not of HAT GCN5 in H9c2 cells. The data indicated that inhibition of Smad4 diminished both AdBMP2 induced and basal histone acetylation levels in the promoter regions of GATA4 and Nkx2.5, suggesting that Smad4 mediated BMP2 signaling pathway was essential for the regulation of GATA4 and Nkx2.5 by affecting the histone H3 acetylation in H9c2 cells.

  1. Tyrosol prevents ischemia/reperfusion-induced cardiac injury in H9c2 cells: involvement of ROS, Hsp70, JNK and ERK, and apoptosis.

    PubMed

    Sun, Liwei; Fan, Hang; Yang, Lingguang; Shi, Lingling; Liu, Yujun

    2015-01-01

    Ischemia-Reperfusion (I/R) injury causes ROS overproduction, creating oxidative stress, and can trigger myocyte death, resulting in heart failure. Tyrosol is an antioxidant abounded in diets and medicine. Our objective was to investigate the protective effect of tyrosol on I/R-caused mortality in H9c2 cardiomyocytes through its influence on ROS, Hsp70, ERK, JNK, Bcl-2, Bax and caspase-8. A simulated I/R model was used, myocytes loss was examined by MTT, and ROS levels were measured using DCFH-DA. Nuclear condensation and caspase-3 activity were assessed by DAPI staining and fluorometric assay. Phosphorylated ERK and JNK were determined by electrochemiluminescent ELISA, and Hsp70, Bcl-2, Bax and caspase-8 were examined by Western blotting. Results show that tyrosol salvaged myocyte loss, inhibited nuclear condensation and caspase-3 activity dose-dependently, indicating its protection against I/R-caused myocyte loss. Furthermore, tyrosol significantly inhibited ROS accumulation and activation of ERK and JNK, augmenting Hsp70 expression. Besides, tyrosol inhibited I/R-induced apoptosis, associated with retained anti-apoptotic Bcl-2 protein, and attenuated pro-apoptotic Bax protein, resulting in a preservation of Bcl-2/Bax ratio. Finally, tyrosol notably decreased cleaved caspase-8 levels. In conclusion, cytoprotection of tyrosol in I/R-caused myocyte mortality was involved with the mitigation of ROS, prohibition of the activation of ERK, JNK and caspase-8, and elevation of Hsp70 and Bcl-2/Bax ratio. PMID:25723850

  2. Antiapoptotic activity of Akt is down-regulated by Ca2+ in myocardiac H9c2 cells. Evidence of Ca(2+)-dependent regulation of protein phosphatase 2Ac.

    PubMed

    Yasuoka, Chie; Ihara, Yoshito; Ikeda, Satoshi; Miyahara, Yoshiyuki; Kondo, Takahito; Kohno, Shigeru

    2004-12-01

    Cell survival signaling of the Akt/protein kinase B pathway was influenced by a change in the cytoplasmic free calcium concentration ([Ca2+]i) for over 2 h via the regulation of a Ser/Thr phosphatase, protein phosphatase 2Ac (PP2Ac), in rat myocardiac H9c2 cells. Akt was down-regulated when [Ca2+]i was elevated by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, but was up-regulated when it was suppressed by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), a cell permeable Ca2+ chelator. The inactivation of Akt was well correlated with the susceptibility to oxidant-induced apoptosis in H9c2 cells. To investigate the mechanism of the Ca(2+)-dependent regulation of Akt via the regulation of PP2A, we examined the transcriptional regulation of PP2Acalpha in H9c2 cells with Ca2+ modulators. Transcription of the PP2Acalpha gene was increased by thapsigargin but decreased by BAPTA-AM. The promoter activity was examined and the cAMP response element (CRE) was found responsible for the Ca(2+)-dependent regulation of PP2Acalpha. Furthermore, phosphorylation of CRE-binding protein increased with thapsigargin but decreased with BAPTA-AM. A long term change of [Ca2+]i regulates PP2Acalpha gene transcription via CRE, resulting in a change in the activation status of Akt leading to an altered susceptibility to apoptosis. PMID:15375154

  3. Stimulating basal mitochondrial respiration decreases doxorubicin apoptotic signaling in H9c2 cardiomyoblasts.

    PubMed

    Deus, Cláudia M; Zehowski, Cheryl; Nordgren, Kendra; Wallace, Kendall B; Skildum, Andrew; Oliveira, Paulo J

    2015-08-01

    Doxorubicin (DOX) is currently used in cancer chemotherapy, however, its use often results in adverse effects highlighted by the development of cardiomyopathy and ultimately heart failure. Interestingly, DOX cardiotoxicity is decreased by resveratrol or by physical activity, suggesting that increased mitochondrial activity may be protective. Conversely, recent studies showed that troglitazone, a PPAR? agonist, increases the cytotoxicity of DOX against breast cancer cells by up-regulating mitochondrial biogenesis. The hypothesis for the current investigation was that DOX cytotoxicity in H9c2 cardiomyoblasts is decreased when mitochondrial capacity is increased. We focused on several end-points for DOX cytotoxicity, including loss of cell mass, apoptotic signaling and alterations of autophagic-related proteins. Our results show that a galactose-based, modified cell culture medium increased H9c2 basal mitochondrial respiration, protein content, and mtDNA copy number without increasing maximal or spare respiratory capacity. H9c2 cardiomyoblasts cultured in the galactose-modified media showed lower DOX-induced activation of the apoptotic pathway, measured by decreased caspase-3 and -9 activation, and lower p53 expression, although ultimately loss of cells was not prevented. Treatment with the PPAR? agonist troglitazone had no effect on DOX toxicity in this cardiac cell line, which agrees with the fact that troglitazone did not increase mitochondrial DNA content or capacity at the concentrations and duration of exposure used in this investigation. Our results show that mitochondrial remodeling caused by stimulating basal rates of oxidative phosphorylation decreased DOX-induced apoptotic signaling and increased DOX-induced autophagy in H9c2 cardiomyoblasts. The differential effect on cytotoxicity in cardiac versus breast cancer cell lines suggests a possible overall improvement in the clinical efficacy for doxorubicin in treating cancer. PMID:25997894

  4. Angiotensin II down-regulates natriuretic peptide receptor-A expression and guanylyl cyclase activity in H9c2 (2-1) cardiac myoblast cells: Role of ROS and NF-?B.

    PubMed

    Gopi, Venkatachalam; Subramanian, Vimala; Manivasagam, Senthamizharasi; Vellaichamy, Elangovan

    2015-11-01

    Atrial natriuretic peptide (ANP)/natriuretic peptide receptor-A (NPR-A) system is suggested as an endogenous anti-hypertrophic protective mechanism of the heart. We have shown previously that Angiotensin II (ANG II), an effector molecule of renin-angiotensin-aldosterone system, down-regulates NPR-A expression and its activity in vivo rat heart. However, the underlying mechanism by which ANG II down-regulates NPR-A expression in the heart is not well understood. Hence, the present investigation was aimed to determine whether ANG II-stimulated reactive oxygen species (ROS) and NF-?B are involved in the down-regulation of NPR-A activity in H9c2 (2-1) cardiac myoblast cells. The H9c2 (2-1) cardiac myoblast cells were exposed to ANG II (10(-7) M for 20 h) with/or without blocker treatment (losartan-10 µM, N-acetyl cysteine (NAC)-10 mM and pyrrolidine dithiocarbamate (PDTC)-100 µM). On exposure, ANG II induced a significant decrease (P < 0.001) in the expression of Npr1 (coding for NPR-A) gene and NPR-A receptor-dependent guanylyl cyclase (GC) activity. The level of expression of proto-oncogenes (c-fos, c-myc, and c-jun) and natriuretic peptides (ANP and BNP) was increased in ANG II-treated cells when compared with control cells. Interestingly, ANG II-dependent repression of Npr1 gene expression and guanylyl cyclase (GC) activity was completely restored on treatment with losartan, while only a partial reversal was observed in NAC- and PDTC-co-treated cells. In conclusion, the results of this study suggest that ROS-mediated NF-?B activation mechanism is critically involved in the ANG II-mediated down-regulation of NPR-A expression and its GC activity. PMID:26215453

  5. Endocytosis?Mediated Invasion and Pathogenicity of Streptococcus agalactiae in Rat Cardiomyocyte (H9C2)

    PubMed Central

    Pooja, Sharma; Pushpanathan, Muthuirulan; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

    2015-01-01

    Streptococcus agalactiae infection causes high mortality in cardiovascular disease (CVD) patients, especially in case of setting prosthetic valve during cardiac surgery. However, the pathogenesis mechanism of S. agalactiae associate with CVD has not been well studied. Here, we have demonstrated the pathogenicity of S. agalactiae in rat cardiomyocytes (H9C2). Interestingly, both live and dead cells of S. agalactiae were uptaken by H9C2 cells. To further dissect the process of S. agalactiae internalization, we chemically inhibited discrete parts of cellular uptake system in H9C2 cells using genistein, chlorpromazine, nocodazole and cytochalasin B. Chemical inhibition of microtubule and actin formation by nocodazole and cytochalasin B impaired S. agalactiae internalization into H9C2 cells. Consistently, reverse? transcription PCR (RT?PCR) and quantitative real time?PCR (RT-qPCR) analyses also detected higher levels of transcripts for cytoskeleton forming genes, Acta1 and Tubb5 in S. agalactiae?infected H9C2 cells, suggesting the requirement of functional cytoskeleton in pathogenesis. Host survival assay demonstrated that S. agalactiae internalization induced cytotoxicity in H9C2 cells. S. agalactiae cells grown with benzyl penicillin reduced its ability to internalize and induce cytotoxicity in H9C2 cells, which could be attributed with the removal of surface lipoteichoic acid (LTA) from S. agalactiae. Further, the LTA extracted from S. agalactiae also exhibited dose?dependent cytotoxicity in H9C2 cells. Taken together, our data suggest that S. agalactiae cells internalized H9C2 cells through energy?dependent endocytic processes and the LTA of S. agalactiae play major role in host cell internalization and cytotoxicity induction. PMID:26431539

  6. 17Beta-estradiol protects against oxidative stress-induced cell death through the glutathione/glutaredoxin-dependent redox regulation of Akt in myocardiac H9c2 cells.

    PubMed

    Urata, Yoshishige; Ihara, Yoshito; Murata, Hiroaki; Goto, Shinji; Koji, Takehiko; Yodoi, Junji; Inoue, Satoshi; Kondo, Takahito

    2006-05-12

    The GSH/glutaredoxin (GRX) system is involved in the redox regulation of certain enzyme activities, and this system protects cells from H2O2-induced apoptosis by regulating the redox state of Akt (Murata, H., Ihara, Y., Nakamura, H., Yodoi, J., Sumikawa, K., and Kondo, T. (2003) J. Biol. Chem. 278, 50226-50233). Estrogens, such as 17beta-estradiol (E2), play an important role in development, growth, and differentiation and appear to have protective effects on oxidative stress mediated by estrogen receptor alpha (ERalpha). However, the role of the ERbeta-mediated pathway in this cytoprotection and the involvement of E2 in the redox regulation are not well understood. In the present study, we demonstrated that E2 protected cardiac H9c2 cells, expressing ERbeta from H2O2-induced apoptosis concomitant with an increase in the activity of Akt. E2 induced the expression of glutaredoxin (GRX) as well as gamma-glutamylcysteine synthetase, a rate-limiting enzyme for the synthesis of GSH. Inhibitors for both gamma-glutamylcysteine synthetase and GRX and ICI182,780, a specific inhibitor of ERs, abolished the protective effect of E2 on cell survival as well as the activity of Akt, suggesting that ERbeta is involved in the cytoprotection and redox regulation by E2. Transcription of the GRX gene was enhanced by E2. The promoter activity of GRX was up-regulated by an ERbeta-dependent element. These results suggest that the GRX/GSH system is involved in the cytoprotective and genomic effects of E2 on the redox state of Akt, a pathway that is mediated, at least in part, by ERbeta. This mechanism may also play an antiapoptotic role in cancer cells during carcinogenesis or chemotherapy. PMID:16549430

  7. Application of novel anodized titanium for enhanced recruitment of H9C2 cardiac myoblast

    PubMed Central

    Behjati, Mohaddeseh; Moradi, Iman; Kazemi, Mohammad

    2015-01-01

    Objective(s): Anodized treated titanium surfaces, have been proposed as potential surfaces with better cell attachment capacities. We have investigated the adhesion and proliferation properties of H9C2 cardiac myoblasts on anodized treated titanium surface. Materials and Methods: Surface topography and anodized tubules were examined by high-resolution scanning electron microscopy (SEM). Control and test substrates were inserted to the bottom of 24-well tissue culture plates. Culture media including H9C2 cells were loaded on the surface of substrate and control wells at the second passage. Evaluation of cell growth, proliferation, viability and surface cytotoxicity was performed using MTT test. After 48 hr, some samples were inspected by SEM. DAPI-staining was used to count attached cells. Results: MTT results for cells cultured on anodized titanium and unanodized titanium surfaces was equal to 1.56 and 0.55 fold change compared to tissue culture polystyrene (TCPS). The surface had no cytotoxic effects on cells. The average cell attachment to TCPS, unanodized and anodized titanium surface was 2497±40.16, 1250±20.11 and 4859.5±54.173, respectively. Cell adhesion to anodized titanium was showed 1.95 and 3.89 fold increase compared to TCPS and unanodized titanium, respectively (P<0.05). Conclusion: Anodized titanium surfaces can be potentially applied for enhanced recruitment of H9C2 cells. This unique property makes these inexpensive anodized surfaces as a candidate surface for attachment of cardiac cells and consequently for cardiac regeneration purposes. PMID:26526098

  8. Cardiomyoblast (h9c2) differentiation on tunable extracellular matrix microenvironment.

    PubMed

    Suhaeri, Muhammad; Subbiah, Ramesh; Van, Se Young; Du, Ping; Kim, In Gul; Lee, Kangwon; Park, Kwideok

    2015-06-01

    Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as ?-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5?kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit ?5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for ?1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis. PMID:25836924

  9. Astragaloside IV prevents lipopolysaccharide-induced injury in H9C2 cardiomyocytes.

    PubMed

    Wang, Shi-Guang; Xu, Yan; Xie, Hao; Wang, Wei; Chen, Xiao-Hu

    2015-02-01

    This study aimed to investigate the protective effects of astragaloside IV (AS IV) on lipopolysaccharide (LPS)-induced injury in H9C2 cardiomyocytes. H9C2 Cardiomyocytes were cultured with LPS (10 ?g·mL(-1)) for 4 h and treated with AS IV at 50, 100, and 150 ?mol·L(-1) for various durations. Cell viability was determined by MTT. The content of released TNF-? and IL-6 from cardiomyocytes were evaluated by enzyme-linked immunosorbent assay (ELISA). The levels of superoxidase dismutase (SOD), malondialdehyde (MDA), lactate dehydrogenase (LDH), and creatine phosphate kinase (CK) were measured by using commercial available kits. The mRNA and protein expression levels of NF-?B p65 were measured by RT-PCR and Western blotting, respectively. And the NF-?B p65 activity was measured by ELISA. Our results demonstrated that AS IV at 50, 100, and 150 ?mol·L(-1) markedly inhibited the release of TNF-? and IL-6 and decreased NF-?B expression, compared with the model group. Moreover, the improved SOD activity and decreased MDA, LDH and CK levels were detected after AS IV treatment. In summary, AS IV could increase the activities of antioxidant enzymes, inhibite lipid peroxidation, and down-regulate the inflammatory mediators involved in the inflammatory responses. These results demonstrated that AS IV could prevent LPS-induced injury in cardiomyocytes. PMID:25769895

  10. Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes

    PubMed Central

    Kohli, Shrey; Chhabra, Aastha; Jaiswal, Astha; Rustagi, Yashika; Sharma, Manish; Rani, Vibha

    2013-01-01

    Background Extracellular matrix (ECM) remodeling facilitates biomechanical signals in response to abnormal physiological conditions. This process is witnessed as one of the major effects of the stress imposed by catecholamines, such as epinephrine and norepinephrine (NE), on cardiac muscle cells. Matrix metalloproteinases (MMPs) are the key proteases involved in degradation of the ECM in heart. Objectives The present study focuses on studying the effect of curcumin on Gelatinase B (MMP-9), an ECM remodeling regulatory enzyme, in NE-induced cardiac stress. Curcumin, a bioactive polyphenol found in the spice turmeric, has been studied for its multi-fold beneficial properties. This study focuses on investigating the role of curcumin as a cardio-protectant. Methods H9c2 cardiomyocytes were subjected to NE and curcumin treatments to study the response in stress conditions. Effect on total collagen content was studied using Picrosirus red staining. Gelatinase B activity was assessed through Gel-Diffusion Assay and Zymographic techniques. RT-PCR, Western Blotting and Immunocytochemistry were performed to study effect on expression of gelatinase B. Further, the effect of curcumin on the localization of NF-?B, known to regulate gelatinase B, was also examined. Results Curcumin suppressed the increase in the total collagen content under hypertrophic stress and was found to inhibit the in-gel and in-situ gelatinolytic activity of gelatinase B. Moreover, it was found to suppress the mRNA and protein expression of gelatinase B. Conclusions The study provides an evidence for an overall inhibitory effect of curcumin on Gelatinase B in NE-induced hypertrophic stress in H9c2 cardiomyocytes which may contribute in the prevention of ECM remodeling. PMID:24116115

  11. Cardioprotective role of Syzygium cumini against glucose-induced oxidative stress in H9C2 cardiac myocytes.

    PubMed

    Atale, Neha; Chakraborty, Mainak; Mohanty, Sujata; Bhattacharya, Susinjan; Nigam, Darshika; Sharma, Manish; Rani, Vibha

    2013-09-01

    Diabetic patients are known to have an independent risk of cardiomyopathy. Hyperglycemia leads to upregulation of reactive oxygen species (ROS) that may contribute to diabetic cardiomyopathy. Thus, agents that suppress glucose-induced intracellular ROS levels can have therapeutic potential against diabetic cardiomyopathy. Syzygium cumini is well known for its anti-diabetic potential, but its cardioprotective properties have not been evaluated yet. The aim of the present study is to analyze cardioprotective properties of methanolic seed extract (MSE) of S. cumini in diabetic in vitro conditions. ROS scavenging activity of MSE was studied in glucose-stressed H9C2 cardiac myoblasts after optimizing the safe dose of glucose and MSE by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide. 2',7'-dichlorfluorescein diacetate staining and Fluorescence-activated cell sorting analysis confirmed the suppression of ROS production by MSE in glucose-induced cells. The intracellular NO and H2O2 radical-scavenging activity of MSE was found to be significantly high in glucose-induced cells. Exposure of glucose-stressed H9C2 cells to MSE showed decline in the activity of catalase and superoxide dismutase enzymes and collagen content. 4',6-diamidino-2-phenylindole, propidium iodide and 10-N-nonyl-3,6-bis (dimethylamino) acridine staining revealed that MSE protects myocardial cells from glucose-induced stress. Taken together, our findings revealed that the well-known anti-diabetic S. cumini can also protect the cardiac cells from glucose-induced stress. PMID:23512199

  12. Difference in protective effects of three structurally similar flavonoid glycosides from Hypericum ascyron against H?O?-induced injury in H9c2 cardiomyoblasts.

    PubMed

    Li, Xiu-Mei; Luo, Xue-Gang; Li, Kun; Wang, Nan; Hua, Er-Bin; Zhang, Yao; Zhang, Tong-Cun

    2015-10-01

    According to previous studies, hyperoside possesses myocardial protective effects. To investigate whether isoquercitrin and isohyperoside have similar functions, the protective effects of isoquercitrin and isohyperoside against H2O2?induced injury in H9c2 rat cardiomyoblasts were evaluated using a 3?(4,5?dimethylthiazol?2?yl)?2,5?diphenyltetrazolium bromide assay. The mechanism of action was investigated by assessing the leakage of lactate dehydrogenase (LDH) of hyperoside and isoquercitrin?pretreated H9c2 cardiomyocytes following H2O2?induced injury, and examining their effects on the levels of malondialdehyde (MDA) and superoxide dismutase (SOD) activity. The isolation of two flavonoid glycosides from H. ascyron was performed, following extraction, using semi?preparative high performance liquid chromatography. Using the spectral characteristics, the structures of these compounds were identified as isoquercitrin and isohyperoside. This was the first time, to the best of our knowledge, that isohyperoside has been identified from H. ascyron. The results revealed that isoquercitrin and isohyperoside possessed similar protective effects to hyperoside against H2O2?induced injury in H9c2 cells. The half maximal inhibitory concentration values of hyperoside, isoquercitrin and isohyperoside were 0.0008, 0.0017 and 0.0002 µM, respectively. Based on these results, isohyperoside possessed more marked protective effects against H2O2?induced injury in the H9c2 cardiomyoblasts. The significant reduction in LDH leakage, decrease in MDA level and increase in SOD activity also provided evidence of the cardioprotective effects of isoquercitrin and isohyperoside. The present study reported for the first time, to the best of our knowledge, the myocardial protective effects of isoquercitrin and isohyperoside. The mechanism of action may involve protection of the cell membrane from oxidative damage. PMID:26177564

  13. Antihistamines modulate the integrin signaling pathway in h9c2 rat cardiomyocytes: Possible association with cardiotoxicity.

    PubMed

    Yun, J S; Kim, S Y

    2015-08-01

    The identification of biomarkers for toxicity prediction is crucial for drug development and safety evaluation. The selective and specific biomarkers for antihistamines-induced cardiotoxicity is not well identified yet. In order to evaluate the mechanism of the life-threatening effects caused by antihistamines, we used DNA microarrays to analyze genomic profiles in H9C2 rat cardiomyocytes that were treated with antihistamines. The gene expression profiles from drug-treated cells revealed changes in the integrin signaling pathway, suggesting that cardiac arrhythmias induced by antihistamine treatment may be mediated by changes in integrin-mediated signaling. It has been reported that integrin plays a role in QT prolongation that may induce cardiac arrhythmia. These results indicate that the integrin-mediated signaling pathway induced by antihistamines is involved in various biological mechanisms that lead to cardiac QT prolongation. Therefore, we suggest that genomic profiling of antihistamine-treated cardiomyocytes has the potential to reveal the mechanism of adverse drug reactions, and this signal pathway is applicable to prediction of in vitro cardiotoxicity induced by antihistamines as a biomarker candidate. PMID:25425550

  14. Methyl donor deficiency in H9c2 cardiomyoblasts induces ER stress as an important part of the proteome response.

    PubMed

    Martinez, Emilie; Deval, Christiane; Jousse, Céline; Mazur, Andrzej; Brachet, Patrick; Comte, Blandine

    2015-02-01

    Deficiency of methyl donors (MDs, folate, vitamin B12, and choline) causes increased plasma level of Hcy, a risk factor for cardiovascular diseases. Previously, we showed that maternal MD deprivation altered the cardiac proteome of rat pups. To better understand its impact on cardiac cells, we exposed rat H9c2 cardiomyoblasts to selectively a synthetic folate- or MD-deficient (FD or MDD) medium. We found that a 4-day exposure to the FD medium, unlike the MDD one, did not cause an abnormal extracellular release of Hcy relatively to similar exposure to the control complete (C) medium. Comparative analyses of the proteomes of FD, MDD, and C cells identified 7 and 6 proteins up- or downregulated by either deficiency, respectively. Most proteins were found interrelated in a single network dealing with "post-translational modification, protein folding and cell death/survival" (FD cells) or "DNA replication/recombination/repair and cell morphology/compromise" (MDD cells). Both deficiencies altered the protein and mRNA levels of the chaperones ?-crystallin B, protein disulfide-isomerase A4, and prohibitin. This was concurrent with rapid induction of several key genes of the ER stress response, notably gadd153/chop, and increased expression of the E3 ubiquitin ligases, Hrd1, and MAFbx. In conclusion, the effects of folate and MD deficiencies on the cardiomyoblast proteome display some dissimilarities possibly related to different cellular production of Hcy. In both cases activation of the ER stress could occur in response to accumulation of ubiquitinated misfolded proteins. PMID:25486180

  15. Cardioprotective effect of carvedilol: inhibition of apoptosis in H9c2 cardiomyocytes via the TLR4/NF-?B pathway following ischemia/reperfusion injury

    PubMed Central

    ZHAO, YONG; XU, YAN; ZHANG, JIANHUA; JI, TINGTING

    2014-01-01

    Carvedilol is a non-selective ?-blocker used in the treatment of cardiovascular disease, including myocardial ischemia. The aim of the present study was to investigate the molecular mechanisms underlying the effects of carvedilol on simulated ischemia/reperfusion (SI/R)-induced cardiomyocyte apoptosis in vitro. H9c2 cardiomyocytes were incubated with either a vehicle or an ischemic buffer during hypoxia followed by reoxygenation with or without carvedilol. In two additional groups, toll-like receptor 4 (TLR4) and nuclear factor ?-light-chain-enhancer of activated B cells (NF-?B) were inhibited by a TLR4 antibody and pyrrolidine dithiocarbamate, respectively. The results revealed that carvedilol markedly decreased SI/R-induced apoptosis in a concentration-dependent manner, as demonstrated by flow-cytometric analysis. This effect was shown to be associated with an increase in the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X (Bax) protein ratio and concurrent reductions in the expression levels of TLR4 and NF-?B. These results suggest that carvedilol provides significant cardioprotection against SI/R-induced injury in H9c2 cardiomyocytes, an effect likely to be mediated through the TLR4/NF-?B signaling pathway. PMID:25187802

  16. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3? signaling pathway in H9c2 cardiomyocytes

    SciTech Connect

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang; Yi, Kyu Yang; Chung, Hun-Jong; Park, Jong Seok; Kim, Bokyung; Feng, Zhong-Ping; Shin, Hwa-Sup

    2013-04-01

    Poly(adenosine 5?-diphosphate ribose) polymerase (PARP) is a nuclear enzyme activated by DNA strand breaks and plays an important role in the tissue injury associated with ischemia and reperfusion. The aim of the present study was to investigate the protective effect of 5-aminoisoquinolinone (5-AIQ), a PARP inhibitor, against oxidative stress-induced apoptosis in H9c2 cardiomyocytes. 5-AIQ pretreatment significantly protected against H{sub 2}O{sub 2}-induced cell death, as determined by the XTT assay, cell counting, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, and Western blot analysis of apoptosis-related proteins such as caspase-3, Bax, and Bcl-2. Upregulation of antioxidant enzymes such as manganese superoxide dismutase and catalase accompanied the protective effect of 5-AIQ on H{sub 2}O{sub 2}-induced cell death. Our data also showed that 5-AIQ pretreatment protected H9c2 cells from H{sub 2}O{sub 2}-induced apoptosis by triggering activation of Akt and glycogen synthase kinase-3? (GSK-3?), and that the protective effect of 5-AIQ was diminished by the PI3K inhibitor LY294002 at a concentration that effectively abolished 5-AIQ-induced Akt and GSK-3? activation. In addition, inhibiting the Akt/GSK-3? pathway by LY294002 significantly attenuated the 5-AIQ-mediated decrease in cleaved caspase-3 and Bax activation and H9c2 cell apoptosis induction. Taken together, these results demonstrate that 5-AIQ prevents H{sub 2}O{sub 2}-induced apoptosis in H9c2 cells by reducing intracellular reactive oxygen species production, regulating apoptosis-related proteins, and activating the Akt/GSK-3? pathway. - Highlights: ? 5-AIQ, a PARP inhibitor, decreased H{sub 2}O{sub 2}-induced H9c2 cell death and apoptosis. ? 5-AIQ upregulated antioxidant Mn-SOD and catalase, while decreasing ROS production. ? 5-AIQ decreased H{sub 2}O{sub 2}-induced increase in cleaved caspase-3 and Bax and decrease in Bcl2. ? 5-AIQ activated Akt and GSK-3?, the effect being abolished by PI3K inhibitor LY294002. ? LY294002 attenuated 5-AIQ-mediated effects on H9c2 apoptosis and related proteins.

  17. Overexpression of ryanodine receptor type 1 enhances mitochondrial fragmentation and Ca2+-induced ATP production in cardiac H9c2 myoblasts

    PubMed Central

    O-Uchi, Jin; Jhun, Bong Sook; Hurst, Stephen; Bisetto, Sara; Gross, Polina; Chen, Ming; Kettlewell, Sarah; Park, Jongsun; Oyamada, Hideto; Smith, Godfrey L.; Murayama, Takashi

    2013-01-01

    Ca+ influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca2+ influx is mainly mediated by the mitochondrial Ca2+ uniporter (MCU). Growing evidence also indicates that mitochondrial Ca2+ influx mechanisms are regulated not solely by MCU but also by multiple channels/transporters. We have previously reported that skeletal muscle-type ryanodine receptor (RyR) type 1 (RyR1), which expressed at the mitochondrial inner membrane, serves as an additional Ca2+ uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca2+ influx mechanism is the dominant regulator of mitochondrial morphology/dynamics and energetics in cardiomyocytes. To investigate the role of mitochondrial RyR1 in the regulation of mitochondrial morphology/function in cardiac cells, RyR1 was transiently or stably overexpressed in cardiac H9c2 myoblasts. We found that overexpressed RyR1 was partially localized in mitochondria as observed using both immunoblots of mitochondrial fractionation and confocal microscopy, whereas RyR2, the main RyR isoform in the cardiac sarcoplasmic reticulum, did not show any expression at mitochondria. Interestingly, overexpression of RyR1 but not MCU or RyR2 resulted in mitochondrial fragmentation. These fragmented mitochondria showed bigger and sustained mitochondrial Ca2+ transients compared with basal tubular mitochondria. In addition, RyR1-overexpressing cells had a higher mitochondrial ATP concentration under basal conditions and showed more ATP production in response to cytosolic Ca2+ elevation compared with nontransfected cells as observed by a matrix-targeted ATP biosensor. These results indicate that RyR1 possesses a mitochondrial targeting/retention signal and modulates mitochondrial morphology and Ca2+-induced ATP production in cardiac H9c2 myoblasts. PMID:24124188

  18. Pachymic acid protects H9c2 cardiomyocytes from lipopolysaccharide-induced inflammation and apoptosis by inhibiting the extracellular signal-regulated kinase 1/2 and p38 pathways.

    PubMed

    Li, Fang-Fang; Yuan, Yuan; Liu, Yuan; Wu, Qing-Qing; Jiao, Rong; Yang, Zheng; Zhou, Meng-Qiao; Tang, Qi-Zhu

    2015-08-01

    Pachymic acid (PA), a lanostane-type triterpenoid and the major component of Poria cocos alcoholic extracts, has various pharmacological effects, including anti-inflammatory, anti-oxidative and anti-apoptotic. However, few studies have investigated the effects of PA on lipopolysaccharide (LPS)-induced H9c2 cell apoptosis and inflammation, or identified the possible mechanisms underlying these effects. In the present study, H9c2 cardiomyocytes were stimulated by LPS and treated with or without PA. The increased mRNA expression levels of tumor necrosis factor-?, interleukin (IL)-1 and IL-6 induced by LPS were attenuated following treatment with PA. PA also attenuated LPS-induced apoptosis, as determined by a terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and regulated the LPS-induced protein expression levels of caspase 3, 8 and 9. Furthermore, the phosphorylations of extracellular-regulated kinase (Erk)1/2 and p38 in the LPS-treated H9c2 cells were inhibited by PA. These results suggested that treatment with PA prevented the LPS-induced inflammatory and apoptotic response in cardiomyocytes, which may be mediated by inhibition of the Erk1/2 and p38 pathways. PMID:25936656

  19. Norepinephrine-induced apoptotic and hypertrophic responses in H9c2 cardiac myoblasts are characterized by different repertoire of reactive oxygen species generation

    PubMed Central

    Thakur, Anita; Alam, Md. Jahangir; Ajayakumar, MR; Ghaskadbi, Saroj; Sharma, Manish; Goswami, Shyamal K.

    2015-01-01

    Despite recent advances, the role of ROS in mediating hypertrophic and apoptotic responses in cardiac myocytes elicited by norepinephrine (NE) is rather poorly understood. We demonstrate through our experiments that H9c2 cardiac myoblasts treated with 2 µM NE (hypertrophic dose) generate DCFH-DA positive ROS only for 2 h; while those treated with 100 µM NE (apoptotic dose) sustains generation for 48 h, followed by apoptosis. Though the levels of DCFH fluorescence were comparable at early time points in the two treatment sets, its quenching by DPI, catalase and MnTmPyP suggested the existence of a different repertoire of ROS. Both doses of NE also induced moderate levels of H2O2 but with different kinetics. Sustained but intermittent generation of highly reactive species detectable by HPF was seen in both treatment sets but no peroxynitrite was generated in either conditions. Sustained generation of hydroxyl radicals with no appreciable differences were noticed in both treatment sets. Nevertheless, despite similar profile of ROS generation between the two conditions, extensive DNA damage as evident from the increase in 8-OH-dG content, formation of ?-H2AX and PARP cleavage was seen only in cells treated with the higher dose of NE. We therefore conclude that hypertrophic and apoptotic doses of NE generate distinct but comparable repertoire of ROS/RNS leading to two very distinct downstream responses. PMID:26070033

  20. Estrogen receptor ? mediates the effects of notoginsenoside R1 on endotoxin-induced inflammatory and apoptotic responses in H9c2 cardiomyocytes.

    PubMed

    Zhong, Lei; Zhou, Xing-Lu; Liu, Yan-Song; Wang, Yi-Min; Ma, Fei; Guo, Bao-Liang; Yan, Zhao-Qi; Zhang, Qing-Yuan

    2015-07-01

    Estrogen receptors (ERs) are important for preventing endotoxin-induced myocardial dysfunction. Therefore, plant-derived phytoestrogens, which target ERs may also affect endotoxin-induced toxicity in cardiomyocytes. Our previous study revealed that notoginsenoside-R1 (NG-R1), a predominant phytoestrogen from Panax notoginseng, protects against cardiac dysfunction. However, the effects of NG-R1 on cardiomyocytes and the precise cellular/molecular mechanisms underlying its action remain to be elucidated. In the present study, pretreatment with NG-R1 suppressed the lipopolysaccharide (LPS)-induced degradation of inhibitor of nuclear factor-?B (NF-?B) ?, the activation of NF-?B and caspase-3, and the subsequent myocardial inflammatory and apoptotic responses in H9c2 cardiomyocytes. An increase in the mRNA and protein expression of ER? was also observed in the NG-R1-treated cardiomyocytes. However, the expression pattern of ER? remained unaltered. Furthermore, the cardioprotective properties of NG-R1 against LPS-induced apoptosis and the inflammatory response in cardiomyocytes were attenuated by ICI 182780, a non-selective ER? antagonist, and methyl-piperidino-pyrazole, a selective ER? antagonist. These findings suggested that NG-R1 reduced endotoxin-induced cardiomyocyte apoptosis and the inflammatory response via the activation of ER?. Therefore, NG-R1 exerted direct anti-inflammatory and anti-apoptotic effects on the cardiomyocytes, representing a potent agent for the treatment of myocardial inflammation during septic shock. PMID:25738436

  1. Dexamethasone effects on creatine kinase activity and insulin-like growth factor receptors in cultured muscle cells

    NASA Technical Reports Server (NTRS)

    Whitson, Peggy A.; Stuart, Charles A.; Huls, M. H.; Sams, Clarence F.; Cintron, Nitza M.

    1989-01-01

    The effect of dexamethasone on the activity of creatine kinase (CK) and the insulin-like growth factor I (IGF-I) binding were investigated using skeletal- and cardiac-muscle-derived cultured cell lines (mouse, C2C12; rat, L6 and H9c2). It was found that, in skeletal muscle cells, dexamethasone treatment during differentiation of skeletal-muscle cells caused dose-dependent increases in CK activity and increases in the degree of myotube formation, whereas cardiac cells (H9c2) exhibited very low CK activity during culture or dexamethasone treatment. Results for IGF-I binding were similar in all three cell lines. The IGF-I binding to dexamethasone-treated cells (50 nM for 24 hr on the day prior to confluence) resulted in an increased number of available binding sites, with no effect on the binding affinities.

  2. CLO: The cell line ontology

    PubMed Central

    2014-01-01

    Background Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO consortium, new cell line cells, upper level alignment with the Cell Ontology (CL) and the Ontology for Biomedical Investigation, and logical extensions. Construction and content Collaboration among the CLO, CL, and OBI has established consensus definitions of cell line-specific terms such as ‘cell line’, ‘cell line cell’, ‘cell line culturing’, and ‘mortal’ vs. ‘immortal cell line cell’. A cell line is a genetically stable cultured cell population that contains individual cell line cells. The hierarchical structure of the CLO is built based on the hierarchy of the in vivo cell types defined in CL and tissue types (from which cell line cells are derived) defined in the UBERON cross-species anatomy ontology. The new hierarchical structure makes it easier to browse, query, and perform automated classification. We have recently added classes representing more than 2,000 cell line cells from the RIKEN BRC Cell Bank to CLO. Overall, the CLO now contains ~38,000 classes of specific cell line cells derived from over 200 in vivo cell types from various organisms. Utility and discussion The CLO has been applied to different biomedical research studies. Example case studies include annotation and analysis of EBI ArrayExpress data, bioassays, and host-vaccine/pathogen interaction. CLO’s utility goes beyond a catalogue of cell line types. The alignment of the CLO with related ontologies combined with the use of ontological reasoners will support sophisticated inferencing to advance translational informatics development. PMID:25852852

  3. Pediatric brain tumor cell lines.

    PubMed

    Xu, Jingying; Margol, Ashley; Asgharzadeh, Shahab; Erdreich-Epstein, Anat

    2015-02-01

    Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research. PMID:25211508

  4. Thyroid cell lines in research on goitrogenesis.

    PubMed

    Gerber, H; Peter, H J; Asmis, L; Studer, H

    1991-12-01

    Thyroid cell lines have contributed a lot to the understanding of goitrogenesis. The cell lines mostly used in thyroid research are briefly discussed, namely the rat thyroid cell lines FRTL and FRTL-5, the porcine thyroid cell lines PORTHOS and ARTHOS, The sheep thyroid cell lines OVNIS 5H and 6H, the cat thyroid cell lines PETCAT 1 to 4 and ROMCAT, and the human thyroid cell lines FTC-133 and HTh 74. Chinese hamster ovary (CHO) cells and COS-7 cells, stably transfected with TSH receptor cDNA and expressing a functional TSH receptor, are discussed as examples for non-thyroidal cells, transfected with thyroid genes. PMID:1726925

  5. Molluscan cells in culture: primary cell cultures and cell lines

    PubMed Central

    Yoshino, T. P.; Bickham, U.; Bayne, C. J.

    2013-01-01

    In vitro cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information unattainable using intact animal models. In vitro cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail Biomphalaria glabrata Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome. PMID:24198436

  6. Immortalization of Mouse Germ Line Stem Cells

    PubMed Central

    Hofmann, Marie-Claude; Braydich-Stolle, Laura; Dettin, Luis; Johnson, Eric; Dym, Martin

    2011-01-01

    In the mammalian testis, the germ line stem cells are a small subpopulation of type A spermatogonia that proliferate and ultimately differentiate into sperm under the control of both endocrine and paracrine factors. To study the early phases of spermatogenesis at the molecular level, an in vitro system must be devised whereby germ line stem cells can be either cultured for a prolonged period of time or expanded as cell lines. In the study reported here, we chose to immortalize type A spermatogonia using the Simian virus large T-antigen gene (LTAg) under the control of an ecdysone-inducible promoter. While the cells escaped the hormonal control after a finite number of generations and expressed the LTAg constitutively, their growth remained slow and the cells exhibited morphological features typical of spermatogonia at the light microscopic level. Moreover, the cells expressed detectable levels of protein markers specific for germ cells such as Dazl, and specific for germ line stem cells such as Oct-4, a transcription factor, and GFR?-1, the receptor for glial cell line–derived neurotrophic factor (GDNF). Further analysis confirmed the spermatogonial phenotype and also revealed the expression of markers expressed in stem cells such as Piwi12 and Prame11. Since the cells respond to GDNF by a marked increase in their rate of proliferation, this cell line represents a good in vitro model for studying aspects of mouse germ line stem cell biology. PMID:15671143

  7. Biophysical Profiling of Tumor Cell Lines

    PubMed Central

    Coffman, Frederick; Hamid, Rachid; Cohen, Marion C.; Garippa, Ralph; Cohen, Stanley

    2011-01-01

    Despite significant differences in genetic profiles, cancer cells share common phenotypic properties, including membrane-associated changes that facilitate invasion and metastasis. The Corning Epic® optical biosensor was used to monitor dynamic mass rearrangements within and proximal to the cell membrane in tumor cell lines derived from cancers of the colon, bone, cervix, lung and breast. Data was collected in real time and required no exogenously added signaling moiety (signal-free technology). Cell lines displayed unique profiles over the time-courses: the time-courses all displayed initial signal increases to maximal values, but the rate of increase to those maxima and the value of those maxima were distinct for each cell line. The rate of decline following the maxima also differed among cell lines. There were correlations between the signal maxima and the observed metastatic behavior of the cells in xenograft experiments; for most cell types the cells that were more highly metastatic in mice had lower time-course maxima values, however the reverse was seen in breast cancer cells. The unique profiles of these cell lines and the correlation of at least one profile characteristic with metastatic behavior demonstrate the potential utility of biophysical tumor cell profiling in the study of cancer biology. PMID:21988886

  8. Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

    PubMed Central

    Penzo-Méndez, Alfredo I.; Chen, Yi-Ju; Li, Jinyang; Witze, Eric S.; Stanger, Ben Z.

    2015-01-01

    Cell competition is a form of cell-cell interaction by which cells compare relative levels of fitness, resulting in the active elimination of less-fit cells, “losers,” by more-fit cells, “winners.” Here, we show that in three routinely-used mammalian cell lines – U2OS, 3T3, and MDCK cells – sub-clones arise stochastically that exhibit context-dependent competitive behavior. Specifically, cell death is elicited when winner and loser sub-clones are cultured together but not alone. Cell competition and elimination in these cell lines is caspase-dependent and requires cell-cell contact but does not require de novo RNA synthesis. Moreover, we show that the phenomenon involves differences in cellular metabolism. Hence, our study demonstrates that cell competition is a common feature of immortalized mammalian cells in vitro and implicates cellular metabolism as a mechanism by which cells sense relative levels of “fitness.” PMID:26200654

  9. SHORT REPORT Open Access Establishment of three cell lines from

    E-print Network

    Gray, Matthew

    , thymus cell line (GSTC), spleen cell line (GSSC) and kidney cell line (GSKC) were initially established, killed and wiped with 75 v/v ethanol, and the thymus, spleen and kidney tissues were removed for primary

  10. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ...part of the Identification of Human Cell Lines Project. All data and corresponding...database. The use of misidentified cell lines in cancer and other biomedical research continues...literature describing studies employing cell lines may be misleading or...

  11. Cancer stem cell-like cells from a single cell of oral squamous carcinoma cell lines

    SciTech Connect

    Felthaus, O.; Department of Oral and Maxillofacial Surgery, University of Regensburg ; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

    Research highlights: {yields} Four oral squamous cancer cell lines (OSCCL) were analyzed for cancer stem cells (CSCs). {yields} Single cell derived colonies of OSCCL express CSC-marker CD133 differentially. {yields} Monoclonal cell lines showed reduced sensitivity for Paclitaxel. {yields} In situ CD133{sup +} cells are slow cycling (Ki67-) indicating a reduced drug sensitivity. {yields} CD133{sup +} and CSC-like cells can be obtained from single colony forming cells of OSCCL. -- Abstract: Resistance of oral squamous cell carcinomas (OSCC) to conventional chemotherapy or radiation therapy might be due to cancer stem cells (CSCs). The development of novel anticancer drugs requires a simple method for the enrichment of CSCs. CSCs can be enriched from OSCC cell lines, for example, after cultivation in serum-free cell culture medium (SFM). In our study, we analyzed four OSCC cell lines for the presence of CSCs. CSC-like cells could not be enriched with SFM. However, cell lines obtained from holoclone colonies showed CSC-like properties such as a reduced rate of cell proliferation and a reduced sensitivity to Paclitaxel in comparison to cells from the parental lineage. Moreover, these cell lines differentially expressed the CSC-marker CD133, which is also upregulated in OSCC tissues. Interestingly, CD133{sup +} cells in OSCC tissues expressed little to no Ki67, the cell proliferation marker that also indicates reduced drug sensitivity. Our study shows a method for the isolation of CSC-like cell lines from OSCC cell lines. These CSC-like cell lines could be new targets for the development of anticancer drugs under in vitro conditions.

  12. Tuft (caveolated) cells in two human colon carcinoma cell lines.

    PubMed Central

    Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

    1988-01-01

    The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:3414781

  13. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W.

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  14. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton (Palos Park, IL); Cook, Glenn M. (Naperville, IL)

    1987-01-01

    Apparatus for processing a metallic fluid containing iron oxide, container for a molten metal including an electrically conductive refractory disposed for contact with the molten metal which contains iron oxide, an electrolyte in the form of a basic slag on top of the molten metal, an electrode in the container in contcat with the slag electrically separated from the refractory, and means for establishing a voltage across the refractory and the electrode to reduce iron oxide to iron at the surface of the refractory in contact with the iron oxide containing fluid. A process is disclosed for refining an iron product containing not more than about 10% by weight oxygen and not more than about 10% by weight sulfur, comprising providing an electrolyte of a slag containing one or more of calcium oxide, magnesium oxide, silica or alumina, providing a cathode of the iron product in contact with the electrolyte, providing an anode in contact with the electrolyte electrically separated from the cathode, and operating an electrochemical cell formed by the anode, the cathode and the electrolyte to separate oxygen or sulfur present in the iron product therefrom.

  15. Antiproliferative efficacy of Tabernaemontana divaricata against HEP2 cell line and Vero cell line

    PubMed Central

    Kumar, Arvind; Selvakumar, S.

    2015-01-01

    Background: Laryngeal cancer may also be called cancer of the larynx or laryngeal carcinoma. Conventional plants are a precious source of novel anticancer agents and are still in performance better role in health concern. The study was intended to estimation of the anticancer activity of the chloroformic extract of Tabernaemontana divaricata on the human epidermoid larynx carcinoma cell line (Hep 2). Materials and Method: The aerial parts (leaves, stem, and flowers) of T. divaricata were tested for its inhibitory effect in 96 microplate formats against Hep 2 cell line. The anticancer activity of samples on Hep 2 and Vero was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and various enzymatic parameters like catalase, reduced glutathione (GSH), GSH peroxidase, and superoxide anion scavenging activity. Viable cells were determined by the absorbance at 540 nm. Measurements were performed, and the concentration required for a 50% inhibition of viability (IC50) was determined graphically. The effect of the samples on the proliferation of Hep 2 and Vero cells was expressed as the % cell viability. Results: The extract on Hep 2 cell line up to 7.8 ?g/ml and that IC50 value on Hep 2 cell line was 112 ?g whereas 94 ?g for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Conclusion: Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line). PMID:26109773

  16. The Clinical Relevance of Cancer Cell Lines

    PubMed Central

    2013-01-01

    Although advances in genomics during the last decade have opened new avenues for translational research and allowed the direct evaluation of clinical samples, there is still a need for reliable preclinical models to test therapeutic strategies. Human cancer-derived cell lines are the most widely used models to study the biology of cancer and to test hypotheses to improve the efficacy of cancer treatment. Since the development of the first cancer cell line, the clinical relevance of these models has been continuously questioned. Based upon recent studies that have fueled the debate, we review the major events in the development of the in vitro models and the emergence of new technologies that have revealed important issues and limitations concerning human cancer cell lines as models. All cancer cell lines do not have equal value as tumor models. Some have been successful, whereas others have failed. However, the success stories should not obscure the growing body of data that motivates us to develop new in vitro preclinical models that would substantially increase the success rate of new in vitro–assessed cancer treatments. PMID:23434901

  17. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect cell lines have been widely used in recombinant baculovirus expression systems and transient gene expression studies. Critical to these applications have been the transfection of foreign DNA. This has been widely done using labor intensive and cytotoxic liposome-based transfection reagents....

  18. Regulatory networks define phenotypic classes of human stem cell lines

    E-print Network

    Shamir, Ron

    LETTERS Regulatory networks define phenotypic classes of human stem cell lines Franz-Josef Mu¨ller1 called stem cells, even though they range from pluripotent cells--typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differenti- ation--to adult stem cell lines, which can

  19. On the ontology based representation of cell lines.

    PubMed

    Ganzinger, Matthias; He, Shan; Breuhahn, Kai; Knaup, Petra

    2012-01-01

    Cell lines are frequently used as highly standardized and reproducible in vitro models for biomedical analyses and assays. Cell lines are distributed by cell banks that operate databases describing their products. However, the description of the cell lines' properties are not standardized across different cell banks. Existing cell line-related ontologies mostly focus on the description of the cell lines' names, but do not cover aspects like the origin or optimal growth conditions. The objective of this work is to develop an ontology that allows for a more comprehensive description of cell lines and their metadata, which should cover the data elements provided by cell banks. This will provide the basis for the standardized annotation of cell lines and corresponding assays in biomedical research. In addition, the ontology will be the foundation for automated evaluation of such assays and their respective protocols in the future. To accomplish this, a broad range of cell bank databases as well as existing ontologies were analyzed in a comprehensive manner. We identified existing ontologies capable of covering different aspects of the cell line domain. However, not all data fields derived from the cell banks' databases could be mapped to existing ontologies. As a result, we created a new ontology called cell culture ontology (CCONT) integrating existing ontologies where possible. CCONT provides classes from the areas of cell line identification, origin, cell line properties, propagation and tests performed. PMID:23144907

  20. A resource for cell line authentication, annotation and quality control.

    PubMed

    Yu, Mamie; Selvaraj, Suresh K; Liang-Chu, May M Y; Aghajani, Sahar; Busse, Matthew; Yuan, Jean; Lee, Genee; Peale, Franklin; Klijn, Christiaan; Bourgon, Richard; Kaminker, Joshua S; Neve, Richard M

    2015-04-16

    Cell line misidentification, contamination and poor annotation affect scientific reproducibility. Here we outline simple measures to detect or avoid cross-contamination, present a framework for cell line annotation linked to short tandem repeat and single nucleotide polymorphism profiles, and provide a catalogue of synonymous cell lines. This resource will enable our community to eradicate the use of misidentified lines and generate credible cell-based data. PMID:25877200

  1. Many are called MDS cell lines: one is chosen.

    PubMed

    Drexler, Hans G; Dirks, Willy G; Macleod, Roderick A F

    2009-08-01

    Myelodysplastic syndromes (MDS) comprise a heterogenous group of clonal disorders of hematopoietic progenitors, showing genetic instability and in many cases progression to acute myeloid leukemia (AML). When MDS progress towards AML (AML/MDS), additional genetic lesions cause a block in differentiation and an accumulation of blast cells. Hence, both pathophysiologically and clinically the MDS and AML/MDS phases are distinguishable. Leukemia cell lines are key resources for modelling hematological malignancies. Characterization of these cell lines has provided a rich vein of insights into the mechanisms underlying malignant transformation. Some 31 cell lines have been described in the literature purportedly established from patients with MDS. However, a significant minority of these has proved false after DNA profiling which revealed their cross-contamination with older established leukemia cell lines. Most remaining ("authentic") MDS cell lines were established during the leukemic phase of the disease progression rather than during the MDS phase. Based on these data we have assigned the 31 candidate MDS cell lines to one of the three categories: (1) false (cross-contaminated) cell lines and non-malignant cell lines; (2) malignant cell lines established in the AML/MDS leukemic phase; and (3) apparently legitimate MDS cell lines established during the MDS phase. While MDS and AML/MDS cell lines both provide singular resources for modelling pathology, mining oncogenically modified macromolecules, and testing druggability, we contend these groups should be considered separately. PMID:19344951

  2. Personalized chemotherapy profiling using cancer cell lines from selectable mice

    PubMed Central

    Kamiyama, Hirohiko; Rauenzahn, Sherri; Shim, Joong Sup; Karikari, Collins A.; Feldmann, Georg; Hua, Li; Kamiyama, Mihoko; Schuler, F. William; Lin, Ming-Tseh; Beaty, Robert M.; Karanam, Balasubramanyam; Liang, Hong; Mullendore, Michael E.; Mo, Guanglan; Hidalgo, Manuel; Jaffee, Elizabeth; Hruban, Ralph H.; Jinnah, H. A.; Roden, Richard B. S.; Jimeno, Antonio; Liu, Jun O.; Maitra, Anirban; Eshleman, James R.

    2013-01-01

    Purpose High-throughput chemosensitivity testing of low-passage cancer cell lines can be used to prioritize agents for personalized chemotherapy. However, generating cell lines from primary cancers is difficult, because contaminating stromal cells overgrow the malignant cells. Experimental Design We produced a series of hypoxanthine phosphoribosyl transferase (hprt)-null immunodeficient mice. During growth of human cancers in these mice, hprt-null murine stromal cells replace their human counterparts. Results Pancreatic and ovarian cancers explanted from these mice were grown in selection media to produce pure human cancer cell lines. We screened one cell line with a 3,131-drug panel and identified seventy-seven FDA approved drugs with activity, including two novel drugs to which the cell line was uniquely sensitive. Xenografts of this carcinoma were selectively responsive to both drugs. Conclusion Chemotherapy can be personalized using patient-specific cell lines derived in biochemically selectable mice. PMID:23340293

  3. Derivation of three new human embryonic stem cell lines.

    PubMed

    Bradley, Cara K; Chami, Omar; Peura, Teija T; Bosman, Alexis; Dumevska, Biljana; Schmidt, Uli; Stojanov, Tomas

    2010-04-01

    Human embryonic stem cells are pluripotent cells capable of extensive self-renewal and differentiation to all cells of the embryo proper. Here, we describe the derivation and characterization of three Sydney IVF human embryonic stem cell lines not already reported elsewhere, designated SIVF001, SIVF002, and SIVF014. The cell lines display typical compact colony morphology of embryonic stem cells, have stable growth rates over more than 40 passages and are cytogenetically normal. Furthermore, the cell lines express pluripotency markers including Nanog, Oct4, SSEA3 and Tra-1-81, and are capable of generating teratoma cells derived from each of the three germ layers in immunodeficient mice. These experiments show that the cell lines constitute pluripotent stem cell lines. PMID:20198447

  4. Stable mammalian producer cell lines for structural biology.

    PubMed

    Büssow, Konrad

    2015-06-01

    The mammalian cell lines HEK293 and CHO have become important expression hosts in structural biology. Generating stable mammalian cell lines remains essential for studying the function and structure of recombinant proteins, despite the emergence of highly efficient transient transfection protocols. Production with stable cell lines can be scaled up easily and high volumetric product yield can be achieved. Protein structure reports of the past two years that used stable cell lines were surveyed for this review. Well-established techniques and novel approaches for generating stable cell lines and stable cell pools are presented, including cell sorting, site-specific recombination, transposons, the Lentivirus system and phage integrases. Host cell line optimization by endoglycosidase overexpression and sequence-specific genome engineering is highlighted. PMID:25804355

  5. Reporter cell lines for skin sensitization testing.

    PubMed

    Natsch, Andreas; Emter, Roger

    2015-10-01

    Skin sensitization has been described as an adverse outcome pathway (AOP), comprising a number of molecular events leading to the final adverse effect. In a new paradigm of toxicology, attempts are made to collect information using single mechanistic tests addressing different targets along such an AOP and to then integrate this information to arrive at a final toxicological prediction. This proposal is strongly influenced by the availability of methods for high-throughput screening of cellular events. Reporter cell lines are a particularly useful tool in such screening paradigms, as they can deliver highly reproducible and easily measureable results, and they can be designed to quantify induction or suppression at the transcription level of very specific molecular targets within cells. The first cell-based assay for skin sensitization, which has recently received ECVAM and OECD endorsement, is the reporter cell assay KeratinoSens™, reflecting activation of the Nrf2 pathway, and other assays measuring the Nrf2 pathway are under development or validation. An alternative approach (THP-G8) was recently developed based on activation of the Interleukin-8 gene. Here, we review these assays, their role in the AOP, their mechanistic interrelationships, their use for hazard and risk assessment, and their application in integrated testing strategies. At the same time, this study reviews (1) other cellular markers for sensitizers, and the potential to develop new reporter gene assays providing additional, non-redundant information, and (2) it presents approaches and new experimental data on attempts to further improve the predictivity of the existing assay. PMID:26194644

  6. The pursuit of ES cell lines of domesticated ungulates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In contrast to differentiated cells, embryonic stem cells (ESC) maintain an undifferentiated state, have the ability to self-renew, and exhibit pluripotency, i.e., they can give rise to most if not all somatic cell types and to the germ cells, egg and sperm. These characteristics make ES cell lines...

  7. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R. (Oakland, CA)

    1989-01-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo[a]pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors.

  8. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

    Substantially genetically stable continuous human cell lines derived from normal human mammary epithelial cells (HMEC) and processes for making and using the same. In a preferred embodiment, the cell lines are derived by treating normal human mammary epithelial tissue with a chemical carcinogen such as benzo(a)pyrene. The novel cell lines serve as useful substrates for elucidating the potential effects of a number of toxins, carcinogens and mutagens as well as of the addition of exogenous genetic material. The autogenic parent cells from which the cell lines are derived serve as convenient control samples for testing. The cell lines are not neoplastically transformed, although they have acquired several properties which distinguish them from their normal progenitors. 2 tabs.

  9. GREG cells, a dysferlin-deficient myogenic mouse cell line

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; Morree, Antoine de; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large ({approx} 200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority ({approx} 66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency.

  10. GREG cells, a dysferlin-deficient myogenic mouse cell line.

    PubMed

    Humphrey, Glen W; Mekhedov, Elena; Blank, Paul S; de Morree, Antoine; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-15

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency. PMID:22020321

  11. GREG cells, a dysferlin-deficient myogenic mouse cell line

    PubMed Central

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S.; de Morree, Antoine; Pekkurnaz, Gulcin; Nagaraju, Kanneboyina; Zimmerberg, Joshua

    2012-01-01

    The dysferlinopathies (e.g. LGMD2b, Myoshi myopathy) are progressive, adult-onset muscle wasting syndromes caused by mutations in the gene coding for dysferlin. Dysferlin is a large (~200 kDa) membrane-anchored protein, required for maintenance of plasmalemmal integrity in muscle fibers. To facilitate analysis of dysferlin function in muscle cells, we have established a dysferlin-deficient myogenic cell line (GREG cells) from the A/J mouse, a genetic model for dysferlinopathy. GREG cells have no detectable dysferlin expression, but proliferate normally in growth medium and fuse into functional myotubes in differentiation medium. GREG myotubes exhibit deficiencies in plasma membrane repair, as measured by laser wounding in the presence of FM1-43 dye. Under the wounding conditions used, the majority (~66%) of GREG myotubes lack membrane repair capacity, while no membrane repair deficiency was observed in dysferlin-normal C2C12 myotubes, assayed under the same conditions. We discuss the possibility that the observed heterogeneity in membrane resealing represents genetic compensation for dysferlin deficiency. PMID:22020321

  12. Analysis of gene amplification in human tumor cell lines

    SciTech Connect

    Fukumoto, M.; Shevrin, D.H.; Roninson, I.B.

    1988-09-01

    Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.

  13. The transcriptional diversity of 25 Drosophila cell lines

    SciTech Connect

    Cherbas, Lucy; Willingham, Aarron; Zhang, Dayu; Yang, Li; Zou, Yi; Eads, Brian D.; Carlson, Joseph W.; Landolin, Jane M.; Kapranov, Philipp; Dumais, Jacqueline; Samsonova, Anastasia; Choi, Jeong-Hyeon; Roberts, Johnny; Davis, Carrie A.; Tang, Haixu; van Baren, Marijke J.; Ghosh, Srinka; Dobin, Alexander; Bell, Kim; Lin, Wei; Langton, Laura; Duff, Michael O.; Tenney, Aaron E.; Zaleski, Chris; Brent, Michael R.; Hoskins, Roger A.; Kaufman, Thomas C.; Andrews, Justen; Graveley, Brenton R.; Perrimon, Norbert; Celniker, Susan E.; Gingeras, Thomas R.; Cherbas, Peter

    2010-11-15

    Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal discderived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. Wereport the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25 lines with emphasis on what those patterns reveal about the origins of the lines and the stability of spatial expression patterns. We also offer an initial analysis of previously unannotated transcripts in the cell lines.

  14. PI Control of Gene Expression in Tumorous Cell Lines 

    E-print Network

    Mendonca, Rouella J.

    2010-01-16

    in different genes in the Human Embryonic Kidney and Human Colon Adenocarcinoma Grade II cell lines. The difference in the gene expressions of the two cell lines motivates the problem in this thesis. The thesis provided intervention methods to make the colon...

  15. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... Cell Lines Project. All data and corresponding information will be posted in a publically held database... will be posted in a publically held database. The use of misidentified cell lines in cancer and other... suitable for use in a standard reference database. STR analysis involves simultaneous amplification...

  16. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines

    PubMed Central

    Eckerle, Isabella; Corman, Victor M.; Müller, Marcel A.; Lenk, Matthias; Ulrich, Rainer G.

    2014-01-01

    Replicative capacity of Middle East respiratory syndrome coronavirus (MERS-CoV) was assessed in cell lines derived from livestock and peridomestic small mammals on the Arabian Peninsula. Only cell lines originating from goats and camels showed efficient replication of MERS-CoV. These results provide direction in the search for the intermediate host of MERS-CoV. PMID:24457147

  17. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma

    Cancer.gov

    This technology describes the UOK 268 cell line, a spontaneously immortalized renal tumor cell line that may be of great interest to industry for studying HLRCC, drug screening, and searching for tumor markers related to diagnosis, prognosis, and drug resistance.

  18. Development of a conditionally immortalized human pancreatic ? cell line

    PubMed Central

    Scharfmann, Raphaël; Pechberty, Severine; Hazhouz, Yasmine; von Bülow, Manon; Bricout-Neveu, Emilie; Grenier-Godard, Maud; Guez, Fanny; Rachdi, Latif; Lohmann, Matthias; Czernichow, Paul; Ravassard, Philippe

    2014-01-01

    Diabetic patients exhibit a reduction in ? cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human ? cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human ? cell line (EndoC-?H1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-?H1 cells display many functional properties of adult ? cells, including expression of ? cell markers and insulin secretion following glucose stimulation; however, unlike primary ? cells, EndoC-?H1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human ? cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-?H2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of ? cell–specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-?H2 cells are highly representative of human ? cells and should be a valuable tool for further analysis of human ? cells. PMID:24667639

  19. Hepatitis C virus infection of neuroepithelioma cell lines

    PubMed Central

    Fletcher, Nicola F; Yang, Jian Ping; Farquhar, Michelle J; Hu, Ke; Davis, Christopher; He, Qiuchen; Dowd, Kimberly; Ray, Stuart C; Krieger, Sophie E; Neyts, Johan; Baumert, Thomas F; Balfe, Peter; McKeating, Jane A; Wong-Staal, Flossie

    2012-01-01

    Background & Aims Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication. Methods We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism. Results Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells. Conclusions Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo. PMID:20538002

  20. Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines.

    PubMed

    Luviano, Axel; Aguińiga-Sánchez, Itzen; Demare, Patricia; Tiburcio, Reynaldo; Ledesma-Martínez, Edgar; Santiago-Osorio, Edelmiro; Regla, Ignacio

    2014-05-01

    In the search for novel chemotherapeutic agents for cancer treatment, capsaicin has been shown to inhibit proliferation and induce apoptosis in various types of cancer cell line, including leukaemia cell lines. The capsaicin analogues, rinvanil and phenylacetylrinvanil (PhAR), share a binding affinity for vanilloid receptors and may have biological activities similar to capsaicin; however, their anticancer potential has not yet been reported. This study analyses the antineoplastic activities of rinvanil and PhAR in leukaemia versus normal cells. P388, J774 and WEHI-3 leukaemia cell lines, as well as mouse bone marrow mononuclear cells, were cultured with varying concentrations of rinvanil and PhAR. Following this, proliferation and apoptosis were determined by the sulforhodamine B (SRB) assay and DNA ladder. Cultured leukaemia cell lines and mouse bone marrow mononuclear cells demonstrated a dose-dependent inhibition of proliferation, while non-diseased cells were less sensitive to the cytotoxic effect of capsaicin, rinvanil and PhAR. Rinvanil and PhAR also induced apoptosis in leukaemia cell lines but not in bone marrow. Given the lower IC50 values for apoptosis induction in leukaemia cells compared with that of normal cells, PhAR is a promising selective anticancer agent. PMID:24765194

  1. A methylation profile of in vitro immortalized human cell lines.

    PubMed

    Liu, Limin; Zhang, Jingmei; Bates, Steven; Li, Jian-Jian; Peehl, Dana M; Rhim, Johng S; Pfeifer, Gerd P

    2005-01-01

    Normal human diploid cells have a limited life span and undergo replicative senescence after various limited population doublings. Cells must pass the senescence barrier to become immortal. The exact mechanisms of immortalization are not clear, although inactivation of the RB pathway, and/or the p53 pathway and activation of telomerase has been shown to be necessary for immortalization of certain cell types with DNA viruses or hTERT. Methylation-associated inactivation of tumor suppressor genes plays an important role in tumor progression. To test if gene-specific methylation contributes to the immortalized and transformed phenotype, we analyzed the methylation status of 17 genes in normal cells immortalized with SV40, hTERT, Ad5, Ad12-SV40 or HPV-18. Some of these immortalized lines were progressively transformed and tumorigenic in nude mice. We observed gene-specific methylation in the in vitro immortalized and transformed cells. SV40 and HPV18 immortalization resulted in different methylation spectra. In SV40- and h-TERT-immortalized prostate epithelial cells, the most frequently methylated gene was RASSF1A, while in HPV18-immortalized cell lines, the RAR-beta2 gene was universally methylated. Immortalization with SV40 resulted in methylation of a greater number of genes than immortalization with HPV. Furthermore, in SV40-immortalized cell lines, methylation affected different genes in fibroblasts compared with epithelial cells, suggesting that different mechanisms may be used by SV40 to immortalize cell lines of different origins. In HPV18-immortalized and subsequently transformed cell lines, the most commonly methylated genes were hormone responsive genes, such as AR, ER-beta and RAR-beta2. In general, more genes were methylated in neoplastically-transformed cell lines than in only immortalized cell lines, indicating that accumulation of epigenetic abnormalities may contribute to oncogenesis. PMID:15586250

  2. Thermoradiotherapy in human head and neck squamous cell carcinoma cell lines.

    PubMed

    Asaumi, Jun-Ichi; Higuchi, Yuzuru; Murakami, Jun; Kuroda, Masahiro; Shibuya, Koichi; Konouchi, Hironobu; Hisatomi, Miki; Matsuzaki, Hidenobu; Shigehara, Hiroshi; Kawasaki, Shoji; Kishi, Kanji; Hiraki, Yoshio

    2002-09-01

    Thermoradiosensitivity of 8 cell lines of head and neck squamous cell carcinoma (HO-1-u-1, HSC2, HSC3, HSC4, SAS, KB, Hep2, and Ca9-22) was investigated. The differences of radiosensitivity between the cell line with the highest radiosensitivity and the cell line with the lowest radiosensitivity were 1.7-, 7.7-, and 41-fold at 2, 6 and 8 Gy, respectively. The differences between the cell line with the highest thermosensitivity and the cell line with the lowest thermosensitivity were 2.4-, 6.2- and 34.4-fold at 43 degrees C for 40, 60 and 100 min, and 2.6-, 4.9- and 127-fold at 44 degrees C for 20, 30 and 50 min, respectively. These findings indicated that there were large differences in both radiosensitivity and thermosensitivity among the 8 cell lines. There was a negative relationship between radiosensitivity and thermosensitivity (43 degrees C: r=-0.600, 44 degrees C: r=-0.848) in 7 of 8 cell lines, the exception being the HSC4 cell line, which was resistant to both therapies. Four of the 8 cell lines at 43 degrees C and 5 at 44 degrees C in the radiotherapy combined with thermotherapy showed actual survival rates smaller than the theoretical survival rates. Thus, thermoradiotherapy was deemed effective in the head and neck carcinoma cell lines, although 1 of 8 cell lines was resistant to both radiotherapy and thermotherapy. PMID:12165802

  3. Radiosensitivity of hepatoma cell lines and human normal liver cell lines exposed to 12C6+ ions

    NASA Astrophysics Data System (ADS)

    Jing, X.; Yang, J.; Li, W.; Guo, C.; Dang, B.; Wang, J.; Zhou, L.; Wei, W.; Gao, Q.

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  4. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We describe the characterization of a spontaneously transformed chicken monocytic cell line that developed as a single colony of cells in a heterophil culture that was inadvertently left in the incubator over a period of 25 days. These cells, hitherto named HTC, grow efficiently at both 37 C or 41 C...

  5. A Stable Cranial Neural Crest Cell Line from Mouse

    PubMed Central

    Ishii, Mamoru; Arias, Athena C.; Liu, Liqiong; Chen, Yi-Bu; Bronner, Marianne E.

    2012-01-01

    Cranial neural crest cells give rise to ectomesenchymal derivatives such as cranial bones, cartilage, smooth muscle, dentin, as well as melanocytes, corneal endothelial cells, and neurons and glial cells of the peripheral nervous system. Previous studies have suggested that although multipotent stem-like cells may exist during the course of cranial neural crest development, they are transient, undergoing lineage restriction early in embryonic development. We have developed culture conditions that allow cranial neural crest cells to be grown as multipotent stem-like cells. With these methods, we obtained 2 independent cell lines, O9-1 and i10-1, which were derived from mass cultures of Wnt1-Cre; R26R-GFP-expressing cells. These cell lines can be propagated and passaged indefinitely, and can differentiate into osteoblasts, chondrocytes, smooth muscle cells, and glial cells. Whole-genome expression profiling of O9-1 cells revealed that this line stably expresses stem cell markers (CD44, Sca-1, and Bmi1) and neural crest markers (AP-2?, Twist1, Sox9, Myc, Ets1, Dlx1, Dlx2, Crabp1, Epha2, and Itgb1). O9-1 cells are capable of contributing to cranial mesenchymal (osteoblast and smooth muscle) neural crest fates when injected into E13.5 mouse cranial tissue explants and chicken embryos. These results suggest that O9-1 cells represent multipotent mesenchymal cranial neural crest cells. The O9-1 cell line should serve as a useful tool for investigating the molecular properties of differentiating cranial neural crest cells. PMID:22889333

  6. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

    Nano, J.L.; Cefai, D.; Rampal, P. )

    1990-02-01

    The effect of exposure of an intestinal epithelial cell line to various concentrations of ethanol (217 mM (1%) to 652 mM (3%)) during 24, 48, and 72 hr was investigated in vitro using a rat intestinal epithelial cell line (IRD 98). Incubation of these cells in the presence of ethanol significantly decreased cell growth. This inhibition was accompanied by a strong increase in cellular protein. Stimulation of specific disaccharidases, gamma-glutamyl transferase, and aminopeptidase activities by ethanol was dose- and time-dependent. Ethanol induces a change in the relative proportions of the different lipid classes synthesized; triglycerides, fatty acids, and cholesterol esters were preferentially synthethysed. Our findings show that cell lines are good models for investigation of the effects of ethanol, and that alcohol considerably modifies the functions of intestinal epithelial cells.

  7. MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES

    SciTech Connect

    Han, Ju; Chang, Hang; Fontenay, Gerald; Wang, Nicholas J.; Gray, Joe W.; Parvin, Bahram

    2009-05-08

    A panel of cell lines of diverse molecular background offers an improved model system for high-content screening, comparative analysis, and cell systems biology. A computational pipeline has been developed to collect images from cell-based assays, segment individual cells and colonies, represent segmented objects in a multidimensional space, and cluster them for identifying distinct subpopulations. While each segmentation strategy can vary for different imaging assays, representation and subpopulation analysis share a common thread. Application of this pipeline to a library of 41 breast cancer cell lines is demonstrated. These cell lines are grown in 2D and imaged through immunofluorescence microscopy. Subpopulations in this panel are identified and shown to correlate with previous subtyping literature that was derived from transcript data.

  8. Human Cell Line for Studies on Signaling and Endocrine Cancer

    Cancer.gov

    The first known immortalized cell line with a naturally-occurring inactivating mutation in PRKAR1A, the regulatory subunit type 1A (R1alpha) of protein kinase A (PKA), which is associated with tumor formation.

  9. Embryonic germ cell lines and their derivation from mouse primordial germ cells.

    PubMed

    Labosky, P A; Barlow, D P; Hogan, B L

    1994-01-01

    When primordial germ cells of the mouse are cultured on feeder layers with the addition of the polypeptide signalling molecules leukaemia inhibitory factor, Steel factor and basic fibroblast growth factor they give rise to cells that resemble undifferentiated blastocyst-derived embryonic stem cells. These primordial germ cell-derived embryonic germ cells (EG cells) can be induced to differentiate extensively in culture and also form teratocarcinomas when injected into nude mice. Additionally, they contribute to chimeras when injected into host blastocysts. We have derived multiple EG cell lines from 8.5 days post coitum (dpc) embryos of C57BL/6 inbred mice. Four independent EG cell lines with normal male karyotypes have formed chimeras (up to 70% coat colour chimerism) when injected into BALB/c host blastocysts. Chimeric mice from all four cell lines are fertile, but only those from one line have transmitted coat colour markers through the germline. Studies have also been carried out to determine whether gonadal primordial germ cells can give rise to pluripotent EG cells. Germ cells from gonads of 15.5 dpc C57BL/6 embryos and newborn mice failed to produce EG cell lines. EG cell lines capable of forming teratocarcinomas and coat colour chimeras have been established from primordial germ cells of 12.5 dpc genital ridges. We are currently testing the genomic imprinting status of the insulin-like growth factor type 2 receptor gene (Igf2r) in our different EG cell lines. PMID:7835148

  10. Metronidazole decreases viability of DLD-1 colorectal cancer cell line.

    PubMed

    Sadowska, Anna; Kr?towski, Rafa?; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

    The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50??g/mL after 24 hours; 0.1, 10, 50, and 250??g/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  11. Metronidazole Decreases Viability of DLD-1 Colorectal Cancer Cell Line

    PubMed Central

    Sadowska, Anna; Kr?towski, Rafa?; Szynaka, Beata; Cechowska-Pasko, Marzanna

    2013-01-01

    Abstract The aim of our study was to evaluate the impact of metronidazole (MTZ) on DLD-1 colorectal cancer cell (CRC) line. Toxicity of MTZ was determined by MTT test. Cells were incubated with MTZ used in different concentrations for 24, 48, and 72 hours. The effect of MTZ on DNA synthesis was measured as [3H]-thymidine incorporation. The morphological changes in human DLD-1 cell line were defined by transmission electron microscope OPTON 900. The influence of MTZ on the apoptosis of DLD-1 cell lines was detected by flow cytometry and fluorescence microscopy, while cell concentration, volume, and diameter were displayed by Scepter Cell Counter from Millipore. Our results show that cell viability was diminished in all experimental groups in comparison with the control, and the differences were statistically significant. We did not find any significant differences in [3H]-thymidine incorporation in all experimental groups and times of observation. Cytofluorimetric assays demonstrated a statistically significant increase of apoptotic rate in MTZ concentrations 10 and 50??g/mL after 24 hours; 0.1, 10, 50, and 250??g/mL after 48 hours; and in all concentrations after 72 hours compared with control groups. In the ultrastructural studies, necrotic or apoptotic cells were occasionally seen. In conclusion, MTZ affects human CRC cell line viability. The reduction of cell viability was consistent with the apoptotic test. PMID:23777253

  12. Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

    SciTech Connect

    Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A. )

    1991-01-01

    1-((4-Amino-2-methylpyrimidin-5-yl)methyl)-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links.

  13. Screening Services – NCI-60 DTP Human Tumor Cell Line Screen

    Cancer.gov

    The In Vitro Cell Line Screening Project (IVCLSP) is a dedicated service providing direct support to the DTP anticancer drug discovery program. The in vitro cell line screen was implemented in fully operational form in April of 1990. It required approximately five years (1985 - 1990) to develop, and persistence in the effort reflected dissatisfaction with the performance of prior in vivo primary screens. This project is designed to screen up to 3,000 compounds per year for potential anticancer activity.

  14. Pharmacogenomic agreement between two cancer cell line data sets.

    PubMed

    2015-12-01

    Large cancer cell line collections broadly capture the genomic diversity of human cancers and provide valuable insight into anti-cancer drug response. Here we show substantial agreement and biological consilience between drug sensitivity measurements and their associated genomic predictors from two publicly available large-scale pharmacogenomics resources: The Cancer Cell Line Encyclopedia and the Genomics of Drug Sensitivity in Cancer databases. PMID:26570998

  15. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  16. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...Comment Request; Identification of Human Cell Lines Project AGENCY: National Institute...repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and...

  17. Two small cell lung cancer cell lines established from rigid bronchoscope biopsies.

    PubMed

    Postmus, P E; de Ley, L; van der Veen, A Y; Mesander, G; Buys, C H; Elema, J D

    1988-04-01

    Two new, good growing cell lines (GLC-8, GLC-11) have been established from biopsies of small cell lung cancer (SCLC). Tumor biopsies were procured by rigid bronchoscopy from tumor recurrences at the site of the primary lesions. Both tumors were clinically resistant to chemotherapy. Cytogenetic analysis revealed deletions in the short arm of chromosome 3. GLC-8 shows amplification of N-myc. Both cell lines show SCLC differentiations; neurosecretory granules were present and the SCLC related hormones dopa-decarboxylase and creatine kinase were elevated. Both cell lines behave as so-called 'classic' SCLC cell lines. PMID:2838297

  18. Exometabolom analysis of breast cancer cell lines: Metabolic signature

    PubMed Central

    Willmann, Lucas; Erbes, Thalia; Halbach, Sebastian; Brummer, Tilman; Jäger, Markus; Hirschfeld, Marc; Fehm, Tanja; Neubauer, Hans; Stickeler, Elmar; Kammerer, Bernd

    2015-01-01

    Cancer cells show characteristic effects on cellular turnover and DNA/RNA modifications leading to elevated levels of excreted modified nucleosides. We investigated the molecular signature of different subtypes of breast cancer cell lines and the breast epithelial cell line MCF-10A. Prepurification of cell culture supernatants was performed by cis-diol specific affinity chromatography using boronate-derivatized polyacrylamide gel. Samples were analyzed by application of reversed phase chromatography coupled to a triple quadrupole mass spectrometer. Collectively, we determined 23 compounds from RNA metabolism, two from purine metabolism, five from polyamine/methionine cycle, one from histidine metabolism and two from nicotinate and nicotinamide metabolism. We observed major differences of metabolite excretion pattern between the breast cancer cell lines and MCF-10A, just as well as between the different breast cancer cell lines themselves. Differences in metabolite excretion resulting from cancerous metabolism can be integrated into altered processes on the cellular level. Modified nucleosides have great potential as biomarkers in due consideration of the heterogeneity of breast cancer that is reflected by the different molecular subtypes of breast cancer. Our data suggests that the metabolic signature of breast cancer cell lines might be a more subtype-specific tool to predict breast cancer, rather than a universal approach. PMID:26293811

  19. BHD Tumor Cell Line and UOK257-2 wild type FLCN-restored Renal Cell Line

    Cancer.gov

    Center for Cancer Research, Urologic Oncology Branch is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize kidney cancer tumor cell lines.

  20. Induction of apoptosis by ubenimex (Bestatin) in human non-small-cell lung cancer cell lines.

    PubMed

    Ezawa, K; Minato, K; Dobashi, K

    1996-01-01

    We studied the direct anti-tumor effects of ubenimex on five human lung cancer cell lines; ABC-1, RERF-LC-OK, RERF-LC-MS (adenocarcinoma) and SQ-5, EBC-1 (squamous cell carcinoma). Ubenimex dose-dependently inhibited the growth of these cancer cell lines except RERF-LC-MS. The results indicated that lung squamous cell carcinoma cell lines were more sensitive to ubenimex than lung adenocarcinoma cell lines. Coincidentally, histological observation by Hematoxylin eosine (HE) staining revealed that ubenimex induced nuclear condensation and apoptic body in the cancer cell lines. Immunohistochemical study showed ubenimex-treated cells expressed LeY antigen which is a useful phenotypic marker predictive of apoptosis. The induction of DNA fragmentation was also observed in the ubenimex treated cancer cell lines by ELISA. We conclude that ubenimex exhibits its direct anti-tumor effect against non-small-cell lung cancer cell lines, more effectively against squamous carcinoma cell lines, through the induction of apoptosis. PMID:8952869

  1. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    EPA Science Inventory

    We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

  2. Continuous production of erythropoietin by an established human renal carcinoma cell line: development of the cell line

    SciTech Connect

    Sherwood, J.B.; Shouval, D.

    1986-01-01

    Establishment of a stable, transformed human renal carcinoma cell line that produces erythropoietin in vitro and has maintained this function continuously since 1981 and for > 150 passages in monolayer culture was accomplished by transplantation of human renal clear cell carcinoma tissue from a patient with erythrocytosis into an immunosuppressed athymic mouse. In addition to its immunocrossreactivity with native human urinary erythropoietin, the tumor erythropoietin demonstrates biological activity in the in vitro mouse erythroid colony-forming unit assay and in tumor-bearing nude mice. The cloned renal carcinoma cell line has an abnormal human karyotype and has ultrastructural features characteristic of human renal clear cell carcinoma. This cell line provides a reproducible model system for the production of an erythropoietin-like material and for the study of its synthesis and secretion.

  3. Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

    PubMed Central

    2010-01-01

    Background The treatment of oral squamous cell carcinomas (OSCC) and human osteosarcoma (HOS) includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang) on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20%) were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50) for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ?80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis. PMID:20840769

  4. Guidelines for the use of cell lines in biomedical research

    PubMed Central

    Geraghty, R J; Capes-Davis, A; Davis, J M; Downward, J; Freshney, R I; Knezevic, I; Lovell-Badge, R; Masters, J R W; Meredith, J; Stacey, G N; Thraves, P; Vias, M

    2014-01-01

    Cell-line misidentification and contamination with microorganisms, such as mycoplasma, together with instability, both genetic and phenotypic, are among the problems that continue to affect cell culture. Many of these problems are avoidable with the necessary foresight, and these Guidelines have been prepared to provide those new to the field and others engaged in teaching and instruction with the information necessary to increase their awareness of the problems and to enable them to deal with them effectively. The Guidelines cover areas such as development, acquisition, authentication, cryopreservation, transfer of cell lines between laboratories, microbial contamination, characterisation, instability and misidentification. Advice is also given on complying with current legal and ethical requirements when deriving cell lines from human and animal tissues, the selection and maintenance of equipment and how to deal with problems that may arise. PMID:25117809

  5. Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines

    PubMed Central

    Crameri, Gary; Todd, Shawn; Grimley, Samantha; McEachern, Jennifer A.; Marsh, Glenn A.; Smith, Craig; Tachedjian, Mary; De Jong, Carol; Virtue, Elena R.; Yu, Meng; Bulach, Dieter; Liu, Jun-Ping; Michalski, Wojtek P.; Middleton, Deborah; Field, Hume E.; Wang, Lin-Fa

    2009-01-01

    Background Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. Methodology/Findings Black flying foxes (Pteropus alecto) were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. Conclusions/Significance The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study. PMID:20011515

  6. Skin Biopsy and Patient-Specific Stem Cell Lines.

    PubMed

    Li, Yao; Nguyen, Huy V; Tsang, Stephen H

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  7. Implantation of Vascular Grafts Lined with Genetically Modified Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Wilson, James M.; Birinyi, Louis K.; Salomon, Robert N.; Libby, Peter; Callow, Allan D.; Mulligan, Richard C.

    1989-06-01

    The possibility of using the vascular endothelial cell as a target for gene replacement therapy was explored. Recombinant retroviruses were used to transduce the lacZ gene into endothelial cells harvested from mongrel dogs. Prosthetic vascular grafts seeded with the genetically modified cells were implanted as carotid interposition grafts into the dogs from which the original cells were harvested. Analysis of the graft 5 weeks after implantation revealed genetically modified endothelial cells lining the luminal surface of the graft. This technology could be used in the treatment of atherosclerosis disease and the design of new drug delivery systems.

  8. Comparative antibiotic eradication of mycoplasma infections from continuous cell lines.

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2002-02-01

    Accumulating data implicate mycoplasma contamination as the single biggest problem in the culture of continuous cell lines. Mycoplasma infection can affect virtually every parameter and functional activity of the eukaryotic cells. A successful alternative to discarding infected cultures is to attempt to eliminate the contaminants by treatment with specific and efficient antimycoplasma antibiotics. The addition of antibiotics to the culture medium during a limited period of time (1-3 wk) is a simple, inexpensive, and very practical approach for decontaminating continuous cell lines. Here, we examined the effectiveness of several antibiotic treatment protocols that we have employed routinely in our cell lines bank. On an aggregate, 673 cultures from 236 chronically mycoplasma-positive cell lines were exposed to one of the following five antibiotic regimens: mycoplasma removal agent (quinolone; a 1-wk treatment), enrofloxacin (quinolone; 1 wk), sparfloxacin (quinolone; 1 wk), ciprofloxacin (quinolone; 2 wk), and BM-Cyclin (alternating tiamulin and minocycline; 3 wk). The mycoplasma infection was permanently (as determined by three solid mycoplasma detection assays) eliminated by the various antibiotics in 66-85% of the cultures treated. Mycoplasma resistance was seen in 7-21%, and loss of the culture as a result of cytotoxically caused cell death occurred in 3-11% of the cultures treated. Overall, 223 of the 236 mycoplasma-positive cell lines could be cured in a first round of antibiotic treatment with at least one regimen. Taken together, 95% of the mycoplasma-infected cell lines were permanently cleansed of the contaminants by antibiotic treatment, which validates this approach as an efficient and technically simple mycoplasma eradication method. PMID:11929000

  9. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

    Radiation effects, such as bystander effects, hyper radiosensitivity/induced radioresistance (HRS/IRR) and adaptive response that are not related to direct DNA damage are now accepted. However the inter-relationship between them and the possible impact on the scientific basis for radiation protection are highly controversial. This thesis attempts to elucidate the mechanisms of some of these well known but little understood effects. Each paper examines some aspect of bystander effects, adaptive responses and HRS/IRR in an effort to understand how they vary with cell type, dose and time of exposure to single or multiple doses. All the effects involve non-linear dose effect curves and are mainly evident following low doses. Overall findings of the thesis include (1) A clear difference was observed between radioresistant, tumorigenic cell lines with mutant p53 gene expression, and radiosensitive, more normal, cell lines with wild type p53. In general death inducing bystander responses are induced in normal cell populations exposed to low doses of radiation while survival inducing IRR and adaptive responses are seen in the radioresistant tumorigenic cell lines. (2) A cohort of fish cell lines which demonstrated survival promoting bystander effects, also did not show a protective adaptive responses. (3) Adaptive responses traditionally occur when a large challenge dose is given 4--6hrs following low (10--100mGy) priming doses but this thesis shows that for the epithelial cell lines tested, the size of the priming dose (range 0.1--2Gy) does not appear to alter the size of the recovery response. Additionally increased survival could be detected in some cases when the challenge dose was given within one hour of the priming dose. The overall conclusion is that cell lines induce either a bystander response or a protective/adaptive response depending on genetic background and other factors. Care is needed in the interpretation of data generated from only one or two cell lines and in the extrapolation of mechanistic ideas based on one or two cell lines to other cell types or to the in vivo situation.

  10. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  11. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  12. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines

    PubMed Central

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Chen, Wei-Ching; Fang, Jung-Hua; Weng, Tzu-Yang; Chen, Yi-Ling

    2015-01-01

    Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines. PMID:25928182

  13. Argininosuccinate synthetase 1 suppression and arginine restriction inhibit cell migration in gastric cancer cell lines.

    PubMed

    Shan, Yan-Shen; Hsu, Hui-Ping; Lai, Ming-Derg; Yen, Meng-Chi; Chen, Wei-Ching; Fang, Jung-Hua; Weng, Tzu-Yang; Chen, Yi-Ling

    2015-01-01

    Gastric cancer metastasis remains a major cause of cancer-related deaths. There is an urgent need to develop new therapeutic approaches targeting metastatic gastric cancer. Argininosuccinate synthetase 1 (ASS1) expression is increased in gastric cancer. We detected the protein expression of ASS1 in human gastric cancer cell lines (AGS, NCI-N87, and MKN45) and in murine gastric cancer cell lines (3I and 3IB2). We used vector-mediated short hairpin RNA (shRNA) expression to silence ASS1 expression in the MKN45 and 3IB2 cell lines, and analyzed the effects of this protein on cell migration and metastasis. We demonstrated that ASS1 silencing suppressed cell migration in the MKN45 and 3IB2 cell lines. ASS1 knockdown significantly reduced liver metastasis in mice after the intrasplenic implantation of 3IB2 cancer cell clones. To determine whether arginine restriction may represent a therapeutic approach to treat gastric cancer, the sensitivity of tumor cells to arginine depletion was determined in gastric cancer cells. Arginine depletion significantly inhibited cell migration in the gastric cancer cell line. The silencing of ASS1 expression in MKN45 and 3IB2 gastric cancer cells markedly decreased STAT3 protein expression. In conclusion, our results indicate that the ASS1 protein is required for cell migration in gastric cancer cell lines. PMID:25928182

  14. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

    Vergara, D; Simeone, P; De Matteis, S; Carloni, S; Lanuti, P; Marchisio, M; Miscia, S; Rizzello, A; Napolitano, R; Agostinelli, C; Maffia, M

    2015-12-15

    Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies. PMID:26588820

  15. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

    Gonda, Steve R. (inventor); Marley, Garry M. (inventor)

    1991-01-01

    Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

  16. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines

    PubMed Central

    Cherbas, Lucy; Hackney, Jennifer; Gong, Lei; Salzer, Claire; Mauser, Eric; Zhang, Dayu; Cherbas, Peter

    2015-01-01

    We describe an adaptation of ?C31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  17. Determination of NAD+ and NADH level in a Single Cell Under H2O2 Stress by Capillary Electrophoresis

    SciTech Connect

    Wenjun Xi

    2008-08-18

    A capillary electrophoresis (CE) method is developed to determine both NAD{sup +} and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD{sup +} and 1 amol NADH on a home-made CE-LIF setup. The method showed good reproducibility and specificity. After an intact cell was injected into the inlet of a capillary and lysed using a Tesla coil, intracellular NAD{sup +} and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD{sup +} levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD{sup +} and NADH levels with and without exposure to oxidative stress induced by H{sub 2}O{sub 2}, it was found that H9c2 cells respond to the stress by reducing both cellular NAD{sup +} and NADH levels, while astrocytes respond by increasing cellular NADH/NAD{sup +} ratio.

  18. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

    Susan Shearer - Stark State College; Gregory Rush - Rolls-Royce Fuel Cell Systems

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to test fuel cell components at a scale and under conditions that can be accurately extrapolated to full system performance. This requires specially designed equipment that replicates the pressure (up to 6.5 bara), temperature (about 910 C), anode and cathode gas compositions, flows and power generation density of the full scale design. The SBTS fuel cell anode gas is produced through the reaction of pipeline natural gas with a mixture of steam, CO2, and O2 in a catalytic partial oxidation (CPOX) reactor. Production of the fuel cell anode gas in this manner provides the capability to test a fuel cell with varying anode gas compositions ranging from traditional reformed natural gas to a coal-syngas surrogate fuel. Stark State College and RRFCS have a history of collaboration. This is based upon SSCAs commitment to provide students with skills for advanced energy industries, and RRFCS need for a workforce that is skilled in high temperature fuel cell development and testing. A key to this approach is the access of students to unique SOFC test and evaluation equipment. This equipment is designed and developed by RRFCS, with the participation of SSC interns. In the near-term, the equipment will be used by RRFCS for technology development. When this stage is completed, and RRFCS has moved to commercial products, SSC will utilize this equipment for workforce training. The RRFCS fuel cell design is based upon a unique ceramic substrate architecture in which a porous, flat substrate (tube) provides the support structure for a network of solid oxide fuel cells that are electrically connected in series. These tubes are grouped into a {approx}350-tube repeat configuration, called a stack/block. Stack/block testing, performed at system conditions, provides data that can be confidently scaled to full scale performance. This is the basis for the specially designed and developed test equipment that is required for advancing and accelerating the RRFCS SOFC power system development program. All contract DE-EE0003229 objectives were achieved and deliverables completed during the peri

  19. Genomic alterations in oral squamous cell carcinoma cell lines detected by two-dimensional gel analysis.

    PubMed

    Yamamoto, K; Konishi, N; Inui, T; Kitahori, Y; Hiasa, Y; Kirita, T; Sugimura, M

    1998-11-01

    To initially analyze the genomic abnormalities in human oral squamous cell carcinoma, DNA extracted from each of four oral carcinoma cell lines (Ca9-22, HO-1-u-1, HSC-2, KB) was examined using restriction landmark genomic scanning (RLGS), a method especially conducive to detection of amplifications and rearrangements of genomic DNA. Isolated cell line and normal oral epithial DNAs were sequentially cleaved with specific restriction enzymes, radiolabelled and separated in two-dimensional gel electrophoreses. Thirteen distinct fragments were commonly amplified in the oral cancer cell lines, six of which were evident in all samples. These results suggest genetic alterations characteristic of oral squamous cell carcinogenesis. PMID:9930363

  20. Engineering retina from human retinal progenitors (cell lines).

    PubMed

    Dutt, Kamla; Cao, Yang

    2009-06-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell-cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr(2)e(3), expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription-polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell line retains its capacity to differentiate into multiple cell types holds great promise for the use of tissue-specific adult stem cells for therapy. PMID:19113950

  1. Validating classical line profile analyses using microbeam diffraction from individual dislocation cell walls and cell interiors

    SciTech Connect

    Levine, Lyle E.; Geantil, P.; Larson, Ben C; Tischler, Jonathan Zachary; Kassner, Michael E.; Liu, Wenjun

    2012-01-01

    Dislocation structures in deformed metals produce broad asymmetric diffraction line profiles. During analysis, these profiles are generally separated into two nearly symmetric subprofiles corresponding to diffraction by dislocation cell walls and cell interiors. These subprofiles are then interpreted using complex models of dislocation-based line broadening. Until now, it has not been possible to test the many assumptions that are made in such an analysis. Here, depth-resolved microbeam diffraction was used to measure diffraction line profiles from numerous individual dislocation cell walls and cell interiors in a heavily deformed Cu single crystal. Summing these profiles directly constructed the cell-interior and cell-wall subprofiles that have been approximated in the line profile analysis literature for the past 30 years. Direct comparison between the reconstructed subprofiles and the macroscopic asymmetric line profile from the same sample allows the first direct tests of many of the assumptions that have been used for interpreting these X-ray measurements.

  2. Comparative proteome analysis across non-small cell lung cancer cell lines.

    PubMed

    Grundner-Culemann, Kathrin; Dybowski, J Nikolaj; Klammer, Martin; Tebbe, Andreas; Schaab, Christoph; Daub, Henrik

    2016-01-01

    Non-small cell lung cancer (NSCLC) cell lines are widely used model systems to study molecular aspects of lung cancer. Comparative and in-depth proteome expression data across many NSCLC cell lines has not been generated yet, but would be of utility for the investigation of candidate targets and markers in oncogenesis. We employed a SILAC reference approach to perform replicate proteome quantifications across 23 distinct NSCLC cell lines. On average, close to 4000 distinct proteins were identified and quantified per cell line. These included many known targets and diagnostic markers, indicating that our proteome expression data represents a useful resource for NSCLC pre-clinical research. To assess proteome diversity within the NSCLC cell line panel, we performed hierarchical clustering and principal component analysis of proteome expression data. Our results indicate that general proteome diversity among NSCLC cell lines supersedes potential effects common to K-Ras or epidermal growth factor receptor (EGFR) oncoprotein expression. However, we observed partial segregation of EGFR or KRAS mutant cell lines for certain principal components, which reflected biological differences according to gene ontology enrichment analyses. Moreover, statistical analysis revealed several proteins that were significantly overexpressed in KRAS or EGFR mutant cell lines. PMID:26361996

  3. Phase transitions in tumor growth: II prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Llanos-Pérez, J. A.; Betancourt-Mar, A.; De Miguel, M. P.; Izquierdo-Kulich, E.; Royuela-García, M.; Tejera, E.; Nieto-Villar, J. M.

    2015-05-01

    We propose a mechanism for prostate cancer cell lines growth, LNCaP and PC3 based on a Gompertz dynamics. This growth exhibits a multifractal behavior and a "second order" phase transition. Finally, it was found that the cellular line PC3 exhibits a higher value of entropy production rate compared to LNCaP, which is indicative of the robustness of PC3, over to LNCaP and may be a quantitative index of metastatic potential tumors.

  4. The antiproliferative effect of coumarins on several cancer cell lines.

    PubMed

    Kawaii, S; Tomono, Y; Ogawa, K; Sugiura, M; Yano, M; Yoshizawa, Y

    2001-01-01

    Twenty-one coumarins were examined for their antiproliferative activity towards several cancer cell lines, namely lung carcinoma (A549), melanin pigment producing mouse melanoma (B16 melanoma 4A5), human T-cell leukemia (CCRF-HSB-2), and human gastric cancer, lymph node metastasized (TGBC11TKB). The structure-activity relationship established from the results revealed that the 6,7-dihydroxy moiety had an important role for their antiproliferative activity. Analysis of cell cycle distribution indicated that esculetin-treated cells accumulated in the G1 (at 400 microM) or in S phase (at 100 microM). PMID:11396185

  5. Fluorescence Assay 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell-

    E-print Network

    Collins, Gary S.

    Fluorescence Assay References 1. 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell- cultures/cell-models-hek-293-cells.html Conclusion A Novel Fluorescence Assay for the Determination of Ligand Binding Constant-therapies in cancer patients. This makes the study of both agonist and antagonist ligands important as the knowledge

  6. Osmotic stress affects functional properties of human melanoma cell lines

    E-print Network

    La Porta, Caterina A M; Pasini, Maria; Laurson, Lasse; Alava, Mikko J; Zapperi, Stefano; Amar, Martine Ben

    2015-01-01

    Understanding the role of microenvironment in cancer growth and metastasis is a key issue for cancer research. Here, we study the effect of osmotic pressure on the functional properties of primary and metastatic melanoma cell lines. In particular, we experimentally quantify individual cell motility and transmigration capability. We then perform a circular scratch assay to study how a cancer cell front invades an empty space. Our results show that primary melanoma cells are sensitive to a low osmotic pressure, while metastatic cells are less. To better understand the experimental results, we introduce and study a continuous model for the dynamics of a cell layer and a stochastic discrete model for cell proliferation and diffusion. The two models capture essential features of the experimental results and allow to make predictions for a wide range of experimentally measurable parameters.

  7. Mouse DRG Cell Line with Properties of Nociceptors

    PubMed Central

    Doran, Ciara; Chetrit, Jonathan; Holley, Matthew C.; Grundy, David; Nassar, Mohammed A.

    2015-01-01

    In vitro cell lines from DRG neurons aid drug discovery because they can be used for early stage, high-throughput screens for drugs targeting pain pathways, with minimal dependence on animals. We have established a conditionally immortal DRG cell line from the Immortomouse. Using immunocytochemistry, RT-PCR and calcium microfluorimetry, we demonstrate that the cell line MED17.11 expresses markers of cells committed to the sensory neuron lineage. Within a few hours under differentiating conditions, MED17.11 cells extend processes and following seven days of differentiation, express markers of more mature DRG neurons, such as NaV1.7 and Piezo2. However, at least at this time-point, the nociceptive marker NaV1.8 is not expressed, but the cells respond to compounds known to excite nociceptors, including the TRPV1 agonist capsaicin, the purinergic receptor agonist ATP and the voltage gated sodium channel agonist, veratridine. Robust calcium transients are observed in the presence of the inflammatory mediators bradykinin, histamine and norepinephrine. MED17.11 cells have the potential to replace or reduce the use of primary DRG culture in sensory, pain and developmental research by providing a simple model to study acute nociception, neurite outgrowth and the developmental specification of DRG neurons. PMID:26053673

  8. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...; Identification of Human Cell Lines Project AGENCY: National Institute of Standards and Technology (NIST...) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All... for Biotechnology Information (NCBI) and will be used to differentiate among cell lines, as...

  9. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

    Heaton, D.; Mustafi, R.; Schwartz, J.L. |

    1992-06-01

    The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

  10. Differential expression of Ia antigens by rheumatoid synovial lining cells.

    PubMed Central

    Burmester, G R; Jahn, B; Rohwer, P; Zacher, J; Winchester, R J; Kalden, J R

    1987-01-01

    The differential expression of Ia antigens was studied in freshly isolated rheumatoid nonlymphoid synovial lining cells (SLC) and rheumatoid synovial fibroblast cell lines cultured in the presence of Interferon-gamma, using a large panel of anti-Ia reagents with monomorphic or polymorphic specificities. All the HLA-DR or -DQ specificities detectable on the corresponding peripheral blood B cells were also expressed in freshly isolated SLC. However, in all instances, the number of DR-positive SLC exceeded the percentage of cells expressing DQ antigens. In addition, the epitope expression of Ia antigens varied within the DR or DQ populations of Ia molecules as revealed by polymorphic reagents. Double-label experiments or using the ingestion of Latex particles as a marker demonstrated that the synovial macrophages (type I SLC) primarily bear the DR+DQ+ phenotype, while there is an additional population of nonphagocytic SLC (previously termed type II SLC) that has a DR+ and monocyte marker negative phenotype but did not have detectable levels of DQ antigens as analyzed by both fluorescence microscopy and cell sorter analysis. This latter population frequently had a morphology showing dendritic processes and rapidly lost the expression of Ia antigens upon culture. Cells with a similar, primarily DR+ phenotype were readily obtained in synovial fibroblast cultures after treatment with Interferon-gamma. These data suggest that there are two populations of Ia+ synovial lining cells: the synovial macrophages (type I cells) with the DR+DQ+ phenotype, and cells probably related to fibroblasts with a DR+ phenotype without detectable DQ antigens (type II cells). The fact that the latter phenotype could be induced by Interferon-gamma treatment of cultured synovial fibroblasts suggests that this mediator may have a similar role in vivo in the activation of certain synovial cell populations. Images PMID:2442194

  11. Mutations to Ku Reveal Differences in Human Somatic Cell Lines

    PubMed Central

    Fattah, Kazi R.; Ruis, Brian L.; Hendrickson, Eric A.

    2008-01-01

    NHEJ (non-homologous end joining) is the predominant mechanism for repairing DNA double-stranded breaks in human cells. One essential NHEJ factor is the Ku heterodimer, which is composed of Ku70 and Ku86. Here we have generated heterozygous loss-of-function mutations for each of these genes in two different human somatic cell lines, HCT116 and NALM-6 using gene targeting. Previous work had suggested that phenotypic differences might exist between the genes and/or between the cell lines. By providing a side-by-each comparison of the four cell lines, we demonstrate that there are indeed subtle differences between loss-of-function mutations for Ku70 versus Ku86, which is accentuated by whether the mutations were derived in the HCT116 or NALM-6 genetic background. Overall, however, the phenotypes of the four lines are quite similar and they provide a compelling argument for the hypothesis that Ku loss-of-function mutations in human somatic cells result in demonstrable haploinsufficiencies. Collectively, these studies demonstrate the importance of proper biallelic expression of these genes for NHEJ and telomere maintenance and they provide insights into why these genes are uniquely essential for primates. PMID:18387344

  12. Propagation of Bombyx mori Nucleopolyhedrovirus in nonpermissive insect cell lines.

    PubMed

    Woo, Soo-Dong; Roh, Jong Yul; Choi, Jae Young; Jin, Byung Rae

    2007-04-01

    This study addresses the susceptibility of Spodoptera frugiperda (Sf9 and Sf21), Trichoplusia ni (Hi5), and S. exigua (Se301) cells to the Bombyx mori nucleopolyhedrovirus (BmNPV). Although these cells have classically been considered nonpermissive to BmNPV, the cytopathic effect, an increase in viral yield, and viral DNA synthesis by BmNPV were observed in Sf9, Sf21, and Hi5 cells, but not in Se301 cells. Very late gene expression by BmNPV in these cell lines was also detected via beta-galactosidase expression under the control of the polyhedrin promoter. Sf9 cells were most susceptible to BmNPV in all respects, followed by Sf21 and Hi5 cells in decreasing order, while the Se301 cells evidenced no distinct viral replication. This particular difference in viral susceptibility in each of the cell lines can be utilized for our understanding of the mechanisms underlying the host specificity of NPVs. PMID:17483798

  13. Phenyl Saligenin Phosphate Induced Caspase-3 and c-Jun N-Terminal Kinase Activation in Cardiomyocyte-Like Cells.

    PubMed

    Felemban, Shatha G; Garner, A Christopher; Smida, Fathi A; Boocock, David J; Hargreaves, Alan J; Dickenson, John M

    2015-11-16

    At present, little is known about the effect(s) of organophosphorous compounds (OPs) on cardiomyocytes. In this study, we have investigated the effects of phenyl saligenin phosphate (PSP), two organophosphorothioate insecticides (diazinon and chlorpyrifos), and their acutely toxic metabolites (diazoxon and chlorpyrifos oxon) on mitotic and differentiated H9c2 cardiomyoblasts. OP-induced cytotoxicity was assessed by monitoring MTT reduction, LDH release, and caspase-3 activity. Cytotoxicity was not observed with diazinon, diazoxon, or chlorpyrifos oxon (48 h exposure; 200 ?M). Chlorpyrifos-induced cytotoxicity was only evident at concentrations >100 ?M. In marked contrast, PSP displayed pronounced cytotoxicity toward mitotic and differentiated H9c2 cells. PSP triggered the activation of JNK1/2 but not ERK1/2, p38 MAPK, or PKB, suggesting a role for this pro-apoptotic protein kinase in PSP-induced cell death. The JNK1/2 inhibitor SP 600125 attenuated PSP-induced caspase-3 and JNK1/2 activation, confirming the role of JNK1/2 in PSP-induced cytotoxicity. Fluorescently labeled PSP (dansylated PSP) was used to identify novel PSP binding proteins. Dansylated PSP displayed cytotoxicity toward differentiated H9c2 cells. 2D-gel electrophoresis profiles of cells treated with dansylated PSP (25 ?M) were used to identify proteins fluorescently labeled with dansylated PSP. Proteomic analysis identified tropomyosin, heat shock protein ?-1, and nucleolar protein 58 as novel protein targets for PSP. In summary, PSP triggers cytotoxicity in differentiated H9c2 cardiomyoblasts via JNK1/2-mediated activation of caspase-3. Further studies are required to investigate whether the identified novel protein targets of PSP play a role in the cytotoxicity of this OP, which is usually associated with the development of OP-induced delayed neuropathy. PMID:26465378

  14. [Establishment and biological characterization of human medulloblastoma cell lines].

    PubMed

    Yamada, M; Shimizu, K; Tamura, K; Okamoto, Y; Matsui, Y; Moriuchi, S; Park, K; Mabuchi, E; Yamamoto, K; Hayakawa, T

    1989-07-01

    Two cell lines of human medulloblastoma (ONS-76 and ONS-81) were established, and their biological characteristics were investigated. The cell line, ONS-76, was established from a tumor specimens obtained from a large cerebellar tumor of a 2-year-old girl. The pathological diagnosis was a typical medulloblastoma. The other cell line, ONS-81, was derived from a metastatic tumor in right frontal lobe of a 9-year-old girl. The tumor specimens were minced into fragments approximately 1 mm in diameter and cultured in plastic culture flasks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50% patients serum. The cells growing as a monolayer were subcultured in RPMI 1640 supplemented with 10% FCS and initially with L-glutamine, sodium pyruvate, and nonessential amino acid. Microscopically, both cultured cells exhibited various morphological appearances, and this morphological heterogeneity seemed to be specific for medulloblastoma cells. The in vitro population doubling time of ONS-76 and ONS-81 were 18.6 and 19.2 hr, respectively. The ONS-76 and ONS-81 cells formed subcutaneous tumors in nude mice as serial transplantable xenograft, and these tumors had a microscopic appearance similar to that of the original medulloblastoma. Ultrastructurally++, the cultured cells showed primitive, undifferentiated appearance, and no neuronal or glial structures were not seen. Immunohistochemical studies showed that both cells expressed neuron-specific enolase (NSE) and neurofilament protein (NFP 200 K, 145 K), but glial fibrillary acidic protein (GFAP) and S-100 protein were not detected. The NFP immunoreactivities of both cultured cells were demonstrated as abnormal perinuclear deposits.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2818910

  15. Identification of lymphoid cell lines bearing receptors for somatostatin.

    PubMed Central

    Nakamura, H; Koike, T; Hiruma, K; Sato, T; Tomioka, H; Yoshida, S

    1987-01-01

    The MT-2, derived from an adult T-cell leukaemia (ATL) cell, the Molt-4F, a human T-cell line, and the Isk, an EB virus-transformed B-cell line, were found to have high-affinity receptors for somatostatin, a cyclic tetradecapeptide that inhibits the release of substances such as growth hormone, TSH, glucagon, insulin, secretin, gastrin and cholecystokinin. The quantity of radioactivity bound varied linearly with the number of cells, and was displaced by non-radioactive somatostatin in a concentration-dependent manner. Specific binding of 125I-somatostatin was time- and temperature-dependent and at 22 degrees reached equilibrium within 120 min. Scatchard analysis demonstrated one class of specific-binding sites on MT-2 cells, Isk cells and Molt-4F cells that had respective densities and dissociation constants of 109 pM and 0.64 nM, 102 pM and 1.1 nM, and 5.8 pM and 0.22 nM. PMID:2892785

  16. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  17. USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY

    EPA Science Inventory

    The need to deploy IN VITRO models to test neurotoxic scribes the use of by industry and government regulatory agencies. his research describes the neuroblastoma cell lines to address the relationship between esterase inhibition and neurotoxic outcome following exposure to organo...

  18. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

    THIS ABSTRACT WAS SUBMITTED ELECTRONICALLY;. SPACE CONSTRAINTS WERE SEVERE)

    Methylation of Arsenite by Some Mammalian Cell Lines.

    Methylation of arsenite is thought to play an important role in the carcinogenicity of arsenic.
    Aim 1: Determine if there is diffe...

  19. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... identification as part of this project will undergo STR profiling, a DNA profiling method that examines/screens for STRs (DNA elements 2-6 bps long repeated in tandem) in the human chromosomes, that has been shown... are expected between cell line DNA samples originating from unrelated individuals. Each unique...

  20. DIVERSITY OF ARSENIC METABOLISM IN CULTURED HUMAN CANCER CELL LINES

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

    Arsenic has been known to cause a variety of malignancies in human. Pentavalent As (As 5+) is reduced to trivalent As (As3+) which is further methylated by arsenic methyltransferase(s) to monomethylarson...

  1. Choosing the right chondrocyte cell line: Focus on nitric oxide.

    PubMed

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; López, Verónica; Pino, Jesús; Gómez, Rodolfo; Gómez-Reino, Juan Jesús; Gualillo, Oreste

    2015-12-01

    Nitric oxide (NO) has been considered a catabolic factor that contributes to OA pathology by inducing chondrocytes apoptosis, matrix metalloproteinases synthesis, and pro-inflammatory cytokines expression. Thus, the research on NO regulation in chondrocytes represents a relevant field which needs to be explored in depth. However, to date, only the murine ATDC-5 cell line and primary chondrocytes are well-established cells to study NO production in cartilage tissues. The goal of this study is to determine whether two commonly used human chondrocytic cell lines: SW-1353 and T/C-28a2 cell lines are good models to examine lipopolysaccharide and/or pro-inflammatory cytokine-driven NO release and iNOS expression. To this aim, we carefully examined NO production and iNOS protein expression in human T/C-28a2 and SW-1353 chondrocytes stimulated with LPS and interleukin (IL)-1 alone or in combination. We also use ATDC-5 cells as a positive control for NO production. NO accumulation has been determined by colorimetric Griess reaction, whereas NOS type II expression was determined by Western Blot analysis. Our results clearly demonstrated that neither human T/C-28a2 nor SW-1353 chondrocytes showed a detectable increase in NO production or iNOS expression after bacterial endotoxin or cytokines challenge with IL-1. Our study demonstrated that T/C-28a2 and SW-1353 human cell lines are not suitable for studying NO release and iNOS expression confirming that ATDC5 and human primary cultured chondrocytes are the best in vitro cell system to study the actions derived from this mediator. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1784-1788, 2015. PMID:26016689

  2. Human small cell lung cancer cell lines expressing the proopiomelanocortin gene have aberrant glucocorticoid receptor function.

    PubMed Central

    Ray, D W; Littlewood, A C; Clark, A J; Davis, J R; White, A

    1994-01-01

    Some human small cell lung carcinomas (SCLC) secrete proopiomelanocortin (POMC) derived peptides, but in contrast to the pituitary, glucocorticoids fail to inhibit this hormone production. We have previously described an in vitro model using human SCLC cell lines that express POMC and are resistant to glucocorticoids. We have now identified the glucocorticoid receptor (GR) in the SCLC cell line COR L24 using a whole cell ligand binding assay (Kd = 5.7 nM; Bmax = 11 fmol/million cells), while another cell line, DMS 79, lacked significant glucocorticoid binding. To analyze GR function both positive (GMCO) and negative (TRE)3-tkCAT), glucocorticoid-regulated reporter gene constructs were transfected into COR L24 cells. In the SCLC cell line, neither hydrocortisone nor dexamethasone (500-2,000 nM) significantly induced chloramphenicol acetyltransferase expression from GMCO; in addition, they did not suppress chloramphenicol acetyltransferase expression from (TRE)3-tkCAT. Similar results were obtained with two other POMC-expressing SCLC cell lines. Expression of wild type GR in COR L24 cells restored glucocorticoid signaling, with marked induction of GMCO reporter gene expression by dexamethasone (9,100 +/- 910%; n = 3), and an estimated EC50 of 10 nM. This failure of the GR explains the resistance of the POMC gene to glucocorticoid inhibition and may have implications for cell growth in SCLC. Images PMID:8163665

  3. Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

    PubMed

    Kniss, Douglas A; Summerfield, Taryn L

    2014-02-11

    Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. PMID:24520087

  4. Reversal of diabetes following transplantation of an insulin-secreting human liver cell line: Melligen cells.

    PubMed

    Lawandi, Janet; Tao, Chang; Ren, Binhai; Williams, Paul; Ling, Dora; Swan, M Anne; Nassif, Najah T; Torpy, Fraser R; O'Brien, Bronwyn A; Simpson, Ann M

    2015-01-01

    As an alternative to the transplantation of islets, a human liver cell line has been genetically engineered to reverse type 1 diabetes (TID). The initial liver cell line (Huh7ins) commenced secretion of insulin in response to a glucose concentration of 2.5 mmol/l. After transfection of the Huh7ins cells with human islet glucokinase, the resultant Melligen cells secreted insulin in response to glucose within the physiological range; commencing at 4.25 mmol/l. Melligen cells exhibited increased glucokinase enzymatic activity in response to physiological glucose concentrations, as compared with Huh7ins cells. When transplanted into diabetic immunoincompetent mice, Melligen cells restored normoglycemia. Quantitative real-time polymerase chain reaction (qRT-PCR) revealed that both cell lines expressed a range of ?-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-?, IL-1?, and IFN-?) affected neither their viability nor their ability to secrete insulin to glucose. Gene expression (microarray and qRT-PCR) analyses indicated the survival of Melligen cells in the presence of known ?-cell cytotoxins was associated with the expression of NF-?B and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial ?-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  5. Generation and characterization of a mouse lymphatic endothelial cell line.

    PubMed

    Sironi, Marina; Conti, Annarita; Bernasconi, Sergio; Fra, Anna M; Pasqualini, Fabio; Nebuloni, Manuela; Lauri, Eleonora; De Bortoli, Maida; Mantovani, Alberto; Dejana, Elisabetta; Vecchi, Annunciata

    2006-07-01

    Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line (MELC) and the generated clones thereof. Cells were isolated from benign lymphangiomas induced by intraperitoneal injections of incomplete Freund's adjuvant. The MELC line expressed molecules typical of lymphatic endothelium, including VEGFR3/Flt-4, podoplanin, Prox-1, and D6, but not LYVE-1. It also expressed CD34, ICAM-1, VCAM, and JAM-A, but not CD31, VE-cadherin, E-selectin, or CX3CL1/fractalkine (both TNFalpha-induced), at variance with vascular endothelial cells tested in parallel. The inflammatory cytokines TNFalpha and IL-4 regulated production of selected adhesion molecules (VCAM), cytokines (IL-6), and chemokines (CCL2/JE). Whole genome transcriptional profiling identified a set of 150 known genes differentially expressed in MELC versus vascular endothelial cells. Thus, the MELC line may represent an invaluable source of lymphatic endothelium. PMID:16534603

  6. Alkylphosphocholines and curcumin induce programmed cell death in cutaneous T-cell lymphoma cell lines.

    PubMed

    Yosifov, Deyan Y; Kaloyanov, Kaloyan A; Guenova, Margarita L; Prisadashka, Kamelia; Balabanova, Maria B; Berger, Martin R; Konstantinov, Spiro M

    2014-01-01

    While most patients with early-stage cutaneous T-cell lymphomas (CTCL) have a very good prognosis, the survival of patients with extensive tumour stage and visceral involvement remains extremely poor and necessitates the development of more effective treatment modalities. In this study, we evaluated the in vitro effects of two alkylphosphocholines (APCs, miltefosine and erufosine) and the polyphenolic compound curcumin on 5 human CTCL cell lines (Hut-78, HH, MJ, My-La CD4+ and My-La CD8+). All tested drugs showed considerable cytotoxic activity, as determined by the MTT dye reduction assay. The IC50 values of both APCs ranged from the low micromolar level (Hut-78 cells) to 60-80?M (HH cells). The IC50 values of curcumin ranged from 12 to 24?M. All tested drugs induced apoptosis, as ascertained by morphological changes, DNA fragmentation and activation of caspase cascades. Miltefosine and erufosine induced dephosphorylation of Akt in My-La CD8+ cells and phosphorylation of JNK in Hut-78 and My-La CD8+ cells. APCs increased the level of the autophagic marker LC3B in Hut-78 and MJ cells. Results from co-treatment with autophagy modulators suggested that the cytotoxicity of APCs in CTCL cells is mediated, at least in part, by induction of autophagy. PMID:24225136

  7. Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

    PubMed Central

    Ding, Xinchun; Wu, Lingyan; Yan, Cong; Du, Hong

    2015-01-01

    Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions. PMID:25807535

  8. Targeted genetic modification of cell lines for recombinant protein production

    PubMed Central

    Piskareva, Olga; Muniyappa, Mohan

    2007-01-01

    Considerable increases in productivity have been achieved in biopharmaceutical production processes over the last two decades. Much of this has been a result of improvements in media formulation and process development. Though advances have been made in cell line development, there remains considerable opportunity for improvement in this area. The wealth of transcriptional and proteomic data being generated currently hold the promise of specific molecular interventions to improve the performance of production cell lines in the bioreactor. Achieving this—particularly for multi-gene modification—will require specific, targeted and controlled genetic manipulation of these cells. This review considers some of the current and potential future techniques that might be employed to realise this goal. PMID:19003191

  9. Plasmids and packaging cell lines for use in phage display

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

    The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, in fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.

  10. Patient sues UCLA over patent on cell line.

    PubMed

    Culliton, B J

    1984-09-28

    A patient's lawsuit against the University of California is raising questions about an individual's rights of ownership in relation to body tissues that have been turned over for biomedical research but are subsequently used commercially. In 1976, John Moore had his spleen removed at the University of California, Los Angeles, in connection with his leukemia treatment. The university and researchers David Golde and Shirley Quan recently received a patent on the biologically interesting Mo cell line, which was derived from Moore's spleen cells. Moore's suit claims that the university misappropriated his tissues and that the researchers failed to obtain a valid informed consent because they did not formally tell him about the potential commercial applications of the cell line. PMID:6474185

  11. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or results from synergistic interactions on the MS-5 cell surface between extracellular matrix proteins and cytokines will require further investigation. PMID:7513651

  12. Immunoglobulin G Locus Events in Soft Tissue Sarcoma Cell Lines

    PubMed Central

    Chen, Zhengshan; Li, Jing; Xiao, Yanna; Zhang, Junjun; Zhao, Yingying; Liu, Yuxuan; Ma, Changchun; Qiu, Yamei; Luo, Jin; Huang, Guowei; Korteweg, Christine; Gu, Jiang

    2011-01-01

    Recently immunoglobulins (Igs) have been found to be expressed by cells other than B lymphocytes, including various human carcinoma cells. Sarcomas are derived from mesenchyme, and the knowledge about the occurrence of Ig production in sarcoma cells is very limited. Here we investigated the phenomenon of immunoglobulin G (IgG) expression and its molecular basis in 3 sarcoma cell lines. The mRNA transcripts of IgG heavy chain and kappa light chain were detected by RT-PCR. In addition, the expression of IgG proteins was confirmed by Western blot and immunofluorescence. Immuno-electron microscopy localized IgG to the cell membrane and rough endoplasmic reticulum. The essential enzymes required for gene rearrangement and class switch recombination, and IgG germ-line transcripts were also identified in these sarcoma cells. Chromatin immunoprecipitation results demonstrated histone H3 acetylation of both the recombination activating gene and Ig heavy chain regulatory elements. Collectively, these results confirmed IgG expression in sarcoma cells, the mechanism of which is very similar to that regulating IgG expression in B lymphocytes. PMID:21731691

  13. Transgenic cell lines for detection of animal viruses.

    PubMed Central

    Olivo, P D

    1996-01-01

    Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses. PMID:8809463

  14. Ecdysone and The Cell Cycle: Investigations in a Mosquito Cell Line

    PubMed Central

    Fallon, Ann M.; Gerenday, Anna

    2010-01-01

    Cell lines provide a tool for investigating basic biological processes that underlie the complex interactions among the tissues and organs of an intact organism. We compare the evolution of insect and mammalian populations as they progress from diploid cell strains to continuous cell lines, and review the history of the well-characterized Aedes albopictus mosquito cell line, C7-10. Like Kc and S3 cells from Drosophila melanogaster, C7-10 cells are sensitive to the insect steroid hormone, 20-hydroxyecdysone (20E), and express 20E-inducible proteins as well as the EcR and USP components of the ecdysteroid receptor. The decrease in growth associated with 20E treatment results in an accumulation of cells in the G1 phase of the cycle, and a concomitant decrease in levels of cyclin A. In contrast, 20E induces a G2 arrest in a well-studied imaginal disc cell line from the moth, Plodia interpunctella. We hypothesize that 20E-mediated events associated with molting and metamorphosis include effects on regulatory proteins that modulate the mitotic cell cycle and that differences between the 20E response in diverse insect cell lines reflect an interplay between classical receptor-mediated effects on gene expression and non-classical effects on signaling pathways similar to those recently described for the vertebrate steroid hormone, estrogen. PMID:20303973

  15. Establishment of an epithelioid malignant schwannoma cell line (YST-1).

    PubMed

    Nagashima, Y; Ohaki, Y; Tanaka, Y; Sumino, K; Funabiki, T; Okuyama, T; Watanabe, S; Umeda, M; Misugi, K

    1990-01-01

    A novel cell line, YST-1, was established from an epithelioid malignant schwannoma (EMS) that occurred in the upper arm of an 8-year-old girl. YST-1 cells were polygonal and stellate in shape, contained abundant free ribosomes, mitochondria, lysosomes and rough-surfaced endoplasmic reticulum, and grew stably with a population doubling time of 40 h. Immunohistochemically, vimentin, S100 protein and S100 protein beta subunit were positive in the cytoplasm. The xeno-transplanted tumor in nude mice was composed of cells with an epithelioid arrangement similar to the original tumor. The borders of the tumor cells were connected intimately without desmosomal junctions, and there were abundant organelles in the cytoplasm. YST-1 cells were considered to be of value for studying the nature and histogenesis of EMS. PMID:1980563

  16. Impairment of cell cycle progression by aflatoxin B1 in human cell lines.

    PubMed

    Ricordy, R; Gensabella, G; Cacci, E; Augusti-Tocco, G

    2002-05-01

    Aflatoxin B1 is a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticum, which may be present as a food contaminant. It is known to cause acute toxic effects and act as a carcinogenic agent. The carcinogenic action has been related to its ability to form unstable adducts with DNA, which represent possible mutagenic sites. On the other hand, the primary cellular target responsible for its toxic action has not yet been clearly identified. Previous data suggested a possible correlation between cell proliferation and responsiveness to aflatoxin toxicity. These observations led us to investigate the effect of the toxin on cell cycle progression of three human cell lines (HepG2, SK-N-MC and SK-N-SH derived from liver and nervous tissue tumours); they were shown to display different responses to toxin exposure and have different growth kinetics. We performed analysis of the cell cycle, DNA synthesis and expression of p21 and p53 in the presence and absence of the toxin in all cell lines exposed. The results of cell cycle cytofluorometric analysis show significant alterations of cell cycle progression as a result of toxin treatment. In all cell lines exposure to a 24 h toxin treatment causes a dose-dependent accumulation in S phase, however, the ability to recover from impairment to traverse S phase varies in the cell lines under study. SK-N-MC cells appear more prone to resume DNA synthesis when the toxin is removed, while the other two cell lines maintain a significant inhibition of DNA synthesis, as indicated by cytofluorimetry and [(3)H]dTR incorporation. The level of p53 and p21 expression in the three cell lines was examined by western blot analysis and significant differences were detected. The ready resumption of DNA synthesis displayed by SK-N-MC cells could possibly be related to the absence of p53 control of cell cycle progression. PMID:11971996

  17. Bioenergetic Analysis of Ovarian Cancer Cell Lines: Profiling of Histological Subtypes and Identification of a Mitochondria-Defective Cell Line

    PubMed Central

    Dier, Usawadee; Shin, Dong-Hui; Hemachandra, L. P. Madhubhani P.; Uusitalo, Larissa M.; Hempel, Nadine

    2014-01-01

    Epithelial ovarian cancer (EOC) is the most lethal of all gynecological cancers, and encompasses distinct histological subtypes that have specific genetic and tissues-of-origin differences. Ovarian clear cell carcinoma (OCCC) represents approximately 10% of cases and has been termed a stress responsive cancer. OCCC is characterized by increased expression of oxidative stress and glycolysis-related genes. In the present study, we hypothesized that bioenergetic profiling might uniquely distinguish OCCC from other EOC histological subtypes. Using an extracellular flux analyzer, OCCC lines (ES-2, TOV-21-G) were shown to be highly metabolically active, with high oxygen consumption rate (OCR) and high extracellular acidification rate (ECAR), indicative of enhanced mitochondrial oxidative phosphorylation and glycolytic rate, respectively. A high bioenergetics profile was associated with the cell lines' ability to form anchorage independent spheroids. Given their high glycolytic and mitochondrial activity, OCCC cells displayed strong sensitivity to 2-deoxy-D-glucose and Rotenone growth inhibition, although this chemosensitivity profile was not specific to only OCCC cells. Bioenergetic profiling also identified a non-OCCC cell line, OVCA420, to have severely compromised mitochondrial function, based on low OCR and a lack of stimulation of maximal respiration following application of the uncoupler FCCP. This was accompanied by mitochondrial morphology changes indicative of enhanced fission, increased expression of the mitochondrial fission protein Drp1, a loss of mitochondrial membrane potential and dependence on glycolysis. Importantly, this loss of mitochondrial function was accompanied by the inability of OVCA420 cells to cope with hypoxic stress, and a compromised ability to stabilize HIF-1? in response to 1% O2 hypoxia. This knowledge may be imperative for researchers planning to utilize this cell line for further studies of metabolism and hypoxia, and suggests that altered mitochondrial fission dynamics represents a phenotype of a subpopulation of EOCs. PMID:24858344

  18. STAT1 signaling is associated with acquired crossresistance to doxorubicin and radiation in myeloma cell lines

    E-print Network

    the 8226/Dox40 cell line to its parental line was performed to identify the underlying molecular mechanisms correlates positively with doxorubicin resistance in a human tumor cell line panel.8 STAT1 signaling has also mechanism that contributes to acquired crossresistance to both drugs and radiation in myeloma cell lines

  19. Proteomic Analysis of HepaRG Cells: A Novel Cell Line That Supports Hepatitis B Virus Infection

    E-print Network

    Proteomic Analysis of HepaRG Cells: A Novel Cell Line That Supports Hepatitis B Virus Infection, the only cell line that is susceptible to hepatitis B virus (HBV) infection and supports a complete virus cellular response to HBV infection. Keywords: Hepatitis B virus · HepaRG cell line · Two dimensional

  20. Toxicity of Calcium Hydroxide Nanoparticles on Murine Fibroblast Cell Line

    PubMed Central

    Dianat, Omid; Azadnia, Sina; Mozayeni, Mohammad Ali

    2015-01-01

    Introduction: One of the major contributing factors, which may cause failure of endodontic treatment, is the presence of residual microorganisms in the root canal system. For years, most dentists have been using calcium hydroxide (CH) as the intracanal medicament between treatment sessions to eliminate remnant microorganisms. Reducing the size of CH particles into nanoparticles enhances the penetration of this medicament into dentinal tubules and increases their antimicrobial efficacy. This in vitro study aimed to compare the cytotoxicity of CH nanoparticles and conventional CH on fibroblast cell line using the Mosmann’s Tetrazolium Toxicity (MTT) assay. Methods and Materials: This study was conducted on L929 murine fibroblast cell line by cell culture and evaluation of the direct effect of materials on the cultured cells. Materials were evaluated in two groups of 10 samples each at 24, 48 and 72 h. At each time point, 10 samples along with 5 positive and 5 negative controls were evaluated. The samples were transferred into tubes and exposed to fibroblast cells. The viability of cells was then evaluated. The Two-way ANOVA was used for statistical analysis and the level of significance was set at 0.05. Results: Cytotoxicity of both materials decreased over time and for conventional CH was lower than that of nanoparticles. However, this difference was not statistically significant (P>0.05). Conclusion: The cytotoxicity of CH nanoparticles was similar to that of conventional CH. PMID:25598810

  1. Serial analysis of gene expression in a microglial cell line.

    PubMed

    Inoue, H; Sawada, M; Ryo, A; Tanahashi, H; Wakatsuki, T; Hada, A; Kondoh, N; Nakagaki, K; Takahashi, K; Suzumura, A; Yamamoto, M; Tabira, T

    1999-12-01

    We used the serial analysis of gene expression (SAGE) method to systematically analyze transcripts present in a microglial cell line. Over 10,000 SAGE tags were sequenced, and shown to represent 6,013 unique transcripts. Among the diverse transcripts that had not been previously detected in microglia were those for cytokines such as endothelial monocyte-activating polypeptide I (EMAP I), and for cell surface antigens, including adhesion molecules such as CD9, CD53, CD107a, CD147, CD162 and mast cell high affinity IgE receptor. In addition, we detected transcripts that were characteristic of hematopoietic cells or mesodermal structures, such as E3 protein, A1, EN-7, B94, and ufo. Furthermore, the profile contained a transcript, Hn1, that is important in hematopoietic cells and neurological development (Tang et al. Mamm Genome 8:695-696, 1997), suggesting the probable neural differentiation of microglia from the hematopoietic system in development. Messenger RNA expression of these genes was confirmed by RT-PCR in primary cultures of microglia. Significantly, this is the first systematic profiling of the genes expressed in a microglial cell line. The identification and further characterization of the genes described here should provide potential new targets for the study of microglial biology. PMID:10559785

  2. Derivation of human embryonic stem cell lines from parthenogenetic blastocysts.

    PubMed

    Mai, Qingyun; Yu, Yang; Li, Tao; Wang, Liu; Chen, Mei-jue; Huang, Shu-zhen; Zhou, Canquan; Zhou, Qi

    2007-12-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen, and the expression of ESC markers was as expected for alkaline phosphatase, octamer-binding transcription factor 4, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81, and there was absence of expression of negative markers such as stage-specific embryonic antigen 1. Expression of genes specific for different embryonic germ layers was detected from the embryoid bodies (EBs) of both hESC lines, suggesting their differentiation potential in vitro. However, in vivo, only hPES-1 formed teratoma consisting of all three embryonic germ layers (hPES-2 did not). Interestingly, after continuous proliferation for more than 100 passages, hPES-1 cells still maintained a normal 46 XX karyotype; hPES-2 displayed abnormalities such as chromosome translocation after long term passages. Short Tandem Repeat (STR) results demonstrated that the hPES lines were genetic matches with the egg donors, and gene imprinting data confirmed the parthenogenetic origin of these ES cells. Genome-wide SNP analysis showed a pattern typical of parthenogenesis. All of these results demonstrated the feasibility to isolate and establish human parthenogenetic ESC lines, which provides an important tool for studying epigenetic effects in ESCs as well as for future therapeutic interventions in a clinical setting. PMID:18071366

  3. Tumorigenic Potential of Mononucleated Small Cells of Hodgkin Lymphoma Cell Lines

    PubMed Central

    Ikeda, Jun-ichiro; Mamat, Suhana; Tian, Tian; Wang, Yi; Rahadiani, Nur; Aozasa, Katsuyuki; Morii, Eiichi

    2010-01-01

    Tumor cells with tumorigenic potential are limited to a small cell population known as cancer stem cells (CSCs). CSCs yield both CSCs and non-CSCs, whereas non-CSCs do not yield CSCs. CSCs have not been identified in any malignant lymphomas. Hodgkin lymphoma (HL) is a mostly B-cell neoplasm that can be diagnosed by the presence of multinucleated (Reed-Sternberg; RS) cells admixed with Hodgkin cells with distinct nucleoli and various inflammatory cells. Here, the tumorigenic potential of cells with a single nucleus (S) and cells with multiple nuclei (M), which may be equivalent to Hodgkin and RS cells, respectively, was examined in HL cell lines L1236 and L428. Cultures of single S cells yielded both S and M cells, whereas M cell cultures yielded only M cells. When either cultured in methylcellulose or inoculated into NOD/SCID mice, the colony number and tumor size were both larger in S than in M cells. Concentrations of intracellular reactive oxygen species (ROS) were at low levels in a portion of S cells that abundantly expressed FoxO3a, a transcription factor that regulates ROS-degrading enzymes. In clinical samples of HL, FoxO3a was expressed in mononuclear Hodgkin cells but not in multinucleated RS cells. These findings suggest that smaller cells or Hodgkin cells that show low-ROS concentrations and high FoxO3a expression levels might be candidates for HL CSCs. PMID:20952592

  4. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

  5. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    PubMed Central

    Kumar, Rahul; Chaudhary, Kumardeep; Singla, Deepak; Gautam, Ankur; Raghava, Gajendra P. S.

    2014-01-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer. PMID:24728108

  6. Biotechnology and the chicken B cell line DT40.

    PubMed

    Bachl, J; Caldwell, R B; Buerstedde, J-M

    2007-01-01

    Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection. PMID:17675859

  7. Hepatitis C virus infection of cholangiocarcinoma cell lines

    PubMed Central

    Fletcher, Nicola F.; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K.; van IJzendoorn, Sven C. D.; Baumert, Thomas F.; Balfe, Peter; Afford, Simon

    2015-01-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo. PMID:25701818

  8. Pseudoislet of hybrid cellular spheroids from commercial cell lines.

    PubMed

    Jo, Y H; Nam, B M; Kim, B Y; Nemeno, J G; Lee, S; Yeo, J E; Yang, W; Park, S H; Kim, Y S; Lee, J I

    2013-10-01

    Investigators conducting diabetes-related research have focused on islet transplantation as a radical therapy for type 1 diabetes mellitus. Pancreatic islet isolation, an essential process, is a very demanding work because of the proteolytic enzymes, species, treatment time, and individual difference. Replacement of primary isolated pancreatic islets must be carried out continuously for various in vitro tests, making primary isolated islets a useful tool for cell transplantation research. Hence, we sought to develop pseudoislets from commercial pancreas-derived cell lines. In this study, we used RIN-5F and RIN-m cells, which secrete insulin, somatostatin, or glucagon. To manufacture hybrid cellular spheroids, the cells were cultured under hanging drop plate and nonadhesive plate methods. We observed that hybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 ?m. Their morphology was similar to naďve islets. Cell line pseudoislets secreted and expressed insulin, glucagon, and somatostatin, as confirmed by reverse transcriptase polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry analyses. Thus, the current artificially manufactured biomimetic pseudoislets resembled pancreatic islets of the endocrine system, appearing as cellular aggregates that secreted insulin, glucagon, and somatostatin. Enhanced immunoisolation techniques may lead to the development of new islet sources for pancreatic transplantation through this pseudoislet strategy. PMID:24157046

  9. HIV-1 latency in actively dividing human T cell lines

    PubMed Central

    Jeeninga, Rienk E; Westerhout, Ellen M; van Gerven, Marja L; Berkhout, Ben

    2008-01-01

    Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNF? treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus. PMID:18439275

  10. New Model for Gastroenteropancreatic Large-Cell Neuroendocrine Carcinoma: Establishment of Two Clinically Relevant Cell Lines

    PubMed Central

    Krieg, Andreas; Mersch, Sabrina; Boeck, Inga; Dizdar, Levent; Weihe, Eberhard; Hilal, Zena; Krausch, Markus; Möhlendick, Birte; Topp, Stefan A.; Piekorz, Roland P.; Huckenbeck, Wolfgang; Stoecklein, Nikolas H.; Anlauf, Martin; Knoefel, Wolfram T.

    2014-01-01

    Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup. PMID:24551139

  11. New model for gastroenteropancreatic large-cell neuroendocrine carcinoma: establishment of two clinically relevant cell lines.

    PubMed

    Krieg, Andreas; Mersch, Sabrina; Boeck, Inga; Dizdar, Levent; Weihe, Eberhard; Hilal, Zena; Krausch, Markus; Möhlendick, Birte; Topp, Stefan A; Piekorz, Roland P; Huckenbeck, Wolfgang; Stoecklein, Nikolas H; Anlauf, Martin; Knoefel, Wolfram T

    2014-01-01

    Recently, a novel WHO-classification has been introduced that divided gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) according to their proliferation index into G1- or G2-neuroendocrine tumors (NET) and poorly differentiated small-cell or large-cell G3-neuroendocrine carcinomas (NEC). Our knowledge on primary NECs of the GEP-system is limited due to the rarity of these tumors and chemotherapeutic concepts of highly aggressive NEC do not provide convincing results. The aim of this study was to establish a reliable cell line model for NEC that could be helpful in identifying novel druggable molecular targets. Cell lines were established from liver (NEC-DUE1) or lymph node metastases (NEC-DUE2) from large cell NECs of the gastroesophageal junction and the large intestine, respectively. Morphological characteristics and expression of neuroendocrine markers were extensively analyzed. Chromosomal aberrations were mapped by array comparative genomic hybridization and DNA profiling was analyzed by DNA fingerprinting. In vitro and in vivo tumorigenicity was evaluated and the sensitivity against chemotherapeutic agents assessed. Both cell lines exhibited typical morphological and molecular features of large cell NEC. In vitro and in vivo experiments demonstrated that both cell lines retained their malignant properties. Whereas NEC-DUE1 and -DUE2 were resistant to chemotherapeutic drugs such as cisplatin, etoposide and oxaliplatin, a high sensitivity to 5-fluorouracil was observed for the NEC-DUE1 cell line. Taken together, we established and characterized the first GEP large-cell NEC cell lines that might serve as a helpful tool not only to understand the biology of these tumors, but also to establish novel targeted therapies in a preclinical setup. PMID:24551139

  12. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  13. Bifurcate effects of glucose on caspase-independent cell death during hypoxia

    SciTech Connect

    Aki, Toshihiko; Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi

    2010-06-04

    We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

  14. Molecular signatures in response to Isoliquiritigenin in lymphoblastoid cell lines

    SciTech Connect

    Lee, Jae-Eun; Hong, Eun-Jung; Nam, Hye-Young; Hwang, Meeyul; Kim, Ji-Hyun; Han, Bok-Ghee; Jeon, Jae-Pil

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer We identified the inhibitory effect of ISL on cell proliferation of LCLs. Black-Right-Pointing-Pointer We found ISL-induced genes and miRNAs through microarray approach. Black-Right-Pointing-Pointer ISL-treated LCLs represented gene expression changes in cell cycle and p53 pathway. Black-Right-Pointing-Pointer We revealed 12 putative mRNA-miRNA functional pairs associated with ISL effect. -- Abstract: Isoliquiritigenin (ISL) has been known to induce cell cycle arrest and apoptosis of various cancer cells. However, genetic factors regulating ISL effects remain unclear. The aim of this study was to identify the molecular signatures involved in ISL-induced cell death of EBV-transformed lymphoblastoid cell lines (LCLs) using microarray analyses. For gene expression and microRNA (miRNA) microarray experiments, each of 12 LCL strains was independently treated with ISL or DMSO as a vehicle control for a day prior to total RNA extraction. ISL treatment inhibited cell proliferation of LCLs in a dose-dependent manner. Microarray analysis showed that ISL-treated LCLs represented gene expression changes in cell cycle and p53 signaling pathway, having a potential as regulators in LCL survival and sensitivity to ISL-induced cytotoxicity. In addition, 36 miRNAs including five miRNAs with unknown functions were differentially expressed in ISL-treated LCLs. The integrative analysis of miRNA and gene expression profiles revealed 12 putative mRNA-miRNA functional pairs. Among them, miR-1207-5p and miR-575 were negatively correlated with p53 pathway- and cell cycle-associated genes, respectively. In conclusion, our study suggests that miRNAs play an important role in ISL-induced cytotoxicity in LCLs by targeting signaling pathways including p53 pathway and cell cycle.

  15. A process-line for large area organic solar cells

    NASA Astrophysics Data System (ADS)

    Alstrup, Jan; Krebs, Frederik C.; Kjćr, Torben; Biancardo, Matteo; Spanggaard, Holger

    2005-10-01

    In this paper we would like to address the key role of fabrication in the performance and lifetime of organic photovoltaics. The realization of a complete process line for the construction of large area organic photovoltaics (250 x 400 mm) is described. Among many of the factors that influence organic solar cell lifetime, oxygen and water exposure is the most important. Multiple processes have to be performed under controlled atmosphere and a glove box (or glove boxes), which involves more volume than commercially available glove boxes, needs to house different instruments. The processes housed in the glove boxes were spin coating, evaporation, lamination/sealing and testing, under an inert atmosphere. The main strategy employed multiply connected glove boxes with one load lock. The first glove box was used for spin coating and lamination/sealing, the second will house a screen printer and the third one accommodate an evaporator completely build in house. The evaporator has 2 thermal evaporation sources and 2 e-beams with 4 and 1 crucibles. The process line should allow the entire device realization from substrate coating, to electrode evaporation including the sealing process avoiding air and water exposure. Organic solar cells from small test cells on ITO glass to big modules (250 x 400 mm) of 91 connected cells on ITO PET substrates were fabricated and characterized.

  16. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Juntendo University School of Medicine, Tokyo ; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  17. SilenciX cell lines for valuable insights into cellular SilenciX cell lines offer a fast and economical approach for performing studies of biological

    E-print Network

    Cai, Long

    that will create cell lines with any silenced gene of a researcher's interest. The cell lines may be HeLa cells in HeLa cells for proteins involved in DNA damage and repair path- ways1. Here we illustrate | Immunocytochemical staining of DNA-PKcs HeLa SilenciX cells 163 days after transfection. (a­d) Control HeLa Silenci

  18. Selected human T cell lines respond to thymopoietin with intracellular cyclic GMP elevations.

    PubMed

    Baker, B; Viamontes, G; Audhya, T; Goldstein, G

    1988-01-01

    Ten established human cell lines were tested for their responsiveness to thymopoietin by measuring their intracellular cyclic nucleotide levels. Three T cell lines (CCRF-CEM, MOLT-4 and CCRF-HSB-2) responded to thymopoietin with elevations of intracellular cGMP but not cAMP; seven other human cell lines did not respond to thymopoietin (three T cell lines, three B cell lines and one erythropoietic stem cell line). Interestingly, only one cell line (MOLT-4) was also responsive to the closely related polypeptide splenin, and this reactivity was restricted to human and not bovine splenin. The detection of human cell lines with distinctive patterns of response to immunoregulatory peptides should provide support for understanding the immunopharmacological mechanisms by which these molecules act. PMID:2849599

  19. Establishment of an ASPL-TFE3 renal cell carcinoma cell line (S-TFE).

    PubMed

    Hirobe, Megumi; Masumori, Naoya; Tanaka, Toshiaki; Kitamura, Hiroshi; Tsukamoto, Taiji

    2013-06-01

    Xp11 translocation renal cell carcinoma is a rare disease diagnosed in children and adolescents in the advanced stage with an aggressive clinical course. Various gene fusions including the transcription factor E3 (TFE3) gene located on chromosome X cause the tumor. We established an Xp11 translocation renal cell carcinoma cell line from a renal tumor in a 18-y-old Japanese female and named it "S-TFE." The cell line and its xenograft demonstrated definite gene fusion including TFE3. They showed strong nuclear staining for TFE3 in immunohistochemistry, TFE3 gene rearrangement in dual-color, break-apart FISH analysis and ASPL-TFE3 type 1 fusion transcripts detected by RT-PCR and direct DNA sequencing. Although many renal cell carcinoma cell lines have been established and investigated, only a few cell lines are recognized as Xp11.2 translocation carcinoma. S-TFE will be useful to examine the characteristics and drug susceptibility of Xp11 translocation renal cell carcinoma. PMID:23760492

  20. Cytotoxic effects of mistletoe (Viscum album L.) in head and neck squamous cell carcinoma cell lines.

    PubMed

    Klingbeil, Ma Fátima G; Xavier, Flávia C A; Sardinha, Luiz R; Severino, Patricia; Mathor, Monica B; Rodrigues, Rodrigo V; Pinto, Décio S

    2013-11-01

    Head and neck squamous cell carcinoma is a complex disease with several etiologic factors and different molecular changes that may trigger certain events; it is also globally one of the most common malignancies in this topography. Extracts from Viscum album L. (VA) (mistletoe) have been used as adjuvant therapies with promising results in several types of cancer, mainly in European countries. In vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells, activating the apoptotic cascade or leading cells to necrosis. This study aimed to verify the effects of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue cell lines SCC9 and SCC25, not previously studied. A concentration of 0.3 mg/ml (IC50) of the drugs induced apoptosis, affecting gene expression and protein levels of AKT, PTEN and CYCLIN D1. It was concluded that VA extracts have a cytotoxic effect on SCC9 and SCC25 cell lines, but while SCC9 cell line was more resistant to the action of the drugs, Iscador Qu Spezial and Iscador M have higher cytotoxic potential in both cell lines compared to Iscador P. PMID:24026291

  1. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    SciTech Connect

    Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

    2010-05-28

    Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

  2. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

    The eye is an immune privileged tissue with multiple mechanisms of immunosuppression to protect the light gathering tissues from the damage of inflammation. One of theses mechanisms involves retinal pigment epithelial cell suppression of phagosome activation in macrophages. The objective of this work is to determine if the human RPE cell line ARPE-19 is capable of suppressing the activation of the phagolysosome in macrophages in a manner similar to primary RPE. The conditioned media of RPE eyecups, sub-confluent, just confluent cultures, or established confluent cultures of human ARPE-19 cells were generated. These condition media were used to treat macrophages phagocytizing pHrodo bioparticles. After 24 hours incubation the macrophages were imaged by fluorescent microscopy, and fluorescence was measured. The fluorescent intensity is proportional to the amount of bioparticles phagocytized and are in an activated phagolysosome. The conditioned media of in situ mouse RPE eyecups significantly suppressed the activation of phagolysosome. The conditioned media from cultures of human ARPE-19 cells, grown to sub-confluence (50%) or grown to confluence had no effect on phagolysosome activation. In contrast, the conditioned media from established confluent cultures significantly suppressed phagolysosome activation. The neuropeptides alpha-MSH and NPY were depleted from the conditioned media of established confluent ARPE-19 cell cultures. This depleted conditioned media had diminished suppression of phagolysosome activation while promoting macrophage cell death. In addition, the condition media from cultures of ARPE-19 monolayers wounded with a bisecting scrape was diminished in suppressing phagolysosome activation. This technical report suggests that like primary RPE monolayers, established confluent cultures of ARPE-19 cells produce soluble factors that suppress the activation of macrophages, and can be used to study the molecular mechanisms of retinal immunobiology. In addition, the results further demonstrate the importance of an intact monolayer of RPE cells to modulate immune cell activity within the eye. PMID:25905107

  3. Complementation analysis of the murine scid cell line

    SciTech Connect

    Zdzienicka, M.Z. |; Priestly, A.; Jeggo, P.A.

    1995-09-01

    It has been shown that several X-ray-sensitive Chinese hamster cell mutants defective in repair of DNA double-strand breaks (DSBs) are also impaired in the process of V(D)J recombination. The hamster mutants with this phenotype represent three distinct complementation groups, represented by the xrs series, XR-1 and V-3. The murine scid cell line also shows the same phenotype, and therefore we examined whether the scid mutant represents a new complementation group or belongs to one of the existing groups. Scid cells were fused with hamster cell mutants representing the three complementation groups. Hybrids between V-3 and scid cells were only partially complemented for X-ray sensitivity, whereas hybrids derived from fusions with the other mutants were resistant to X rays. These results suggest that V-3 and scid cells are defective in the same gene. To confirm this finding, a single human chromosome 8, which is known to carry the scid gene, was introduced into V-3 cells by microcell-mediated chromosome transfer. Nine hybrid clones derived from V-3 and carrying human chromosome 8 were obtained, and seven were found to be partially complemented for X-ray sensitivity. When human chromosome 8 was introduced into scid cells, seven of eight hybrid clones became resistant to X rays. The results indicate that the defective genes in V-3 and scid are both localized on human chromosome 8. This supports the results from the fusion analysis that V-3 and scid cells are defective in the same gene. 53 refs., 4 figs., 1 tab.

  4. Functional inhibition of endogenously produced urokinase decreases cell proliferation in a human melanoma cell line

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Christ, G.; Binder, B.R. )

    1989-07-01

    Binding of urokinase-type plasminogen activator (u-PA) to its receptor has been shown not only to focus proteolytic activity to the cell surface but also to exert a mitogenic effect on the human epidermal tumor cell line CCL 20.2. This report shows that u-PA is an autocrine mitogen in the human melanoma cell line GUBSB and that inhibition of receptor-bound u-PA by specific anti-u-PA antibodies causes a significant suppression of cell proliferation in this system. The GUBSB cell line secretes 70-80% of the u-Pa in its active form and expresses high-affinity u-PA receptors. Approximately 70% of the u-Pa receptors on these cells are occupied by endogenously secreted u-PA. Addition of the monoclonal antiu-PA antibody MPW5UK (10 nM), directed against the active site of u-PA, twice daily to the cell cultures resulted in a significant decrease of ({sup 3}H)thymidine incorporation by the tumor cells, whereas a 10 times higher concentration of the monoclonal antibody MPW4UK, which does not inhibit plasminogen activator activity of u-PA, was necessary to achieve the same effect. Therefore, inhibition of receptor-bound u-PA might represent a tool not only to inactivate cell-bound proteolytic activity, necessary for invasion, but also to exert a specific antiproliferative effect on certain tumor cells.

  5. Discriminating Normal and Cancerous Thyroid Cell Lines using Implicit Context Representation Cartesian Genetic Programming

    E-print Network

    Fernandez, Thomas

    Discriminating Normal and Cancerous Thyroid Cell Lines using Implicit Context Representation a method for discrimi- nating between thyroid cell lines. Five commercial thyroid cell lines were obtained common thyroid malignancy, followed by follicular carcinoma. Both of these cancers have a high chance

  6. Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research

    E-print Network

    Bandettini, Peter A.

    Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH Funded Research NIH Form for use in NIH funded research in accord with the NIH Guidelines for Human Stem Cell Research (74 FR 32170 in NIH funded research, the stem cell lines, and provider information if the lines are available

  7. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  8. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  9. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  10. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  11. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  12. LETTER doi:10.1038/nature11003 The Cancer Cell Line Encyclopedia enables predictive

    E-print Network

    Kaski, Samuel

    LETTER doi:10.1038/nature11003 The Cancer Cell Line Encyclopedia enables predictive modelling and pharmacological annotation is available1 . Here we describe the Cancer Cell Line Encyclopedia (CCLE cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479of

  13. CHALLENGES AND PROSPECTS FOR THE ESTABLISHMENT OF EMBRYONIC STEM CELL LINES OF DOMESTICATED UNGULATES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The establishment of embryonic stem (ES) cell lines of domesticated ungulates, e.g., the pig, sheep, goat, cow or horse, is of interest for similar reasons to those of mouse and primate ES cell lines. Several applied research initiatives await the establishment of ungulates ES cell lines. These inc...

  14. LINE-1 induces hTERT and ensures telomere maintenance in tumour cell lines.

    PubMed

    Aschacher, T; Wolf, B; Enzmann, F; Kienzl, P; Messner, B; Sampl, S; Svoboda, M; Mechtcheriakova, D; Holzmann, K; Bergmann, M

    2016-01-01

    A hallmark of cancer cells is an activated telomere maintenance mechanism, which allows prolonged survival of the malignant cells. In more than 80% of tumours, telomeres are elongated by the enzyme telomerase, which adds de novo telomere repeats to the ends of chromosomes. Cancer cells are also characterized by expression of active LINE-1 elements (L1s, long interspersed nuclear elements-1). L1 elements are abundant retrotransposons in the eukaryotic genome that are primarily known for facilitating aberrant recombination. Using L1-knockdown (KD), we show for the first time that L1 is critical for telomere maintenance in telomerase-positive tumour cells. The reduced length of telomeres in the L1-KD-treated cells correlated with an increased rate of telomere dysfunction foci, a reduced expression of shelterin proteins and an increased rate of anaphase bridges. The decreased telomere length was associated with a decreased telomerase activity and decreased telomerase mRNA level; the latter was increased upon L1 overexpression. L1-KD also led to a decrease in mRNA and protein expression of cMyc and KLF-4, two main transcription factors of telomerase and altered mRNA levels of other stem-cell-associated proteins such as CD44 and hMyb, as well as a corresponding reduced growth of spheroids. The KD of KLF-4 or cMyc decreased the level of L1-ORF1 mRNA, suggesting a specific reciprocal regulation with L1. Thus, our findings contribute to the understanding of L1 as a pathogenicity factor in cancer cells. As L1 is only expressed in pathophysiological conditions, L1 now appears to be target in the rational treatment of telomerase-positive cancer. PMID:25798839

  15. The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

    PubMed Central

    Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

    2013-01-01

    Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

  16. Susceptibility of human bone marrow cells and hematopoietic cell lines to coxsackievirus B3 infection.

    PubMed Central

    Vuorinen, T; Vainionpää, R; Vanharanta, R; Hyypiä, T

    1996-01-01

    Viremia is commonly observed in association with enterovirus infections, and during this phase viruses can be transmitted to secondary target organs in the body. It is not known, however, whether blood cells play a role in the pathogenesis of enterovirus infection supporting virus replication. Our earlier work (T. Vuorinen, R. Vainionpää, H. Kettinen, and T. Hyypiä, Blood 84:823-829, 1994) demonstrated that coxsackievirus B3 is able to replicate in representatives of B- and T-cell lines but not in a monocytic cell line or peripheral blood mononuclear cells, indicating that virus replication may depend on the differentiation and maturation stages of the cells. Therefore, we have broaden our studies and analyzed the susceptibility of granulocyte-macrophage CFU and hematopoietic cell lines with various differentiation and maturation stages to coxsackievirus B3 infection. Virus replication was detected in B- and T-cell lines with no direct correlation to the maturation stage. Granulocyte-macrophage CFU were also able to support virus multiplication. PMID:8971035

  17. Characterization of a mantle cell lymphoma cell line resistant to the Chk1 inhibitor PF-00477736.

    PubMed

    Restelli, Valentina; Chilŕ, Rosaria; Lupi, Monica; Rinaldi, Andrea; Kwee, Ivo; Bertoni, Francesco; Damia, Giovanna; Carrassa, Laura

    2015-11-10

    Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the chromosomal translocation t(11;14) that leads to constitutive expression of cyclin D1, a master regulator of the G1-S phase. Chk1 inhibitors have been recently shown to be strongly effective as single agents in MCL. To investigate molecular mechanisms at the basis of Chk1 inhibitor activity, a MCL cell line resistant to the Chk1 inhibitor PF-00477736 (JEKO-1 R) was obtained and characterized. The JEKO-1 R cell line was cross resistant to another Chk1 inhibitor (AZD-7762) and to the Wee1 inhibitor MK-1775. It displayed a shorter doubling time than parental cell line, likely due to a faster S phase. Cyclin D1 expression levels were decreased in resistant cell line and its re-overexpression partially re-established PF-00477736 sensitivity. Gene expression profiling showed an enrichment in gene sets involved in pro-survival pathways in JEKO-1 R. Dasatinib treatment partly restored PF-00477736 sensitivity in resistant cells suggesting that the pharmacological interference of pro-survival pathways can overcome the resistance to Chk1 inhibitors. These data further corroborate the involvement of the t(11;14) in cellular sensitivity to Chk1 inhibitors, fostering the clinical testing of Chk1 inhibitors as single agents in MCL. PMID:26439697

  18. Establishment and characterization of triple drug resistant head and neck squamous cell carcinoma cell lines.

    PubMed

    Govindan, Sindhu Valiyaveedan; Kulsum, Safeena; Pandian, Ramanan Somasundara; Das, Debashish; Seshadri, Mukund; Hicks, Wesley; Kuriakose, Moni Abraham; Suresh, Amritha

    2015-08-01

    Resistance to chemotherapy leading to poor outcome and survival remains a challenge for developing strategies for therapeutic interventions in all types of cancer, including head and neck cancer. In vitro chemoresistant cell line models are an indispensable resource towards delineating the mechanisms involved in drug resistance/response and for the development of novel drugs. Current treatment for head and neck cancer includes chemotherapy with cisplatin, docetaxel and 5-fluorouracil (5-FU) and the response rates to these drugs in patients is 60-80%. The present study aimed to generate head and neck cancer triple drug-resistant cell lines in an effort towards elucidating the mechanisms underlying chemoresistance and providing a resourceful tool for drug design. Using two head and neck squamous cell carcinoma cell lines, Hep-2 (larynx) and CAL-27 (oral cavity), the present study sequentially exposed these cells to increasing concentrations of the combination of docetaxel, cisplatin and 5-FU (TPF) to generate triple drug-resistant cells, termed Hep-2 TPF resistant (TPFR) and CAL-27 TPFR. The effect of the drug treatments on the cell viability, apoptosis, cell cycle and the expression of genes associated with multidrug resistance were analyzed in the parental cells and drug-resistant counterparts. PMID:25962396

  19. PACAP protects against TNF?-induced cell death in olfactory epithelium and olfactory placodal cell lines

    PubMed Central

    Kanekar, Shami; Gandham, Mahendra; Lucero, Mary T

    2010-01-01

    In mouse olfactory epithelium (OE), pituitary adenylate cyclase activating peptide (PACAP) protects against axotomy-induced apoptosis. We used mouse OE to determine whether PACAP protects neurons during exposure to the inflammatory cytokine TNF?. Live slices of neonatal mouse OE were treated with 40 ng/ml TNF? ± 40 nM PACAP for 6 hours and dying cells were live-labeled with 0.5% propidium iodide. TNF? significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNF?-mediated cell death in the olfactory placodal (OP) cell lines, OP6 and OP27. Although OP cell lines express all three PACAP receptors (PAC1, VPAC1,VPAC2), PACAP’s protection of these cells from TNF? was mimicked by the specific PAC1 receptor agonist maxadilan and abolished by the PAC1 antagonist PACAP6–38. Treatment of OP cell lines with blockers or activators of the PLC and AC/MAPKK pathways revealed that PACAP-mediated protection from TNF? involved both pathways. PACAP may therefore function through PAC1 receptors to protect neurons from cell death during inflammatory cytokine release in vivo as would occur upon viral infection or allergic rhinitis-associated injury. PMID:20654718

  20. Cell lines with extended in vitro growth potential from human renal proximal tubule: characterization, response to inducers, and comparison with established cell lines.

    PubMed

    Racusen, L C; Monteil, C; Sgrignoli, A; Lucskay, M; Marouillat, S; Rhim, J G; Morin, J P

    1997-03-01

    Few model systems exist for the study of injury to human renal proximal tubule epithelium. Optimized differentiated human renal epithelial cell lines with extended in vitro growth potential would provide an alternative model system to primary culture or other available non-human mammalian kidney cell lines. For this purpose, human renal tubule epithelial cells were isolated from normal kidney cortex and exposed in culture to a hybrid immortalizing virus, adenovirus 12-SV40. Cell lines were developed by limiting dilution, and three selected cell lines were screened for growth pattern, production of immortalizing virus, tumorigenicity, and ploidy. Cell lines were also monitored for response to inducer agents and matrix factors and were screened for expression of biochemical properties and differentiation markers of renal epithelium. All three are nonproducers of the immortalizing virus and are nontumorigenic. They grow in monolayer, have intermediate growth kinetics, and express markers of renal proximal tubular epithelium by immunohistochemistry. They also express biochemical properties comparable to other widely used proximal tubular cell lines including LLC-RK1, OK, and HK-2 and comparable to human tubular cells in stable culture. Growth medium containing low levels of fetal calf serum, or epidermal growth factor combined with parathyroid hormone, produced optimal growth characteristics, brush border enzyme expression, biochemical properties, and glucose transport in a selected cell line. The addition of dimethyl sulfoxide allows maintenance in morphologically intact monolayers for prolonged periods. These cell lines should be useful model systems for the study of human renal proximal tubular injury or disease. PMID:9042817

  1. Carbon nanotube biocompatibility with cardiac muscle cells

    NASA Astrophysics Data System (ADS)

    Garibaldi, Silvano; Brunelli, Claudio; Bavastrello, Valter; Ghigliotti, Giorgio; Nicolini, Claudio

    2006-01-01

    Purified carbon nanotubes are new carbon allotropes, sharing similarities with graphite, that have recently been proposed for their potential use with biological systems as probes for in vitro research and for diagnostic and clinical purposes. However the biocompatibility of carbon nanotubes with cells represents an important problem that, so far, remains largely uninvestigated. The objective of this in vitro study is to explore the cytocompatibility properties of purified carbon nanofibres with cardiomyocytes. Cardiac muscle cells from a rat heart cell line H9c2 (2-1) have been used. Highly purified single-walled nanotubes (SWNTs) were suspended at the concentration of 0.2 mg ml-1 by ultrasound in complete Dulbecco's modified Eagle's medium, and administered to cells to evaluate cell proliferation and shape changes by light microscopy, cell viability by trypan blue exclusion, and apoptosis, determined flow cytometrically by annexin/PI staining. Microscopic observation evidenced that carbon nanotubes bind to the cell membrane, causing a slight modification in cell shape and in cell count only after three days of treatment. Cell viability was not affected by carbon nanotubes in the first three days of culture, while after this time, cell death was slightly higher in nanotube-treated cells (p = ns). Accordingly, nanotube treatment induced little and non-significant change in the apoptotic cell number at day 1 and 3. The effect of nanotubes bound to cells was tested by reseeding treated cardiomyocytes. Cells from a trypsinized nanotube-treated sample showed a limited ability to proliferate, and a definite difference in shape, with a high degree of cell death: compared to reseeded untreated ones, in SWNT-treated samples the annexin-positive/PI-negative cells increased from 2.9% to 9.3% in SWNT (p<0.05, where p<0.05 defines a statistically significant difference with a probability above 95%), and the annexin-positive/PI-positive cells increased from 5.2% to 18.7% (p<0.05). However, overtime cells from a trypsinized nanotube-treated sample continued to grow, and partially recovered the original shape. In conclusion our results demonstrate that highly purified carbon nanotubes possess no evident short-term toxicity and can be considered biocompatible with cardiomyocytes in culture, while the long-term negative effects, that are evidenced after reseeding, are probably due to physical rather than chemical interactions.

  2. Sourcing human embryos for embryonic stem cell lines: problems & perspectives.

    PubMed

    Mehta, Rajvi H

    2014-11-01

    The ability to successfully derive human embryonic stem cells (hESC) lines from human embryos following in vitro fertilization (IVF) opened up a plethora of potential applications of this technique. These cell lines could have been successfully used to increase our understanding of human developmental biology, transplantation medicine and the emerging science of regenerative medicine. The main source for human embryos has been 'discarded' or 'spare' fresh or frozen human embryos following IVF. It is a common practice to stimulate the ovaries of women undergoing any of the assisted reproductive technologies (ART) and retrieve multiple oocytes which subsequently lead to multiple embryos. Of these, only two or maximum of three embryos are transferred while the rest are cryopreserved as per the decision of the couple. in case a couple does not desire to 'cryopreserve' their embryos then all the embryos remaining following embryo transfer can be considered 'spare' or if a couple is no longer in need of the 'cryopreserved' embryos then these also can be considered as 'spare'. But, the question raised by the ethicists is, "what about 'slightly' over-stimulating a woman to get a few extra eggs and embryos? The decision becomes more difficult when it comes to 'discarded' embryos. As of today, the quality of the embryos is primarily assessed based on morphology and the rate of development mainly judged by single point assessment. Despite many criteria described in the literature, the quality assessment is purely subjective. The question that arises is on the decision of 'discarding' embryos. What would be the criteria for discarding embryos and the potential 'use' of ESC derived from the 'abnormal appearing' embryos? This paper discusses some of the newer methods to procure embryos for the derivation of embryonic stem cell lines which will respect the ethical concerns but still provide the source material. PMID:25673530

  3. Effect of Quercetin on Cell Cycle and Cyclin Expression in Ovarian Carcinoma and Osteosarcoma Cell Lines.

    PubMed

    Catanzaro, Daniela; Ragazzi, Eugenio; Vianello, Caterina; Caparrotta, Laura; Montopoli, Monica

    2015-08-01

    Resistance to chemotherapeutic drugs is a major problem in cancer treatment. The search for new interventions able to overcome this resistance may involve compounds of natural origin, such as flavonoids, ubiquitously present in many foods. In the present study, the cytotoxic effects and cell cycle modulation of the flavonoid quercetin were investigated in ovarian carcinoma (SKOV3) and osteosarcoma (U2OS) human cell lines and in their cisplatin (CDDP)-resistant counterparts (SKOV3/CDDP and U2OSPt cells, respectively). Quercetin (10-50 ?M) caused evident changes in the distribution of cell cycle phases in the CDDP-resistant SKOV3/CDDP ovarian cell line. The levels of cyclin D1 and cyclin B1 were determined by means of Western blot in all cell lines incubated with quercetin (50 ?M) for 48 hours. The cyclin D1 expression was significantly decreased following the treatment with quercetin in SKOV3 and U2OSPt cells, but not in SKOV3/CDDP and U2OS cells. The reduction of cyclin D1 level could be linked to the G1/S phase alteration found in quercetin-treated cells. Although cyclin B1 is required for G2/M phase, and despite our observation that quercetin influenced the G2/M phase of cell cycle, the flavonoid did not affect cyclin B1 levels in all cell lines, indicating the involvement of other possible mechanisms. These results suggest that quercetin, exceeding the resistance to CDDP, might become an interesting tool to evaluate cytotoxic activity in combination with chemotherapy drugs. PMID:26434118

  4. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  5. Single-cell printing to form three-dimensional lines of olfactory ensheathing cells.

    PubMed

    Othon, Christina M; Wu, Xingjia; Anders, Juanita J; Ringeisen, Bradley R

    2008-09-01

    Biological laser printing (BioLP) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ( approximately microLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 microm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth. PMID:18689930

  6. Control of Differentiation of a Mammary Cell Line by Lipids

    NASA Astrophysics Data System (ADS)

    Dulbecco, Renato; Bologna, Mauro; Unger, Michael

    1980-03-01

    A rat mammary cell line (LA7) undergoes spontaneous differentiation into domes due to production of specific inducers by the cells. Some of these inducers may be lipids, and we show that lipids regulate this differentiation as both inducers and inhibitors. One inhibitor is the tumor promoter tetradecanoyl-13 phorbol 12-acetate. The inducers are saturated fatty acids of two groups: butyric acid and acids with chain lengths from C13 to C16, especially myristic acid (C14). Other inducers are myristoyl and palmitoyl lysolecithins, myristic acid methyl ester, and two cationic detergents with a tetradecenyl chain. We propose that the lipids with a C14-C16 alkyl chain affect differentiation by recognizing specific receptors through their alkyl chains and that the effects obtained depend on the head groups. These lipids may be physiological regulators in the mammary gland.

  7. Identification of a mitotic death signature in cancer cell lines.

    PubMed

    Sakurikar, Nandini; Eichhorn, Joshua M; Alford, Sarah E; Chambers, Timothy C

    2014-02-28

    This study examined the molecular mechanism of action of anti-mitotic drugs. The hypothesis was tested that death in mitosis occurs through sustained mitotic arrest with robust Cdk1 signaling causing complete phosphorylation of Mcl-1 and Bcl-xL, and conversely, that mitotic slippage is associated with incomplete phosphorylation of Mcl-1/Bcl-xL. The results, obtained from studying six different cancer cell lines, strongly support the hypothesis and identify for the first time a unique molecular signature for mitotic death. The findings represent an important advance in understanding anti-mitotic drug action and provide insight into cancer cell susceptibility to such drugs which has important clinical implications. PMID:24099917

  8. Quantitative assessment of telomerase components in cancer cell lines.

    PubMed

    Ak?nc?lar, Semih Can; Low, Kee Chung; Liu, Chia Yi; Yan, Ting Dong; Oji, Asami; Ikawa, Masahito; Li, Shang; Tergaonkar, Vinay

    2015-04-13

    Besides its canonical function of catalyzing the formation of telomeric repeats, many groups have recently reported non-canonical functions of hTERT in particular, and telomerase in general. Regulating transcription is the central basis of non-canonical functions of telomerase. However, unlike reverse transcriptase activity of telomerase that requires only a few molecules of enzymatically active hTERT, non-canonical functions of hTERT or other telomerase components theoretically require several hundred copies. Here, we provide the first direct quantification of all the telomerase components in human cancer cell lines. We demonstrate that telomerase components do not exist in a 1:1 stoichiometric ratio, and there are several hundred copies of hTERT in cells. This provides the molecular basis of hTERT to function in other signaling cascades, including transcription. PMID:25749370

  9. Development of cell lines from the sheep used to construct the CHORI-243 ovine BAC library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.OVSM and MARC.OKF, were initiated from the aorta and kidney, respectively, obtained from the Texel ram used to make the CHORI-243 Ovine BAC library. These cell lines have been submitted to the NIA Aging Cell Repository at the Coriell Cell Respositories, Camden, NJ, U...

  10. Research paper Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line

    E-print Network

    Rubel, Edwin

    Research paper Cisplatin-induced hair cell loss in zebrafish (Danio rerio) lateral line Henry C. Ou hair cell loss in the lateral line in a dose-dependent fashion. The kinetics of hair cell loss during online 19 July 2007 Abstract We have used time-lapse imaging to study cisplatin-induced hair cell death

  11. High-Resolution Genomic Profiles of Breast Cancer Cell Lines Assessed by Tiling BAC Array

    E-print Network

    Lunds Universitet,

    High-Resolution Genomic Profiles of Breast Cancer Cell Lines Assessed by Tiling BAC Array Physics,Lund University,Lund,Sweden 6 Lund Strategic Research Center for Stem Cell Biologyand Cell Therapy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1

  12. Photodynamic therapy-induced programmed cell death in carcinoma cell lines

    NASA Astrophysics Data System (ADS)

    He, Xiao-Yan; Sikes, Robert A.; Thomsen, Sharon L.; Chung, L.; Jacques, Steven L.

    1993-06-01

    The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal apoptosis at the LD50 but had a marked response at the LD85. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (< 1 hr). H322a cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

  13. Gene expression pattern of insect fat body cells from in vitro challenge to cell line establishment.

    PubMed

    Zhang, Huan; Meng, Qian; Tang, Ping; Li, Xuan; Zhu, Wei; Zhou, Guiling; Shu, Ruihao; Zhang, Jihong; Qin, Qilian

    2014-12-01

    The cell lines provided excellent tools to understand the mechanism of biological phenomenon at the cellular and molecular levels. The continuous development of new cell culture technology is both of interest for use in biochemical, immunology, and virological studies. The transformation of cells of the primary culture is a key procedure for insect cell line establishment but little is known about the molecular basis of these changes. Here, we found that the cell cycle progression of the cells of the primary culture was delayed or arrested in G2/M by fluorescence-activated cell sorting analysis. In this study, two subtractive cDNA libraries were constructed to screen for immortal-related genes of Spodoptera exigua (Lepidoptera: Noctuidae). Gene ontology and pathway analysis indicated that members of the oxidative phosphorylation, PI3K-Akt signaling pathway, and the ubiquitin proteasome pathway are involved in processes leading toward cell immortalization merit further investigation. Our findings suggest that tumor-related genes or target genes of these pathways may contribute to the transformation of primary cell through regulation of G2/M cell cycle progression. PMID:25213689

  14. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  15. Atrazine exposure decreases cell proliferation in Chinese Hamster Ovary (CHO-K1) cell line.

    PubMed

    Kmetic, I; Gaurina Srcek, V; Slivac, I; Simi?, B; Kniewald, Z; Kniewald, J

    2008-08-01

    The aim of this study was to determine the toxic effect of atrazine at the ovarian cellular level. Chinese Hamster Ovary (CHO-K1) cell line was used to evaluate the degree of in vitro atrazine cytotoxicity and the morphological changes were followed during the cell death. Application of four bioassays confirmed that atrazine decreases ovarian cell proliferation and IC(50) were determined with each assay after 72 h of exposure. The level of apoptosis in atrazine treated cells was low. PMID:18465069

  16. Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

    PubMed Central

    Hamilton, Gerhard

    2014-01-01

    Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. PMID:24608973

  17. Relating hepatocellular carcinoma tumor samples and cell lines using gene expression data in translational research

    PubMed Central

    2015-01-01

    Cancer cell lines are used extensively to study cancer biology and to test hypotheses in translational research. The relevance of cell lines is dependent on how closely they resemble the tumors being studied. Relating tumors and cell lines, and recognizing their similarities and differences are thus very important for translational research. Rapid advances in genomics have led to the generation of large volumes of genomic and transcriptomic data for a diverse set of primary cancer samples, normal tissue samples and cancer cell lines. Hepatocellular Carcinoma (HCC) is one of the most common tumors worldwide, with high occurrence in Asia and sub-Saharan regions. The current effective treatments of HCC remain limited. In this work, we compared the gene expression measurements of 200 HCC tumor samples from The Cancer Genome Atlas and over 1000 cancer cell lines including 25 HCC cancer cell lines from Cancer Cell Line Encyclopedia. We showed that the HCC tumor samples correlate closely with HCC cell lines in comparison to cell lines derived from other tumor types. We further demonstrated that the most commonly used HCC cell lines resemble HCC tumors, while we identified nearly half of the cell lines that do not resemble primary tumors. Interestingly, a substantial number of genes that are critical for disease development or drug response are either expressed at low levels or absent among highly correlated cell lines; additional attention should be paid to these genes in translational research. Our study will be used to guide the selection of HCC cell lines and pinpoint the specific genes that are differentially expressed in either tumors or cell lines. PMID:26043652

  18. A comparison of heat and radiation sensitivity of three human glioma cell lines

    SciTech Connect

    Raaphorst, G.P.; Feeley, M.M.; Da Silva, V.F.; Danjoux, C.E.; Gerig, L.H. )

    1989-09-01

    Three human glioma cell lines were tested for radiation and hyperthermia sensitivity and compared to the responses of a normal human fibroblast cell line. The radiation response of the glioma cell lines exhibited a large shoulder on the radiation survival curve indicating radioresistance when compared to the more radiosensitive fibroblast cell line. The hyperthermia response for the glioma cell lines was qualitatively similar to responses reported for other cell lines. When compared to normal human fibroblasts the glioma cells were found to be more sensitive to hyperthermia than the normal fibroblasts indicating hyperthermia may be a promising method or adjunct to radiotherapy in the treatment of resistant glioma cells or tumors. The results also show that both the radiation and thermal response is influenced by cell culture conditions and growth status. Two of the cell lines grown to confluency and treated in confluency showed an increased radiation resistance at low doses and the cell lines showed decreased resistance at high doses compared to cells plated to confluency. An increased thermal resistance, especially at the lower heating temperatures, was also observed for cells grown to confluency. Measurements of residual glucose in the culture medium at the time of irradiation was about the same for the two culture methods (55%-65%). Cell cycle analysis showed that the differences were not related to changes in cell cycle distribution.

  19. The telomerase inhibitor imetelstat depletes cancer stem cells in breast and pancreatic cancer cell lines.

    PubMed

    Joseph, Immanual; Tressler, Robert; Bassett, Ekaterina; Harley, Calvin; Buseman, Christen M; Pattamatta, Preeti; Wright, Woodring E; Shay, Jerry W; Go, Ning F

    2010-11-15

    Cancer stem cells (CSC) are rare drug-resistant cancer cell subsets proposed to be responsible for the maintenance and recurrence of cancer and metastasis. Telomerase is constitutively active in both bulk tumor cell and CSC populations but has only limited expression in normal tissues. Thus, inhibition of telomerase has been shown to be a viable approach in controlling cancer growth in nonclinical studies and is currently in phase II clinical trials. In this study, we investigated the effects of imetelstat (GRN163L), a potent telomerase inhibitor, on both the bulk cancer cells and putative CSCs. When breast and pancreatic cancer cell lines were treated with imetelstat in vitro, telomerase activity in the bulk tumor cells and CSC subpopulations were inhibited. Additionally, imetelstat treatment reduced the CSC fractions present in the breast and pancreatic cell lines. In vitro treatment with imetelstat, but not control oligonucleotides, also reduced the proliferation and self-renewal potential of MCF7 mammospheres and resulted in cell death after <4 weeks of treatment. In vitro treatment of PANC1 cells showed reduced tumor engraftment in nude mice, concomitant with a reduction in the CSC levels. Differences between telomerase activity expression levels or telomere length of CSCs and bulk tumor cells in these cell lines did not correlate with the increased sensitivity of CSCs to imetelstat, suggesting a mechanism of action independent of telomere shortening for the effects of imetelstat on the CSC subpopulations. Our results suggest that imetelstat-mediated depletion of CSCs may offer an alternative mechanism by which telomerase inhibition may be exploited for cancer therapy. PMID:21062983

  20. 188Rhenium-induced cell death and apoptosis in a panel of tumor cell lines

    NASA Astrophysics Data System (ADS)

    Antoccia, Antonio; Banzato, Alessandra; Bello, Michele; Bollini, Dante; De Notaristefani, Francesco; Giron, Cecilia; Mazzi, Ulderico; Alafort, Laura Melendez; Moschini, Giuliano; Nadali, Anna; Navarria, Francesco; Perrotta, Andrea; Rosato, Antonio; Tanzarella, Caterina; Uzunov, Nikolay

    2007-02-01

    Assessment of "in vitro" tumor growth inhibition and radiobiological effects, such as apoptosis, have been evaluated in human neoplastic cells of different histotypes (H460 lung cancer cells, U87 glioblastoma, LnCaP prostate tumor cells) treated using solutions of 188Rhenium-perrhenate. The MTT assay, which measures mitochondrial metabolism in the entire cell culture is a recognized test for cytotoxicity and was used in cells exposed 48-72 h to specific activities ranged from 37 to 148 GBq/l. Whereas H460 and LnCaP were particularly sensitive to treatment, U87 glioblastoma cells behaved as radioresistant ones. However, evaluation of 188Re-induced apoptosis indicated that this kind of cell death contributed only marginally to the reduction in cell viability of H460 and LNCaP lines, suggesting the existence of protective mechanisms against apoptosis. In this respect, the membrane receptor, CD44, whose expression is dysregulated in most malignant cell types has proven to alter the response of cancer cells to apoptotic stimuli, including ionizing radiation. Cell samples decorated with a FITC-labelled CD44 antibody indicated, that in H460 and U87 cells the CD44(+) correlated well with an apoptosis-resistant response. Conversely, LnCap cells proven as CD44(-) did not display however sensitivity to radio-induced apoptosis.

  1. Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

    PubMed

    Zou, Jianhua; Ma, Xiaoqiong; Zhang, Guangji; Shen, Li; Zhou, Liting; Yu, Yu; Zhu, Fanfan; Chen, Zhe

    2015-11-01

    Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI-MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile-water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1-5?M iAs(III)(As(III) ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAs(III) is 1.0?M. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. PMID:26454796

  2. Enhancement of Radiation Response in Osteosarcoma and Rhabomyosarcoma Cell Lines by Histone Deacetylase Inhibition

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

    Purpose: Histone deacetylase inhibitors (HDACIs) can enhance the sensitivity of cells to photon radiation treatment (XRT) by altering numerous molecular pathways. We investigated the effect of pan-HDACIs such as suberoylanilide hydroxamic acid (SAHA) on radiation response in two osteosarcoma (OS) and two rhabdomyosarcoma (RMS) cell lines. Methods and Materials: Clonogenic survival, cell cycle analysis, and apoptosis were examined in OS (KHOS-24OS, SAOS2) and RMS (A-204, RD) cell lines treated with HDACI and HDACI plus XRT, respectively. Protein expression was investigated via immunoblot analysis, and cell cycle analysis and measurement of apoptosis were performed using flow cytometry. Results: SAHA induced an inhibition of cell proliferation and clonogenic survival in OS and RMS cell lines and led to a significant radiosensitization of all tumor cell lines. Other HDACI such as M344 and valproate showed similar effects as investigated in one OS cell line. Furthermore, SAHA significantly increased radiation-induced apoptosis in the OS cell lines, whereas in the RMS cell lines radiation-induced apoptosis was insignificant with and without SAHA. In all investigated sarcoma cell lines, SAHA attenuated radiation-induced DNA repair protein expression (Rad51, Ku80). Conclusion: Our results show that HDACIs enhance radiation action in OS and RMS cell lines. Inhibition of DNA repair, as well as increased apoptosis induction after exposure to HDACIs, can be mechanisms of radiosensitization by HDACIs.

  3. Gene expression profiling analysis of osteosarcoma cell lines

    PubMed Central

    SUN, LU; LI, JIE; YAN, BING

    2015-01-01

    Osteosarcoma (OS) is the most common type of primary bone malignancy and has a poor prognosis. To investigate the mechanisms of osteosarcoma, the present analyzed the GSE28424 microarray. GSE28424 was downloaded from the Gene Expression Omnibus, and included a collective of 19 OS cell lines and four normal bone cell lines, which were used as controls. Subsequently, the differentially expressed genes (DEGs) were screened using the Limma package in Bioconductor. Gene Ontology (GO) and pathway enrichment analysis of the DEGs was performed using the Database for Annotation, Visualization and Integrated Discovery, interactions between the proteins encoded by the DEGs were identified using STRING, and the protein-protein interaction (PPI) network was visualized using Cytoscape. In addition, modular analysis of the PPI network was performed using the Clique Percolation Method (CPM) in CFinder. A total of 1,170 DEGs were screened, including 530 upreguated and 640 downregulated genes. The enriched functions included organelle fission, immune response and response to wounding. In addition, RPL8 was observed to be involved with the ribosomal pathway in module A of the PPI network of the DEGs. PLCG1, SYK and PLCG2 were also involved in the B-cell receptor signaling pathway in module B and the Fc-epsilon RI signaling pathway in module C. In addition, AURKA (degree=39), MAD2L1 (degree=38), CDCA8 (degree=38), BUB1 (degree=37) and MELK (degree=37) exhibited higher degrees of connectivity in module F. The results of the present study suggested that the RPL8, PLCG1, PLCG2, SYK, MAD2L1, AURKA, CDCA8, BUB1 and MELK genes may be involved in OS. PMID:26096802

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

    SciTech Connect

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee; Jeon, Jae-Pil

    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.

  5. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  6. Phytoestrogens regulate the proliferation and expression of stem cell factors in cell lines of malignant testicular germ cell tumors

    PubMed Central

    HASIBEDER, ASTRID; VENKATARAMANI, VIVEK; THELEN, PAUL; RADZUN, HEINZ-JOACHIM; SCHWEYER, STEFAN

    2013-01-01

    Phytoestrogens have been shown to exert anti-proliferative effects on different cancer cells. In addition it could be demonstrated that inhibition of proliferation is associated with downregulation of the known stem cell factors NANOG, POU5F1 and SOX2 in tumor cells. We demonstrate the potential of Belamcanda chinensis extract (BCE) and tectorigenin as anticancer drugs in cell lines of malignant testicular germ cell tumor cells (TGCT) by inhibition of proliferation and regulating the expression of stem cell factors. The TGCT cell lines TCam-2 and NTera-2 were treated with BCE or tectorigenin and MTT assay was used to measure the proliferation of tumor cells. In addition, the expression of stem cell factors was analyzed by quantitative PCR and western blot analysis. Furthermore, global expression analysis was performed by microarray technique. BCE and tectorigenin inhibited proliferation and downregulated the stem cell factors NANOG and POU5F1 in TGCT cells. In addition, gene expression profiling revealed induction of genes important for the differentiation and inhibition of oncogenes. Utilizing connectivity map in an attempt to elucidate mechanism underlying BCE treatments we found highly positive association to histone deacetylase inhibitors (HDACi) amongst others. Causing no histone deacetylase inhibition, the effects of BCE on proliferation and stem cell factors may be based on histone-independent mechanisms such as direct hyperacetylation of transcription factors. Based on these findings, phytoestrogens may be useful as new agents in the treatment of TGCT. PMID:23969837

  7. Differential sensitivity of telomerase from human hematopoietic stem cells and leukemic cell lines to mild hyperthermia.

    PubMed

    Deezagi, Abdolkhaleg

    2014-07-01

    We have investigated the effects of hyperthermia (HT) on cell proliferation and telomerase activity of human hematopoietic stem cells (HSCs) and compared with human leukemic cell lines (TF-1, K562 and HL-60). The cells were exposed to HT at 42 and 43 °C up to 120 min. The cells were incubated at 37 °C for 96 h. Then the cells were collected and assayed for cell proliferation, viability, telomerase activity, and terminal restriction fragment (TRF) lengths. The enzyme activity from HSCs was decreased up to 68.6 at 42 and 85.1 % at 43 °C for 120 min. This inhibition in leukemic cells was up to 28.9 and 53.6 % in TF-1; 53 and 63.9 % in K562; 45.2 and 61.1 % in HL-60 cells. The treated cells showed TRF lengths about 5.3 kb for control HL-60 cells, 5.0 kb for HL-60 cells treated at 42 and 4.5 kb at 43 °C for 120 min. In HSCs, the TRF length was about 4.5 kb for untreated cells and 4.0-4.5 kb for treated cells at 42 and 43 °C for 120 min. The time response curves indicated that, inhibition of the enzyme activity in leukemic cells was dependent to the time of exposure to HT. But in HSCs, the inhibition was reached to steady state at 15 min exposure to 43 °C heat stress. TRF length was constant at treated two types of cells, which implies that in cells subjected to mild HT no telomere shortening was observed. PMID:24590263

  8. Bub1 is required for maintaining cancer stem cells in breast cancer cell lines.

    PubMed

    Han, Jeong Yoon; Han, Yu Kyeong; Park, Ga-Young; Kim, Sung Dae; Kim, Joong Sun; Jo, Wol Soon; Geun Lee, Chang

    2015-01-01

    Breast cancer is a leading cause of death among women worldwide due to therapeutic resistance and cancer recurrence. Cancer stem cells are believed to be responsible for resistance and recurrence. Many efforts to overcome resistance and recurrence by regulating cancer stem cells are ongoing. Bub1 (Budding uninhibited by benzimidazoles 1) is a mitotic checkpoint serine/threonine kinase that plays an important role in chromosome segregation. Bub1 expression is correlated with a poor clinical prognosis in patients with breast cancer. We identified that depleting Bub1 using shRNAs reduces cancer stem cell potential of the MDA-MB-231 breast cancer cell line, resulting in inhibited formation of xenografts in immunocompromised mice. These results suggest that Bub1 may be associated with cancer stem cell potential and could be a target for developing anti-breast cancer stem cell therapies. PMID:26522589

  9. Bub1 is required for maintaining cancer stem cells in breast cancer cell lines

    PubMed Central

    Han, Jeong Yoon; Han, Yu Kyeong; Park, Ga-Young; Kim, Sung Dae; Kim, Joong Sun; Jo, Wol Soon; Geun Lee, Chang

    2015-01-01

    Breast cancer is a leading cause of death among women worldwide due to therapeutic resistance and cancer recurrence. Cancer stem cells are believed to be responsible for resistance and recurrence. Many efforts to overcome resistance and recurrence by regulating cancer stem cells are ongoing. Bub1 (Budding uninhibited by benzimidazoles 1) is a mitotic checkpoint serine/threonine kinase that plays an important role in chromosome segregation. Bub1 expression is correlated with a poor clinical prognosis in patients with breast cancer. We identified that depleting Bub1 using shRNAs reduces cancer stem cell potential of the MDA-MB-231 breast cancer cell line, resulting in inhibited formation of xenografts in immunocompromised mice. These results suggest that Bub1 may be associated with cancer stem cell potential and could be a target for developing anti-breast cancer stem cell therapies. PMID:26522589

  10. More similar than different: Host cell protein production using three null CHO cell lines.

    PubMed

    Yuk, Inn H; Nishihara, Julie; Walker, Donald; Huang, Eric; Gunawan, Feny; Subramanian, Jayashree; Pynn, Abigail F J; Yu, X Christopher; Zhu-Shimoni, Judith; Vanderlaan, Martin; Krawitz, Denise C

    2015-10-01

    To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ?80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (?1-2?g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis. PMID:25894672

  11. Comparison of betanodavirus replication efficiency in ten Indian fish cell lines.

    PubMed

    Sarath Babu, V; Abdul Majeed, S; Nambi, K S N; Taju, G; Madan, N; Sundar Raj, N; Sahul Hameed, A S

    2013-06-01

    Ten cell lines established from Indian marine, brackishwater and freshwater fish were tested for their susceptibility to fish nodavirus. In addition, the efficiency of betanodavirus replication was tested in these cell lines. Multiple vacuolation, a typical cytopathic effect for virus infection, was observed in infected SISK, SISS, SIGE and ICF cells. Infection of the different fish cell lines was confirmed by RT-PCR, immunodot blot assay and indirect ELISA. The virus concentration in culture supernatant collected from infected sea bass and grouper cell lines increased progressively from 10(3) at day 1 postinfection to 10(8) TCID50 ml(-1) at day 9. The amount of virus in different cell lines was also quantified by real-time PCR. These results indicate the suitability of the SISK, SISS, and SIGE cell lines for fish nodavirus propagation for developing viral diagnostics and vaccines. PMID:23392632

  12. Nuclear Motility in Glioma Cells Reveals a Cell-Line Dependent Role of Various Cytoskeletal Components

    PubMed Central

    Kiss, Alexa; Horvath, Peter; Rothballer, Andrea; Kutay, Ulrike; Csucs, Gabor

    2014-01-01

    Nuclear migration is a general term for the movement of the nucleus towards a specific site in the cell. These movements are involved in a number of fundamental biological processes, such as fertilization, cell division, and embryonic development. Despite of its importance, the mechanism of nuclear migration is still poorly understood in mammalian cells. In order to shed light on the mechanical processes underlying nuclear movements, we adapted a micro-patterning based assay. C6 rat and U87 human glioma cells seeded on fibronectin patterns - thereby forced into a bipolar morphology - displayed oscillatory movements of the nucleus or the whole cell, respectively. We found that both the actomyosin system and microtubules are involved in the nuclear/cellular movements of both cell lines, but their contributions are cell-/migration-type specific. Dynein activity was necessary for nuclear migration of C6 cells but active myosin-II was dispensable. On the other hand, coupled nuclear and cellular movements of U87 cells were driven by actomyosin contraction. We explain these cell-line dependent effects by the intrinsic differences in the overall mechanical tension due to the various cytoskeletal elements inside the cell. Our observations showed that the movements of the nucleus and the centrosome are strongly correlated and display large variation, indicating a tight but flexible coupling between them. The data also indicate that the forces responsible for nuclear movements are not acting directly via the centrosome. Based on our observations, we propose a new model for nuclear oscillations in C6 cells in which dynein and microtubule dynamics are the main drivers of nuclear movements. This mechanism is similar to the meiotic nuclear oscillations of Schizosaccharomyces pombe and may be evolutionary conserved. PMID:24691067

  13. Honokiol induces cell cycle arrest and apoptosis in human gastric carcinoma MGC-803 cell line

    PubMed Central

    Yan, Bin; Peng, Zhi-Yong

    2015-01-01

    Objective: Gastric carcinoma is a malignant tumor that responds poorly to both chemotherapy and radiation therapy. In our study, we investigated the anti-cancer effect of honokiol, an active component isolated and purified from the Magnolia officinalis, in human gastric carcinoma MGC-803 cell line. Methods: The cell viability was detected by the CCK8 assay. The cell apoptosis and cell cycle arrest were assessed by flow cytometer. The protein expression of cell cycle regulators and tumor suppressors were analyzed by western blotting. Results: Treatment of human gastric carcinoma cells with honokiol induced cell death in a dose-and time-dependent manner by using CCK8 assay. Consistent with the CCK8 assay, the flow cytometry results showed that the proportion of apoptosis cells had gained when the cells were exposed to honokiol. Moreover, Cyclin B1, CDC2 and cdc25C were downregulated, and the expression of p-CDC2 and p-cdc25c was significantly upregulated upon honokiol treatment. P53 and p21 were significantly upregulated by honokiol treatment. Treatment of MGC-803 cells with honokiol significantly increased the pro-apoptotic Bax level and decreased the anti-apoptotic Bcl-2 level. Conclusions: These results confirmed that honokiol could induce apoptosis and cell cycle arrest, the underlying molecular mechanisms, at least partially, through activation p53 signaling and downregulation CDC2/cdc25C expression. PMID:26131123

  14. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    SciTech Connect

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z. . E-mail: hzsheng2003@yahoo.com

    2006-11-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.

  15. Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines.

    PubMed

    Moriwaki, S; Ohba, H; Nakamura, O; Sallay, I; Suzuki, M; Tsubouchi, H; Yamasaki, N; Itoh, K

    2000-02-01

    The cytoagglutination by abrin-a against human cultured cell lines derived from acute lymphoblastic leukemia (ALL) and human peripheral blood lymphocytes obtained from normal adults and from patients with adult T cell leukemia (ATL) was investigated. Among acute T lymphoblastic leukemia (T-ALL) cell lines, abrin-a showed strong cytoagglutination against relatively differentiated cell lines, such as Jurkat and CCRF-HSB-2. Among acute B lymphoblastic leukemia (B-ALL) cell lines, abrin-a strongly agglutinated an immature cell line, NALM6. In comparison with ALL cell lines, cytoagglutination by abrin-a against normal lymphocytes was weak. Abrin-a showed higher cytoagglutination against lymphocytes derived from ATL than lymphocytes derived from normal adults. In connection with the cytoagglutination, abrin-a-induced cytotoxicity against human cultured leukemic cell lines was evaluated. In proportion to the extent of cytoagglutination, abrin-a induced cytotoxicity in Jurkat, CCRF-HSB-2, MOLT-4, RPMI8402, and BALL-1 as well. Although CCRF-CEM and BALM-1 were both weakly agglutinated by abrin-a, these cell lines were very sensitive to the abrin-a-induced cytotoxicity. NALM6 was strongly agglutinated by abrin-a, but abrin-a exhibited less strong cytotoxicity against this cell line. These results suggest the feasible application of abrin-a as a tool to distinguish the human leukemic cells and its potential for clinical application. PMID:10738971

  16. Designing cell lines for viral vaccine production: Where do we stand?

    PubMed

    Genzel, Yvonne

    2015-05-01

    Established animal cells, such as Vero, Madin Darby canine kidney (MDCK) or chicken embryo fibroblasts (CEFs), are still the main cell lines used for viral vaccine production, although new "designer cells" have been available for some years. These designer cell lines were specifically developed as a cell substrate for one application and are well characterized. Later screening for other possible applications widened the product range. These cells grow in suspension in chemically defined media under controlled conditions and can be used for up to 100 passages. Scale-up is easier and current process options allow cultivation in disposable bioreactors at cell concentrations higher than 1 × 10(7) cells/mL. This review covers the limitations of established cell lines and discusses the requirements and screening options for new host cells. Currently available designer cells for viral vaccine production (PER.C6, CAP, AGE1.CR, EB66 cells), together with other new cell lines (PBS-1, QOR/2E11, SogE, MFF-8C1 cells) that were recently described as possible cell substrates are presented. Using current process knowledge and cell line development tools, future upstream processing could resemble today's Chinese hamster ovary (CHO) cell processes for monoclonal antibody production: small scale bioreactors (disposable) in perfusion or fed-batch mode with cell concentrations above 1 × 10(8) cells/mL. PMID:25903999

  17. Effect of 9-cis-retinoic acid on oral squamous cell carcinoma cell lines.

    PubMed

    Hayashi, K; Yokozaki, H; Naka, K; Yasui, W; Yajin, K; Lotan, R; Tahara, E

    2000-04-14

    Retinoic acid (RA) has been shown to be effective in suppressing premalignant lesions and preventing second primary malignancies in patients cured of squamous cell carcinoma of the head and neck. However, the precise mechanisms of these effects are still uncertain. In the present study, we examined the effect of 9-cis-RA on the growth of six oral cancer cell lines (HSC-2, HSC-3, HSC-4, Ca9-22, Ho-1-N-1 and Ho-1-u-1). In addition, the relationship among growth and differentiation of tumor cells, RA responsiveness and the expression of nuclear retinoic acid receptors were also investigated. Among the six cell lines examined, five (HSC-2, HSC-3, HSC-4, Ca9-22 and Ho-1-u-1) displayed growth inhibition after treatment with 1x10(-6) M 9-cis-RA, while Ho-1-N-1 cells were resistant to 9-cis-RA. The expression level of RARbeta in 9-cis-RA resistant Ho-1-N-1 cells was very low in comparison with the sensitive cell lines. On the other hand, all of the six the cell lines expressed RARalpha, RARgamma, and RXRalpha at various levels. 9-cis-RA induced accumulation of cell population in G1 phase in HSC-3 cells on the 6th day of the treatment, followed by a marked reduction in the levels of hyperphosphorylated pRB, whereas p53 level was not altered. Interestingly, 9-cis-RA induced transiently the expression of p21(Waf1/Cip1), p27(Kip1), p300, CBP, BAX, Bak and bcl-2 proteins, respectively. This effect was associated with reduction of cyclin D1, cdk4 and CDK-activating kinase (cyclin H and cdk7) protein in HSC-3 cells. These results suggest that the growth inhibitory effect of 9-cis-RA on oral squamous cell carcinoma may depend on the expression levels of RARs, especially RARbeta proteins and RXRalpha proteins, and that 9-cis-RA may provide a powerful therapeutic agent for head and neck cancers. PMID:10738115

  18. Cell-type specific adhesive interactions of skeletal myoblasts with thrombospondin-1.

    PubMed Central

    Adams, J C; Lawler, J

    1994-01-01

    Thrombospondin-1 (TSP-1) is an extracellular matrix glycoprotein that may play important roles in the morphogenesis and repair of skeletal muscle. To begin to explore the role of thrombospondin-1 in this tissue, we have examined the interactions of three rodent skeletal muscle cell lines, C2C12, G8, and H9c2, with platelet TSP-1. The cells secrete thrombospondin and incorporate it into the cell layer in a distribution distinct from that of fibronectin. Myoblasts attach and spread on fibronectin- or thrombospondin-coated substrates with similar time and concentration dependencies. Whereas cells adherent on fibronectin organize actin stress fibers, cells adherent on TSP-1 display prominent membrane ruffles and lamellae that contain radial actin microspikes. Attachment to thrombospondin-1 or the 140-kDa tryptic fragment is mediated by interactions with the type 1 repeats and the carboxy-terminal globular domain. Attachment is not inhibited by heparin, GRGDSP peptide, or VTCG peptide but is inhibited by chondroitin sulphate A. Integrins of the beta 1 or alpha V subgroups do not appear to be involved in myoblast attachment to TSP-1; instead, this process depends in part on cell surface chondroitin sulphate proteoglycans. Whereas the central 70-kDa chymotryptic fragment of TSP-1 does not support myoblast attachment, the carboxy-terminal domain of TSP-1 expressed as a fusion protein in the bacterial expression vector, pGEX, supported myoblast attachment to 30% the level of intact TSP-1. Thrombospondin-4 (TSP-4) is also present in skeletal muscle and a fusion protein containing the carboxy-terminal domain of TSP-4 also supported myoblast adhesion, although this protein was less active on a molar basis than the TSP-1 fusion protein. Thus, the carboxyterminal domain of TSP-1 appears to contain a primary attachment site for myoblasts, and this activity is present in a second member of the thrombospondin family. Images PMID:7519904

  19. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines

    PubMed Central

    Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G.; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G.; MacLeod, Roderick A. F.

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (?4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings highlight biallelic deletions as a major class of chromosomal lesion in PMBL cell lines, while endorsing the latter as preclinical models for hunting and testing new biomarkers and actionable targets. PMID:26599546

  20. Cytochemical comparison of immunologically characterized human leukaemia/lymphoma cell lines representing different levels of maturation.

    PubMed Central

    Srivastava, B. I.; Rossowski, W.; Minowada, J.

    1983-01-01

    Forty-seven human leukaemia/lymphoma cell lines belonging to myelocytic, monocytic, non-T/non-B, T-, and B-lineage and representing different levels of maturation as well as fresh cells from normal and leukaemic subjects were examined for immunological markers and cytochemically for acid phosphatase, alkaline phosphatase, alpha-naphthyl acetate esterase (pH 5.8 and 8.0), alpha-naphthyl butyrate esterase (pH 5.8 and 8.0), non-specific esterase, chloroacetate esterase, chymotrypsin-like protease, deoxyribonuclease II, beta-glucuronidase, sudan black, and periodic acid Schiff's staining. Strong sudan black, nonspecific esterase, and chloroacetate esterase reaction was obtained only for myelocytic and monocytic cell lines with the reaction intensity increasing progressively in more mature cells. Focal acid phosphatase reaction like T-ALL was found in all T-ALL cell lines, whereas myeloid/monocytoid lines had semicircular distribution and B-cell lines cytoplasmic distribution of activity. Acid phosphatase activity appeared to decline with maturation along both myeloid and T-cell lineage. High activity of alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase both at pH 5.8 and 8.0 and of beta-glucuronidase was found in myeloid/monocytoid lines although both B- and T-cell lines in contrast to peripheral blood B-cells also had significant esterase activity. alpha-Naphthyl butyrate esterase activity declined with increasing cell maturation along myeloid lineage. Except for weak activity in two B-cell lines alkaline phosphatase was not detected in any cell lines. Monocyte esterase activity was inhibited by sodium fluoride whereas acid phosphatase, only from hairy cell leukaemia line, was resistant to L-tartarate. Although periodic acid Schiff's staining could not distinguish myeloid, T-, B-, or non-T/non-B cell lines it gave characteristic reaction (large number of coarse granules against a clear background forming a ring around the nucleus) with erythroblastic leukaemia cell line and along myeloid series its intensity increased in more mature cells. Deoxyribonuclease II and chymotrypsin-like protease staining were not discriminatory. The results of this study show that cytochemical staining characteristics of various leukaemia/lymphoma cell lines are comparable to those of corresponding cells from patients and that the intensity and pattern of expression of these activities are related to cell type and degree of cell maturation. These studies give further credence to the use of these cell lines in cell differentiation, differential drug cytotoxicity, and many other studies. Images Figure 1 Figure 2 PMID:6190491

  1. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    SciTech Connect

    Youakim, A.; Herscovics, A.

    1985-11-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-(2-TH)mannose or L-(5,6-TH)fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with (2-TH)mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with (2-TH)mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-(1,6-TH)glucosamine and L-(1- UC)fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced TH-labeled N-acetylglucosamine and N-acetylgalactosamine.

  2. Antitumor activity of D-mannosamine in vitro: different sensitivities among human leukemia cell lines possessing T-cell properties.

    PubMed

    Onoda, T; Morikawa, S; Harada, T; Suzuki, Y; Inoue, K; Nishigami, K

    1982-07-01

    D-Mannosamine is toxic to human malignant T-lymphoid cell lines derived from patients with T-cell leukemia. We observed heterogeneity of mannosamine susceptibility among those cell lines. The leukemic T-cell lines, subgrouped according to the degree of mannosamine inhibition on nucleic acid biosyntheses, were: Subgroup 1, HPB-MLT cells; Subgroup 2, CCRF-HSB-2 and HPB-ALL cells; and Subgroup 3, MOLT-4 cells. The most sensitive line, HPB-MLT, originated from the patient with adult T-cell leukemia. The cytotoxicity of mannosamine was potentiated by a fatty acid, sodium oleate, at concentrations that were noncytolytic, and the interaction between the two drugs was synergistic. These results would suggest that mannosamine induces changes in the membrane structure of the leukemia cells. Thus, the primary target of the tumoricidal activity of mannosamine may also be the cellular membranes. PMID:6979385

  3. Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

    PubMed Central

    2011-01-01

    Background We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance. Methods Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin. Results Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated). Conclusion Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance. PMID:21575166

  4. ISOLATION AND CHARACTERIZATION OF PORCINE VISCERAL ENDODERM CELL LINES DERIVED FROM IN VIVO 11-DAY BLASTOCYSTS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two porcine cell lines of yolk-sac visceral endoderm, designated PE-1 and PE-2, were derived from in vivo 11-day porcine blastocysts that were either ovoid (PE-1) or at the early tubular stage of elongation (PE-2). Primary and secondary culture of cell lines was done on STO feeder cells. The PE-1 ...

  5. Unexpectedly Low Loss of Heterozygosity in Genetically Unstable Werner Syndrome Cell Lines

    E-print Network

    Monnat, Ray

    -immortalized Werner syndrome (WS) and control fibroblast cell lines. Five microsatellite loci were genotyped in two WS and two control SV40-immortalized fibroblast cell lines and in 154 independent primary. A second, unexpected conclusion is that WS cells may possess a novel means of either suppressing or masking

  6. Establishment and maintenance of three Chinese human embryonic stem cell lines.

    PubMed

    Zhou, Di; Lin, Ge; Xie, Chang-Qing; Xiong, Bo; Zhou, Xiao-Ying; Qian, Xiao-Bing; Liao, Yi; Lu, Guang-Xiu

    2010-04-01

    To establish a potential resource for cell therapy and a developmental model for human diseases, we had isolated three Chinese human embryonic stem cell lines from the inner cell mass of human blastocysts in 2002. All the three cell lines were grown on mouse embryonic fibroblasts as feeder cells; one of these cell lines, chHES-3, has maintained its normal karyotype even after being cultured in vitro for more than 100 passages, after the standardization of mouse feeder preparation. Each hES cell line has been completely characterized. All the three cell lines expressed hES-specific markers and pluripotency-related genes. These cells maintained their normal karyotype during long-term culture and displayed a high telomerase activity. When differentiated in vivo and in vitro, the derivatives representing the three germ layers could be observed. Human leukocyte antigen, ABO blood type, and DNA fingerprinting were also performed to provide a unique identity to each cell line. By establishing these hES cell lines, we provide an appropriate in vitro model to study human development and regeneration. All the three cell lines can be obtained for research purposes by placing a request at our website at www.hescbank.cn. PMID:20224969

  7. Development and characterization of two porcine monocyte-derived macrophage cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell lines Cdelta2+ and Cdelta2- were developed from monocytes obtained from a 10-month-old, crossbred, female pig. These cells morphologically resembled macrophages, stained positively for a-naphthyl esterase and negatively for peroxidase. The cell lines were bactericidal and highly phagocytic. ...

  8. Exploring the Transcriptome Space of a Recombinant BHK Cell Line Through Next

    E-print Network

    Karypis, George

    Abstract: Baby Hamster Kidney (BHK) cell lines are used in the production of veterinary vaccines Periodicals, Inc. KEYWORDS: BHK; Baby Hamster Kidney cells; Syrian hamster; transcriptome; RNA-seq; sequence variants Introduction Baby hamster kidney (BHK) cell lines, which originated from primary cultures

  9. Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish

    E-print Network

    Rubel, Edwin

    Neomycin-Induced Hair Cell Death and Rapid Regeneration in the Lateral Line of Zebrafish (Danio ABSTRACT Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after

  10. Rheotaxis in Larval Zebrafish Is Mediated by Lateral Line Mechanosensory Hair Cells

    E-print Network

    Rubel, Edwin

    Rheotaxis in Larval Zebrafish Is Mediated by Lateral Line Mechanosensory Hair Cells Arminda Suli1 and trunk of developing larvae. Mechanosensory hair cells in the center of each neuromast respond location of mechanosensory hair cells on the body surface has made the lateral line a valuable system

  11. Unexpected expression of intermediate filament protein genes in human oligodendroglioma cell lines

    SciTech Connect

    Kashima, Tsuyoshi; Vinters, H.V.; Campagnoni, A.T.

    1995-01-01

    From a human oligodendroglioma cell line cDNA library, ten intermediate filament (IF) cDNA clones were isolated. Five clones corresponded to vimentin mRNA, two corresponded to cytokeratin K7 mRNA, and two corresponded to cytokeratin K8 mRNA. One clone encoded a novel IF mRNA. The expression of these and other IF protein genes was examined in five cell lines derived from human oligodendroglioma, astrocytoma and neuroblastoma tumors. Vimentin mRNA and K18 mRNA were expressed in all the cell lines. The K7 and K8 genes were expressed only in the oligodendroglioma cell lines. Surprisingly, nestin mRNA was expressed in the astrocytoma lines and the neuroblastoma line, but was not expressed in the oligodendroglioma lines. These results indicate that oligodendroglioma cell lines express Types I and II cytokeratin genes. This pattern of IF gene expression was different from that of the astrocytoma and neuroblastoma cell lines, which expressed IF genes usually associated with the mature cell types or with differentiating fetal neural precursor cells, i.e. GFAP and neurofilament-L. The results also suggest that the oligodendroglioma cell lines are more epithelial in character and do not reflect the gene expression of mature oligodendrocytes. 46 refs., 8 figs., 2 tabs.

  12. Melatonin inhibits cell proliferation and induces caspase activation and apoptosis in human malignant lymphoid cell lines.

    PubMed

    Sánchez-Hidalgo, Marina; Lee, Melanie; de la Lastra, Catalina A; Guerrero, Juan M; Packham, Graham

    2012-11-01

    Melatonin exerts strong anti-tumour activity via several mechanisms, including anti-proliferative and pro-apoptotic effects in addition to its potent antioxidant activity. Several studies have investigated the effects of melatonin on haematological malignancies. However, the previous studies investigating lymphoid malignancies have been largely restricted to a single type of malignancy, Burkitt's lymphoma (BL). Thus, we examined the actions of melatonin on the growth and apoptosis in a small panel of cell lines representing different human lymphoid malignancies including Ramos (Epstein-Barr virus-negative BL), SU-DHL-4 (diffuse large B cell lymphoma), DoHH2 (follicular B non-Hodgkin lymphoma) and JURKAT (acute T cell leukaemia). We showed that melatonin promotes cell cycle arrest and apoptosis in all these cells, although there was marked variations in responses among different cell lines (sensitivity; Ramos/DoHH2 > SU-DHL-4 > JURKAT). Melatonin-induced apoptosis was relatively rapid, with increased caspase 3 and PARP cleavage detected within 0.5-1 h following melatonin addition. Moreover, there was evidence for rapid processing of both caspase 9, as well as a breakdown of the mitochondrial inner transmembrane potential. On the contrary, caspase activation was detected only in SU-DHL-4 and Ramos cells following melatonin treatment suggesting that the extrinsic pathway does not make a consistent contribution to melatonin-induced apoptosis in malignant lymphocytes. Although all cell lines expressed the high-affinity melatonin receptors, MT1 and MT2, melatonin-induced caspase activation appeared to be independent these receptors. Our findings confirm that melatonin could be a potential chemotherapeutic/preventive agent for malignant lymphocytes. However, it is necessary to take into account that different lymphoid malignancies may differ in their response to melatonin. PMID:22582944

  13. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line

    PubMed Central

    Sluch, Valentin M.; Davis, Chung-ha O.; Ranganathan, Vinod; Kerr, Justin M.; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A.; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S.; Mao, Hai-Quan; Zack, Donald J.

    2015-01-01

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation. PMID:26563826

  14. Differential responses to genotoxic agents between induced pluripotent stem cells and tumor cell lines.

    PubMed

    Lu, Yinghua; Xu, Dazhong; Zhou, Jing; Ma, Yupo; Jiang, Yongping; Zeng, Wenxian; Dai, Wei

    2013-01-01

    Given potential values of induced pluripotent stem (iPS) cells in basic biomedical research and regenerative medicine, it is important to understand how these cells regulate their genome stability in response to environmental toxins and carcinogens. The present study characterized the effect of Cr(VI), a well-known genotoxic agent and environmental carcinogen, on major molecular components of DNA damage response pathways in human iPS cells. We compared the effect of Cr(VI) on human iPS cells with two established cell lines, Tera-1 (teratoma origin) and BEAS-2B (lung epithelial origin). We also studied the effect of hydrogen peroxide and doxorubicin on modulating DNA damage responses in these cell types. We demonstrated that ATM and p53 phosphorylation is differentially regulated in human iPS cells compared with Tera-1 and BEAS-2B cells after exposure to various genotoxic agents. Moreover, we observed that inhibition of CK2, but not p38, promotes phosphorylation of p53S392 in iPS cells. Combined, our data reveal some unique features of DNA damage responses in human iPS cells. PMID:24283650

  15. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line.

    PubMed

    Sluch, Valentin M; Davis, Chung-Ha O; Ranganathan, Vinod; Kerr, Justin M; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S; Mao, Hai-Quan; Zack, Donald J

    2015-01-01

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation. PMID:26563826

  16. Treatment of prostate cancer cell lines and primary cells using low temperature plasma

    NASA Astrophysics Data System (ADS)

    O'Connell, Deborah; Hirst, Adam; Frame, Fiona F.; Maitland, Norman J.

    2014-10-01

    The mechanisms of cell death after plasma treatment of both benign and cancerous prostate epithelial cells are investigated. Prostate cancer tissue was obtained with patient consent from targeted needle core biopsies following radical prostatectomy. Primary cells were cultured from cancer tissue and plated onto a chamber slide at a density of 10,000 cells per well in 200 microliter of stem cell media (SCM). The treated sample was previously identified as Gleason grade 7 cancer through tissue histo-pathology. A dielectric barrier discharge (DBD) jet configuration, with helium as a carrier gas, and 0.3% O2 admixture was used for treating the cells. Reactive oxygen and nitrogen species (RONS) produced by the plasma are believed to be the main mediators of the plasma-cell interaction and response. We found the concentration of reactive oxygen species (ROS) induced inside the cells increased with plasma exposure. Exposure to the plasma for >3 minutes showed high levels of DNA damage compared to untreated and hydrogen peroxide controls. Cell viability and cellular recovery are also investigated and will be presented. All findings were common to both cell lines, suggesting the potential of LTP therapy for both benign and malignant disease.

  17. Cell lines used for microbeam and track segment studies at RARAF Experiments conducted at RARAF have used a host of adherent cell lines for various experiments. While

    E-print Network

    Brest Cancer Amundson's group MES Mouse embryonic stem cells Lieberman's group H1299 Human non 2002/03/15. PubMed PMID: 11893236. Cell line Description Source Reference WI-38 Human lung fibroblasts Coriell cell repositories (Camden, NJ, USA) (1) V79-4 Chinese hamster lung fibroblasts American Type

  18. Opioid binding site in EL-4 thymoma cell line

    SciTech Connect

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  19. Characterization of twenty-five ovarian tumour cell lines that phenocopy primary tumours

    PubMed Central

    Ince, Tan A.; Sousa, Aurea D.; Jones, Michelle A.; Harrell, J. Chuck; Agoston, Elin S.; Krohn, Marit; Selfors, Laura M.; Liu, Wenbin; Chen, Ken; Yong, Mao; Buchwald, Peter; Wang, Bin; Hale, Katherine S.; Cohick, Evan; Sergent, Petra; Witt, Abigail; Kozhekbaeva, Zhanna; Gao, Sizhen; Agoston, Agoston T.; Merritt, Melissa A.; Foster, Rosemary; Rueda, Bo R.; Crum, Christopher P.; Brugge, Joan S.; Mills, Gordon B.

    2015-01-01

    Currently available human tumour cell line panels consist of a small number of lines in each lineage that generally fail to retain the phenotype of the original patient tumour. Here we develop a cell culture medium that enables us to routinely establish cell lines from diverse subtypes of human ovarian cancers with >95% efficiency. Importantly, the 25 new ovarian tumour cell lines described here retain the genomic landscape, histopathology and molecular features of the original tumours. Furthermore, the molecular profile and drug response of these cell lines correlate with distinct groups of primary tumours with different outcomes. Thus, tumour cell lines derived using this methodology represent a significantly improved platform to study human tumour pathophysiology and response to therapy. PMID:26080861

  20. Drug-Resistant Urothelial Cancer Cell Lines Display Diverse Sensitivity Profiles to Potential Second-Line Therapeutics12

    PubMed Central

    Vallo, Stefan; Michaelis, Martin; Rothweiler, Florian; Bartsch, Georg; Gust, Kilian M.; Limbart, Dominik M.; Rödel, Franz; Wezel, Felix; Haferkamp, Axel; Cinatl, Jindrich

    2015-01-01

    Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUPrGEMCI20), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer. PMID:26055179

  1. Evasion mechanisms to tumor necrosis factor alpha (TNF-alpha) of small cell lung carcinoma and non-small cell lung carcinoma cell lines: comparison with the erythroleukaemia K-562 cell line.

    PubMed

    López-González, J S; Hernández García, A; Noyola, M I; Cázares, D A; Mandoki, J J; Morales, F M; Mendieta, I C; Caloca, J V

    2000-03-01

    The tumour necrosis factor alpha (TNF-alpha) is produced by mononuclear phagocytes as a defence mechanism against malignant cells. However, these cells can evade destruction by TNF-alpha. The present study evaluates in three lung cancer cell lines (small cell carcinoma NCI-H69, adenocarcinoma A-427, squamous carcinoma SK-MES-1) and one erythroleukaemia (K-562) cell line the following evasion mechanisms: (1) inhibition of TNF-alpha production, in indirect and direct co-cultures with monocytes; (2) the expression of type I and type II receptors for TNF-alpha (TNFRI and TNFRII) by tumour cell lines, using indirect immunofluorescence and flow cytometry; (3) the sensitivity of tumour cell lines to the toxic action of recombinant human TNF-alpha (rhTNF-alpha). With the exception of cell line NCI-H69, the other tumour cell lines liberated soluble factors that inhibited TNF-alpha production in monocytes. This effect occurred even after membrane contact with the A-427 and SK-MES-1 cell lines. Erythroleukaemia K-562 cells expressed both types of receptors for TNF-alpha, whereas the NCI-H69 cells expressed only TNFRI, and the A-427 and SK-MES-1 cells expressed no receptors. Lines NCI-H69, A-427 and K-562 were insensitive to the cytotoxic action of rhTNF-alpha. In conclusion, different lung cancer cell lines may evade destruction by TNF-alpha by various mechanisms that range from blocking TNF-alpha production by monocytes to blocking the cytotoxic action of this molecule. For selecting the most effective immunotherapy, knowledge of the evasion mechanisms would be useful. PMID:10699691

  2. Production of Primary Human CD4+ T Cell Lines and Clones

    PubMed Central

    Matthis, Jessica; Reijonen, Helena

    2014-01-01

    Summary Tetramer staining of CD4+ T cells is a valuable technique in immunology for detecting rare auto-reactive T cells. Generating clones or cell lines from auto-antigen tetramer positive CD4+ T cells allows further characterization and phenotyping of auto-reactive cells. PMID:23329513

  3. Capacity for epithelial differentiation in synovial sarcoma: analysis of a new human cell line

    PubMed Central

    Yakushiji, T; Yonemura, K; Tsuruta, J; Nishida, K; Kato, T; Takagi, K

    2000-01-01

    Aim—To analyse the capacity for epithelial differentiation in synovial sarcoma using a new human cell line. Methods—A new human cell line, KU-SS-1, was established from a monophasic, spindle cell type of synovial sarcoma by grafting those cells on to severe combined immunodeficient (SCID) mice and then transferring them to in vitro culture systems. The KU-SS-1 cells were characterised by light and electron microscopy, and by immunohistochemical, flow cytometric, and cytogenetic analysis. Results—Primary tumour and cultured cells at passage 20 showed a positive reaction for vimentin, which is a mesenchymal marker. After 40 passages, subcultured cells were injected into SCID mice to induce further tumours. These advanced subcultured cells and the tumour cells that they induced were positive for cytokeratin, an epithelial marker, and exhibited epithelial ultrastructural features such as intermediate junctions. Furthermore, two colour immunofluorescent analysis for proliferating nuclear cell antigen (PCNA) and intermediate filaments showed that a large number of PCNA expressing cells were positive for vimentin, and that part of this fraction also expressed cytokeratin. The existence of cells with reactivity for these three markers indicated that, in this cell line, a fraction with high proliferating capacity had both mesenchymal and epithelial markers. In addition, cytogenetically, this cell line expressed the SYT–SSX chimaeric transcript as a result of the t(X;18)(p11;q11) translocation. Conclusions—A human synovial sarcoma cell line was established and stably maintained in cell culture for more than 70 passages. In addition, this cell line showed epithelial differentiation, which supports the hypothesis that synovial sarcoma is a carcinosarcoma like tumour with true epithelial differentiation. This cell line will be a useful tool for investigating the nature of this tumour and will contribute to clinical studies. Key Words: synovial sarcoma • cell line • carcinosarcoma • differentiation PMID:10961176

  4. A Bovine Cell Line That Can Be Infected by Natural Sheep Scrapie Prions

    PubMed Central

    Oelschlegel, Anja M.; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schaetzl, Hermann; Groschup, Martin H.

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases. PMID:25565633

  5. A bovine cell line that can be infected by natural sheep scrapie prions.

    PubMed

    Oelschlegel, Anja M; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schatzl, Herman; Schaetzl, Hermann; Groschup, Martin H

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases. PMID:25565633

  6. Chemical Degradation of Cathode Linings in Hall-Héroult Cells—An Autopsy Study of Three Spent Pot Linings

    NASA Astrophysics Data System (ADS)

    Tschöpe, Kati; Schřning, Cristian; Rutlin, Jřrn; Grande, Tor

    2012-04-01

    Cathode autopsies are used frequently in the aluminum industry to investigate pot failure and the degradation of the cathode lining. The materials observed in spent pot lining (SPL) has so far been assumed to reflect the sequence of layers from the cathode to the nonreacted refractory lining as present during the operation of the cell. Here, we demonstrate that the thermal gradient in the lining is reversed during cooling and that the physical appearance of the SPL is caused both by processes taking place during operation and cooling of the shutdown cell. X-ray diffraction and microscopy of the SPL from three shutdown cells revealed that sodium metal is the main component responsible for the chemical degradation of the refractory lining. Two distinct reaction fronts were identified in the three SPL showing that sodium is penetrating deeper down into the lining than the molten fluorides from the electrolyte. The mechanisms for the transport of sodium and bath components in the refractory lining are proposed based on the experimental observations. The sodium penetration is inhibited by the formation of a viscous barrier as suggested previously, but the current findings suggest that the barrier retards diffusion of O2- and F- anions rather than Na+ as proposed previously.

  7. FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

    NASA Astrophysics Data System (ADS)

    Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.

    2012-06-01

    The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

  8. Tick Cell Lines for Study of Crimean-Congo Hemorrhagic Fever Virus and Other Arboviruses

    PubMed Central

    Kohl, Alain; Bente, Dennis A.; Fazakerley, John K.

    2012-01-01

    Abstract Continuous cell lines derived from many of the vectors of tick-borne arboviruses of medical and veterinary importance are now available. Their role as tools in arbovirus research to date is reviewed and their potential application in studies of tick cell responses to virus infection is explored, by comparison with recent progress in understanding mosquito immunity to arbovirus infection. A preliminary study of propagation of the human pathogen Crimean-Congo hemorrhagic fever virus (CCHFV) in tick cell lines is reported; CCHFV replicated in seven cell lines derived from the ticks Hyalomma anatolicum (a known vector), Amblyomma variegatum, Rhipicephalus (Boophilus) decoloratus, Rhipicephalus (Boophilus) microplus, and Ixodes ricinus, but not in three cell lines derived from Rhipicephalus appendiculatus and Ornithodoros moubata. This indicates that tick cell lines can be used to study growth of CCHFV in arthropod cells and that there may be species-specific restriction in permissive CCHFV infection at the cellular level. PMID:21955214

  9. Production Process for Stem Cell Based Therapeutic Implants: Expansion of the Production Cell Line and Cultivation of Encapsulated Cells

    NASA Astrophysics Data System (ADS)

    Weber, C.; Pohl, S.; Poertner, R.; Pino-Grace, Pablo; Freimark, D.; Wallrapp, C.; Geigle, P.; Czermak, P.

    Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

  10. Cell cycle control by natural phenols in cisplatin-resistant cell lines.

    PubMed

    Catanzaro, Daniela; Vianello, Caterina; Ragazzi, Eugenio; Caparrotta, Laura; Montopoli, Monica

    2014-10-01

    Fifteen plant polyphenols, including flavonoids, cinnamic acids, coumarins and capsaicin, were investigated for their capacity to suppress cell growth and regulate the cell cycle of in vitro human ovarian carcinoma (2008 cell line) and cervix squamous carcinoma cells (A431), and their cisplatin (CDDP)-resistant subclones (C13 and A431Pt, respectively). Evaluation of the cytotoxic effects of the polyphenols (0.01-100 ?M) indicated that especially rhein and quercetin were almost equiactive in wild type and CDDP-resistant cells, indicating lack of cross-resistance with cisplatin. Capsaicin was more potent in CDDP-resistant subclones than in wild type cells. The order of their potencies is flavonoids > anthraquinones > vanilloids > coumarins > phenols, cinnamic acids. The natural phenols which were most cytotoxic (rhein, quercetin and capsaicin) were able to cause the arrest of the cancer cell cycle, suggesting that specific cell cycle regulatory proteins are possibly involved in their intracellular mechanism of action. In particular, the natural compounds were revealed to be more active in CDDP-resistant cells than in wild types, especially inducing apoptotic death. PMID:25522537

  11. A cell line with multinucleated giant cell formation established from a human giant cell tumor of tendon sheath--preliminary report.

    PubMed

    Hosaka, M; Hatori, M; Smith, R A; Kokubun, S

    2001-01-01

    We first established a cell line with unique giant cell formation properties from a human giant cell tumor of tendon sheath (GCTTS) arising in the right ankle of a 7-year-old girl. The specimen for cell culture taken from the tumor was heterotransplanted into the back of a BALB/c (nu/nu) nude mouse. An in-vitro cell line was established from a tumor that grew after this heterotransplantation. Only mononuclear cells were observed in the primary culture, and these remained constant in growth. Multinucleated giant cells appeared at passage 3 and were constantly observed thereafter. The fusion of mononuclear cells into giant cells was verified by light and phase-contrast microscopy. This cell line was confirmed to be derived from a human by karyotype analysis and DNA fingerprinting. The cell-doubling time was 150 h. This cell line should be useful for studies of the mechanism of multinucleation in giant cell tumors. PMID:11845350

  12. Identification of embryonic stem cell activities in an embryonic cell line derived from marine medaka (Oryzias dancena).

    PubMed

    Lee, Dongwook; Ryu, Jun Hyung; Lee, Seung Tae; Nam, Yoon Kwon; Kim, Dong Soo; Gong, Seung Pyo

    2015-12-01

    This study was conducted to identify embryonic stem cell (ESC) activities of a long-term cultured embryonic cell line previously derived from blastula-stage Oryzias dancena embryos. Five sub-cell lines were established from the embryonic cell line via clonal expansion of single cells. ESC activities, including clonogenicity, alkaline phosphatase (AP) activity, and differentiation capacity, were examined in the five sub-cell lines. We observed both clonogenicity and AP activity in all five sub-cell lines, but the proportion of cells that exhibited both properties was significantly different among them. Even though we detected different formation rates and sizes of embryoid body (EB) among these cells, all lines were stably able to form EBs and further induction for differentiation showed their capability to differentiate into other cell types in a spontaneous manner. From this study, we determined that the embryonic cell lines examined possessed heterogeneous ESC activities and can be utilized as a marine model system for fish ESC-based research. PMID:26239820

  13. Annona squamosa Linn: cytotoxic activity found in leaf extract against human tumor cell lines.

    PubMed

    Wang, De-Shen; Rizwani, Ghazala H; Guo, Huiqin; Ahmed, Mansoor; Ahmed, Maryam; Hassan, Syed Zeeshan; Hassan, Amir; Chen, Zhe-Sheng; Xu, Rui-Hua

    2014-09-01

    Cancer is a common cause of death in human populations. Surgery, chemotherapy and radiotherapy still remain the corner stone of treatment. However, herbal medicines are gaining popularity on account of their lesser harmful side effects on non-targeted human cells and biological environment. Annona squamosa Linn is a common delicious edible fruit and its leaf have been used for the treatment in various types of diseases. The objective of present study is to determine the anticancer potential of the organic and aqueous extracts of leaf of Annona squamosa L. MTT (3-(4, 5-dimethylthiazole-2yl)-2, 5-biphenyl tetrazolium bromide) assay against hepatocellular carcinoma cell line BEL-7404, lung cancer line H460, human epidermoid carcinoma cell line KB-3-1, prostatic cancer cell line DU145, breast carcinoma cell line MDA-MB-435, and colon cancer cell line HCT-116 Human primary embryonic kidney cell line HEK293 as control were used for the study. The crude extract (Zcd) and Ethyl acetate extract (ZE) were found significant anticancer activity only on human epidermoid carcinoma cell line KB-3-1 and colon cancer cell line HCT-116. PMID:25176251

  14. Anti-leukemic properties of deferasirox via apoptosis in murine leukemia cell lines

    PubMed Central

    Jeon, Sol-Rim; Lee, Jae-Wook; Jang, Pil-Sang; Cho, Bin; Jeong, Dae-Chul

    2015-01-01

    Background Although deferasirox (DFX) is reported to have anti-tumor effects, its anti-leukemic activity remains unclear. We evaluated the effect of DFX treatment on two murine lymphoid leukemia cell lines, and clarified the mechanisms underlying its potential anti-leukemic activity. Methods L1210 and A20 murine lymphoid leukemia cell lines were treated with DFX. Cell viability and apoptosis were evaluated by the 3-(4,5-dimethylthaizol-2-yl)-5-(3-carboxymethylphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and fluorescence-activated cell sorting (FACS) analysis, respectively. Immunoblotting was performed to detect the expression of key apoptotic proteins. Results In dose- and time-dependent manner, DFX decreased viability and increased apoptosis of murine leukemic cells. Fas expression was significantly higher in A20 cells than in L1210 cells at all DFX concentrations tested. Although both cell lines exhibited high caspase 3 and caspase 9 expression, a critical component of the intrinsic mitochondrial apoptotic pathway, expression was greater in L1210 cells. In contrast, caspase 8, a key factor in the extrinsic apoptotic pathway, showed greater expression in A20 cells. Cytochrome c expression was significantly higher in L1210 cells. In both cell lines, co-treatment with ferric chloride and DFX diminished the expression of these intracellular proteins, as compared to DFX treatment alone. Conclusion Treatment with DFX increased caspase-dependent apoptosis in two murine lymphoid leukemia cell lines, with differing apoptotic mechanisms in each cell line. PMID:25830128

  15. Efficient production of a gene mutant cell line through integrating TALENs and high-throughput cell cloning.

    PubMed

    Sun, Changhong; Fan, Yu; Li, Juan; Wang, Gancheng; Zhang, Hanshuo; Xi, Jianzhong Jeff

    2015-02-01

    Transcription activator-like effectors (TALEs) are becoming powerful DNA-targeting tools in a variety of mammalian cells and model organisms. However, generating a stable cell line with specific gene mutations in a simple and rapid manner remains a challenging task. Here, we report a new method to efficiently produce monoclonal cells using integrated TALE nuclease technology and a series of high-throughput cell cloning approaches. Following this method, we obtained three mTOR mutant 293T cell lines within 2 months, which included one homozygous mutant line. PMID:25524489

  16. Distinct population of highly malignant cells in a head and neck squamous cell carcinoma cell line established by xenograft model

    PubMed Central

    2009-01-01

    The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC), have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation. PMID:19917110

  17. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling.

    PubMed

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  18. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    PubMed Central

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  19. Immortalized Human Schwann Cell Lines Derived From Tumors of Schwannomatosis Patients

    PubMed Central

    Ostrow, Kimberly Laskie; Donaldson, Katelyn; Blakeley, Jaishri; Belzberg, Allan; Hoke, Ahmet

    2015-01-01

    Schwannomatosis, a rare form of neurofibromatosis, is characterized predominantly by multiple, often painful, schwannomas throughout the peripheral nervous system. The current standard of care for schwannomatosis is surgical resection. A major obstacle to schwannomatosis research is the lack of robust tumor cell lines. There is a great need for mechanistic and drug discovery studies of schwannomatosis, yet appropriate tools are not currently available. Schwannomatosis tumors are difficult to grow in culture as they survive only a few passages before senescence. Our lab has extensive experience in establishing primary and immortalized human Schwann cell cultures from normal tissue that retain their phenotypes after immortalization. Therefore we took on the challenge of creating immortalized human Schwann cell lines derived from tumors from schwannomatosis patients. We have established and fully characterized 2 schwannomatosis cell lines from 2 separate patients using SV40 virus large T antigen. One patient reported pain and the other did not. The schwannomatosis cell lines were stained with S100B antibodies to confirm Schwann cell identity. The schwannomatosis cells also expressed the Schwann cell markers, p75NTR, S100B, and NGF after multiple passages. Cell morphology was retained following multiple passaging and freeze/ thaw cycles. Gene expression microarray analysis was used to compare the cell lines with their respective parent tumors. No differences in key genes were detected, with the exception that several cell cycle regulators were upregulated in the schwannomatosis cell lines when compared to their parent tumors. This upregulation was apparently a product of cell culturing, as the schwannomatosis cells exhibited the same expression pattern of cell cycle regulatory genes as normal primary human Schwann cells. Cell growth was also similar between normal primary and immortalized tumor cells in culture. Accurate cell lines derived directly from human tumors will serve as invaluable tools for advancing schwannomatosis research, including drug screening. PMID:26657314

  20. Prediction of epigenetically regulated genes in breast cancer cell lines

    SciTech Connect

    Loss, Leandro A; Sadanandam, Anguraj; Durinck, Steffen; Nautiyal, Shivani; Flaucher, Diane; Carlton, Victoria EH; Moorhead, Martin; Lu, Yontao; Gray, Joe W; Faham, Malek; Spellman, Paul; Parvin, Bahram

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the panel of breast cancer cell lines. Subnetwork enrichment of these genes has identifed 35 common regulators with 6 or more predicted markers. In addition to identifying epigenetically regulated genes, we show evidence of differentially expressed methylation patterns between the basal and luminal subtypes. Our results indicate that the proposed computational protocol is a viable platform for identifying epigenetically regulated genes. Our protocol has generated a list of predictors including COL1A2, TOP2A, TFF1, and VAV3, genes whose key roles in epigenetic regulation is documented in the literature. Subnetwork enrichment of these predicted markers further suggests that epigenetic regulation of individual genes occurs in a coordinated fashion and through common regulators.

  1. Characterization of PICM-19H porcine liver stem cell line for potential use in a bioartificial liver

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A hepatocyte cell line is needed as the biological component of a bioartificial liver (BAL). One candidate is the PICM-19 pig liver stem cell line. These cells have many normal hepatocyte functions often lacking in tumor-derived liver cell lines. The study characterized a PICM-19 derivative cell ...

  2. EPSTEIN-BARR VIRUS-NEGATIVE HUMAN MALIGNANT T-CELL LINES

    PubMed Central

    Kaplan, Joseph; Shope, Thomas C.; Peterson, Ward D.

    1974-01-01

    Two lymphoblastoid lines, CCRF-CEM and HSB-2, with properties of malignant cells, derived from children with leukemia secondary to lymphosarcoma, have T-cell properties and lack Epstein-Barr virus antigens. PMID:4363409

  3. Openness and the governance of human stem cell lines: a conceptual approach 

    E-print Network

    George, Carol Charlene

    2013-07-03

    My research examines the extent to which features of ‘openness’ might usefully contribute to mechanisms of governance of human stem cell lines, with a view to the production of therapeutic stem cell treatments for the ...

  4. Chlorpyrifos inhibits cell proliferation through ERK1/2 phosphorylation in breast cancer cell lines.

    PubMed

    Ventura, Clara; Venturino, Andrés; Miret, Noelia; Randi, Andrea; Rivera, Elena; Núńez, Mariel; Cocca, Claudia

    2015-02-01

    It is well known the participation of oxidative stress in the induction and development of different pathologies including cancer, diabetes, neurodegeneration and respiratory disorders among others. It has been reported that oxidative stress may be induced by pesticides and it could be the cause of health alteration mediated by pollutants exposure. Large number of registered products containing chlorpyrifos (CPF) is used to control pest worldwide. We have previously reported that 50 ?M CPF induces ROS generation and produces cell cycle arrest followed by cell death. The present investigation was designed to identify the pathway involved in CPF-inhibited cell proliferation in MCF-7 and MDA-MB-231 breast cancer cell lines. In addition, we determined if CPF-induced oxidative stress is related to alterations in antioxidant defense system. Finally we studied the molecular mechanisms underlying in the cell proliferation inhibition produced by the pesticide. In this study we demonstrate that CPF (50 ?M) induces redox imbalance altering the antioxidant defense system in breast cancer cells. Furthermore, we found that the main mechanism involved in the inhibition of cell proliferation induced by CPF is an increment of p-ERK1/2 levels mediated by H2O2 in breast cancer cells. As PD98059 could not abolish the increment of ROS induced by CPF, we concluded that ERK1/2 phosphorylation is subsequent to ROS production induced by CPF but not the inverse. PMID:25180937

  5. Amygdalin induces apoptosis in human cervical cancer cell line HeLa cells.

    PubMed

    Chen, Yu; Ma, Jinshu; Wang, Fang; Hu, Jie; Cui, Ai; Wei, Chengguo; Yang, Qing; Li, Fan

    2013-02-01

    Amygdalin, a naturally occurring substance, has been suggested to be efficacious as an anticancer substance. The effect of amygdalin on cervical cancer cells has never been studied. In this study, we found that the viability of human cervical cancer HeLa cell line was significantly inhibited by amygdalin. 4,6-Diamino-2-phenyl indole (DAPI) staining showed that amygdalin-treated HeLa cells developed typical apoptotic changes. The development of apoptosis in the amygdalin-treated HeLa cells were confirmed by double staining of amygdalin-treated HeLa cells with annexin V-FITC and propidium iodide (PI) along with increase in caspase-3 activity in these cells. Further studies indicated that antiapoptotic protein Bcl-2 was downregulated whereas proapoptotic Bax protein was upregulated in the amygdalin-treated HeLa cells implying involvement of the intrinsic pathway of apoptosis. In vivo, amygdalin administration inhibited the growth of HeLa cell xenografts through a mechanism of apoptosis. The results in the present study suggest that amygdalin may offer a new therapeutic option for patients with cervical cancer. PMID:23137229

  6. Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

    SciTech Connect

    Booth, Brian W.; Boulanger, Corinne A.; Anderson, Lisa H.; Jimenez-Rojo, Lucia; Brisken, Cathrin; Smith, Gilbert H.

    2010-02-01

    Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D {beta}-geo (CD{beta}geo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CD{beta}geo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG{sup -/-} mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.

  7. Escin reduces cell proliferation and induces apoptosis on glioma and lung adenocarcinoma cell lines.

    PubMed

    Çiftçi, Gül?en Akalin; I?can, Arzu; Kutlu, Mehtap

    2015-10-01

    Aesculus hippocastanum (the horse chestnut) seed extract has a wide variety of biochemical and pharmacological effects including anti-inflammatory, antianalgesic, and antipyretic activities. The main active compound of this plant is escin. It is known that several medicinal herbs with anti-inflammatory properties have been found to have a role in the prevention and treatment of cancer. In the present study, the cytotoxic effects of escin in the C6 glioma and A549 cell lines were analyzed by MTT. Apoptotic effects of escin on both cell lines were evaluated by Annexin V binding capacity with flow cytometric analysis. Structural and ultrastructural changes were also evaluated using transmission electron microscopy. The results indicated that escin has potent antiproliferative effects against C6 glioma and A549 cells. These effects are both dose and time dependent. Taken together, escin possesses cell cycle arrest on G0/G1 phase and selective apoptotic activity on A549 cells as indicated by increased Annexin V-binding capacity, bax protein expression, caspase-3 activity and morphological changes obtained from micrographs by transmission electron microscopy. PMID:25906387

  8. The Tribolium castaneum cell line TcA: a new tool kit for cell biology

    PubMed Central

    Silver, Kristopher; Jiang, Hongbo; Fu, Jinping; Phillips, Thomas W.; Beeman, Richard W.; Park, Yoonseong

    2014-01-01

    The red flour beetle, Tribolium castaneum, is an agriculturally important insect pest that has been widely used as a model organism. Recently, an adherent cell line (BCIRL-TcA-CLG1 or TcA) was developed from late pupae of the red flour beetle. Next generation transcriptome sequencing of TcA cells demonstrated expression of a wide variety of genes associated with specialized functions in chitin metabolism, immune responses and cellular and systemic RNAi pathways. Accordingly, we evaluated the sensitivity of TcA cells to dsRNA to initiate an RNAi response. TcA cells were highly sensitive to minute amounts of dsRNA, with a minimum effective dose of 100?pg/mL resulting in significant suppression of gene expression. We have also developed a plasmid containing two TcA-specific promoters, the promoter from the 40S ribosomal protein subunit (TC006550) and a bi-directional heat shock promoter (TcHS70) from the intergenic space between heat shock proteins 68a and b. These promoters have been employed to provide high levels of either constitutive (TC006550) or inducible (TcHS70) gene expression of the reporter proteins. Our results show that the TcA cell line, with its sensitivity to RNAi and functional TcA-specific promoters, is an invaluable resource for studying basic molecular and physiological questions. PMID:25354547

  9. The Ah receptor regulates growth factor expression in head and neck squamous cell carcinoma cell lines.

    PubMed

    John, Kaarthik; Lahoti, Tejas S; Wagner, Kelly; Hughes, Jarod M; Perdew, Gary H

    2014-10-01

    Previous studies in head and neck squamous cell carcinoma (HNSCC) cell lines have revealed that the Ah receptor (AHR) plays a significant role in mediating the "aggressive" phenotype of these cells, which includes enhanced inflammatory signaling (e.g., IL6) and migratory potential. Here we sought to identify putative novel targets of the AHR associated with enhanced tumor invasiveness. Global gene expression analysis identified a number of genes that are repressed upon treatment of OSC-19 or HN30 cells with an AHR antagonist. Three growth factors were targets of AHR activity; amphiregulin (AREG), epiregulin (EREG), and platelet-derived growth factor A (PDGFA) were repressed by an AHR antagonist and further examined. Quantitative PCR analysis, ELISA, and siRNA-mediated knock down of AHR revealed an attenuation of basal and/or induced levels of expression of these growth factors in two HNSCC lines, following AHR antagonism. In silico analysis revealed that these growth factors possess dioxin-like response elements. Two other AHR ligands, 6-formylindolo[3,2-b]carbazole and benzo(a)pyrene (BP) also elicited similar responses. In conclusion, this study identified AREG, EREG, and PDGFA as growth factor targets of AHR activity associated with metastatic phenotype of HNSCC cells, suggesting that attenuation of AHR activity may be a therapeutic strategy. PMID:23625689

  10. Cytotoxic activity of some lichen extracts on murine and human cancer cell lines.

    PubMed

    Bézivin, C; Tomasi, S; Lohézic-Le Dévéhat, F; Boustie, J

    2003-01-01

    Eight lichens were extracted successively with n-hexane, diethyl ether and methanol using a Soxhlet process. The cytotoxic activity of the 24 lichen extracts was evaluated in vitro using two murine (the L1210: lymphocytic leukaemia, and the 3LL: Lewis lung carcinoma) and four human (the K-562: chronic myelogenous leukaemia, the U251: glioblastoma, the DU145: prostate carcinoma, and the MCF7: breast adenocarcinoma) cancer cell lines and non-cancerous cells, the Vero cell line (African green monkey kidney cell line). The MTT assay revealed significant cytotoxicity (IC50 < or = 20 microg/ml) on one of the tested cancer cell lines for at least one extract of each lichen species. Some extracts of Cladonia convoluta, Cladonia rangiformis, Parmelia caperata, Platismatia glauca and Ramalina cuspidata demonstrated interesting activities particularly on human cancer cell lines as good selectivity indices were recorded (SI > 3). PMID:13678234

  11. Cell line development for biomanufacturing processes: recent advances and an outlook.

    PubMed

    Le, Huong; Vishwanathan, Nandita; Jacob, Nitya M; Gadgil, Mugdha; Hu, Wei-Shou

    2015-08-01

    At the core of a biomanufacturing process for recombinant proteins is the production cell line. It influences the productivity and product quality. Its characteristics also dictate process development, as the process is optimized to complement the producing cell to achieve the target productivity and quality. Advances in the past decade, from vector design to cell line screening, have greatly expanded our capability to attain producing cell lines with certain desired traits. Increasing availability of genomic and transcriptomic resources for industrially important cell lines coupled with advances in genome editing technology have opened new avenues for cell line development. These developments are poised to help biosimilar manufacturing, which requires targeting pre-defined product quality attributes, e.g., glycoform, to match the innovator's range. This review summarizes recent advances and discusses future possibilities in this area. PMID:25971160

  12. Expression of Tropomyosin 1 Gene Isoforms in Human Breast Cancer Cell Lines

    PubMed Central

    Dube, Syamalima; Yalamanchili, Santhi; Lachant, Joseph; Abbott, Lynn; Benz, Patricia; Mitschow, Charles; Dube, Dipak K.; Poiesz, Bernard J.

    2015-01-01

    Nine malignant breast epithelial cell lines and 3 normal breast cell lines were examined for stress fiber formation and expression of TPM1 isoform-specific RNAs and proteins. Stress fiber formation was strong (++++) in the normal cell lines and varied among the malignant cell lines (negative to +++). Although TPM1? and TPM1? were the dominant transcripts of TPM1, there was no clear evidence for TPM1? protein expression. Four novel human TPM1 gene RNA isoforms were discovered (?, ?, ?, and ?), which were not identified in adult and fetal human cardiac tissues. TPM1? was the most frequent isoform expressed in the malignant breast cell lines, and it was absent in normal breast epithelial cell lines. By western blotting, we were unable to distinguish between TPM1?, ?, and ? protein expression, which were the only TPM1 gene protein isoforms potentially expressed. Some malignant cell lines demonstrated increased or decreased expression of these isoforms relative to the normal breast cell lines. Stress fiber formation did not correlate with TPM1? RNA expression but significantly and inversely correlated with TPM1? and TPM1? expression, respectively. The exact differences in expression of these novel isoforms and their functional properties in breast epithelial cells will require further study. PMID:26171250

  13. Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos.

    PubMed

    Li, Chunliang; Yang, Ying; Lu, Xiaowei; Sun, Yijuan; Gu, Junjie; Feng, Yun; Jin, Ying

    2010-04-01

    Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn. PMID:20186511

  14. Induction of Neuronal Morphology in the 661W Cone Photoreceptor Cell Line with Staurosporine

    PubMed Central

    Thompson, Alex F.; Crowe, Megan E.; Lieven, Christopher J.; Levin, Leonard A.

    2015-01-01

    Purpose RGC-5 cells undergo differentiation into a neuronal phenotype with low concentrations of staurosporine. Although the RGC-5 cell line was initially thought to be of retinal ganglion cell origin, recent evidence suggests that the RGC-5 line could have been the result of contamination with 661W mouse cone photoreceptor cells. This raised the possibility that a cone photoreceptor cell line could be multipotent and could be differentiated to a neuronal phenotype. Methods 661W and RGC-5 cells, non-neuronal retinal astrocytes, retinal endothelial cells, retinal pericytes, M21 melanoma cells, K562 chronic myelogenous leukemia cells, and Daudi Burkitt lymphoma cells, were differentiated with staurosporine. The resulting morphology was quantitated using NeuronJ with respect to neurite counts and topology. Results Treatment with staurosporine induced similar-appearing morphological differentiation in both 661W and RGC-5 cells. The following measures were not significantly different between 661W and RGC-5 cells: number of neurites per cell, total neurite field length, number of neurite branch points, and cell viability. Neuronal-like differentiation was not observed in the other cell lines tested. Conclusions 661W and RGC-5 cells have virtually identical and distinctive morphology when differentiated with low concentrations of staurosporine. This result demonstrates that a retinal neuronal precursor cell with cone photoreceptor lineage can be differentiated to express a neuronal morphology. PMID:26684837

  15. CD133 Expression and Identification of CD133/nestin Positive Cells in Rhabdomyosarcomas and Rhabdomyosarcoma Cell Lines

    PubMed Central

    Sana, Jiri; Zambo, Iva; Skoda, Jan; Neradil, Jakub; Chlapek, Petr; Hermanova, Marketa; Mudry, Peter; Vasikova, Alzbeta; Zitterbart, Karel; Hampl, Ales; Sterba, Jaroslav; Veselska, Renata

    2011-01-01

    Background: Co-expression of CD133, cell surface glycoprotein, and nestin, an intermediate filament protein, was determined to be a marker of neural stem cells and of cancer stem cells in neurogenic tumors. Methods: We examined the expression of CD133 and nestin in ten tumor tissue samples taken from patients with rhabdomyosarcomas and in five rhabdomyosarcoma cell lines. Immunohistochemistry and immunofluorescence were used to examine FFPE tumor tissue samples. Cell lines were analyzed by immunofluorescence, immunoblotting, flow cytometry, and RT-PCR. Functional assays (clonogenic in vitro assay and tumorigenic in vivo assay) were also performed using these cell lines. Results: CD133 and nestin were detected in all 10 tumor tissue samples and in all 5 cell lines; however, the frequency of CD133+, Nes+, and CD133+/Nes+ cells, as well as the intensity of fluorescence varied in individual samples or cell lines. The expression of CD133 and nestin was subsequently confirmed in all cell lines by immunoblotting. Furthermore, we observed an increasing expression of CD133 in relation to the cultivation. All cell lines were positive for Oct3/4 and nucleostemin; NSTS-11 cells were also able to form xenograft tumors in mice. Conclusion: Our results represent the first evidence of CD133 expression in rhabdomyosarcoma tissue and in rhabdomyosarcoma cell lines. In addition, the co-expression of CD133 and nestin as well as results of the functional assays suggest a possible presence of cancer cells with a stem-like phenotype in these tumors. PMID:22156015

  16. Comparison of thermoradiosensitization in two human melanoma cell lines and one fibroblast cell line by concurrent mild hyperthermia and low-dose-rate irradiation

    SciTech Connect

    Raaphorst, G.P.; Bussey, A.; Heller, D.P.; Ng, C.E. )

    1994-03-01

    Two human melanoma cell lines, one radioresistant (Sk-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41[degrees]C). None of the cell lines expressed chronic thermotolerance during heating at 39-41[degrees]C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40[degrees]C but HT-144 cells were more sensitive at 41[degrees]C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD. 24 refs., 5 figs., 1 tab.

  17. Comparative study of the photodynamic effect in tumor and nontumor animal cell lines

    NASA Astrophysics Data System (ADS)

    Stoykova, Elena V.; Alexandrova, R.; Shurulinkov, Stanislav; Sabotinov, O.; Minchev, Georgi

    2004-09-01

    In this study we evaluate the cytotoxicity of two photosensitisers with absorption peaks in the green and red part of the spectrum on animal cell lines. The cytotoxicity assessment was performed for a tumor cell line LSCC-SF-Mc29, obtained from a transplantable chicken hepatoma induced by the myelocytomatosis virus Mc29, a tumor line LSR-SF-SR, obtained from a transplantable sarcoma in rat induced by Rous sarcoma virus strain Schmidt-Ruppin and for normal mouse and bovine cell lines. Up to now the effect of the photodynamic therapy on virus-induced cancers has not been clarified. The cells were treated with 5,10,15,20 - tetra (4-sulfophenyl) porphyrin with main absorption peak at 519 nm and a dye activated with a red light. The cells were seeded in 96-well plates at 2 x 104 cells/well. The cells were exposed to irradiation from a pulsed CuBr vapor laser at 510.6 nm and 578.2 nm and exposure rate 50 mW/cm2, from an Ar-ion laser at 514 nm and 1 mW/cm2 and to 655 nm-irradiation from a semiconductor laser at 10 mW/cm2. The biological activity of the tested compounds was measured by the neutral red uptake cytotoxicity test. The light dose-response curves and light exposures that ensure 50% drop in the treated cells viability in comparison with the cells grown in non-modified medium were obtained for each cell line. The cytotoxic effect of both photosensitisers is most distinguished for the tumor line LSCC-SF-Mc29. The 2-4 times higher viability of the normal cell lines in comparison with the tumor lines is established. The bovine cell lines are more vulnerable than the mouse lines.

  18. Efficient gene transfer into the human natural killer cell line, NKL, using the Amaxa nucleofection system.

    PubMed

    Maasho, Kerima; Marusina, Alina; Reynolds, Nicole M; Coligan, John E; Borrego, Francisco

    2004-01-01

    Natural killer (NK) cell lines are useful for studying facets of NK cell biology. Such cell lines are notoriously difficult to transfect by traditional methods, a fact that has hampered NK cell biology studies for a long time. To overcome this, we investigated the use of the Amaxa nucleofection system that directly transfers DNA into the nucleus of the cell. This technology has revolutionized transfection studies with heretofore relatively transfection resistant cell types such as T cells, B cells and dendritic cells. Despite these advances, NK cells and NK cell lines have remained relatively resistant to transfection, including nucleofection. In this study we employed cDNA for SHP1 and various Rab proteins cloned in enhanced green/yellow fluorescent protein (EGFP/EYFP) expression plasmids for transient transfections into NKL cells. The expression of EGFP/EYFP fusion proteins was analyzed by flow cytometry, immunoblot and confocal microscopic analyses. We achieved 40-70% transfection efficiency with high levels of expression in this cell line with 85-90% viability. The method used in this report proves to be far superior to existing methods for delivering DNA into this well studied NK cell line and, consequently, provides new experimental opportunities. PMID:14736423

  19. Differential quantitative zinc-induced expression of human metallothionein isogenes in haematopoietic precursor cell lines.

    PubMed

    Maghdooni Bagheri, Pegah; Rahman, Mohammad Tariqur; Van Soest, Sofie; De Ley, Marc

    2009-01-01

    The expression pattern of functional members of the metallothionein (MT) gene family was studied in the haematopoietic precursor cell lines, K562, DAMI, MEG-01, and ELF-153 in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 with 10% (v/v) foetal calf serum, with or without different zinc supplements. Expression of MT isogenes was analysed by quantitative real-time PCR (RT-PCR) using mRNA extracted from cultured cells. The more mature K562, DAMI, and MEG-01 cell lines exhibited transcription of all MT isogenes, except MT-3 and MT-4. Relative quantitative expression of MT isogenes in the mature cell lines such as K562, DAMI, and MEG-01 was higher than in the immature ELF-153 cell line. Immunohistochemical staining (IHC) reveals an increased MT protein biosynthesis in more mature cell lines such as K562, DAMI and MEG-01 greater than in the immature ELF-153 cell line. Real-time PCR and immunohistochemical staining for investigating the effect of phorbol ester and hemin (haematopoietic differentiation stimuli) on expression of MT isogenes in K562 cells reveals that phorbol ester induces increased MT transcription and biosynthesis. Therefore, to our knowledge, the role of MT in differentiation in human haematopoietic precursor cell lines is here reported for the first time. PMID:19398060

  20. Establishment of an immortal chicken embryo liver-derived cell line.

    PubMed

    Lee, Jeongyoon; Foster, Douglas N; Bottje, Walter G; Jang, Hyeon-Min; Chandra, Yohanna G; Gentles, Lauren E; Kong, Byung-Whi

    2013-06-01

    A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV. PMID:23687157

  1. Fibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines

    E-print Network

    Athanasiou, Kyriacos

    , both adult and embryonic stem cells may have the capacity to overcome these issues, but they also bringFibrochondrogenesis in Two Embryonic Stem Cell Lines: Effects of Differentiation Timelines · Human embryonic stem cells · Tissue engineering ABSTRACT Human embryonic stem cells (h

  2. Baculovirus Infection Influences Host Protein Expression in Two Established Insect Cell Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified host proteins that changed in response to host cell susceptibility to baculovirus infection. We used three baculovirus–host cell systems utilizing two cell lines derived from pupal ovaries, Hz-AM1 (from Helicoverpa zea) and Hv-AM1 (from Heliothis virescens). Hv-AM1 cells are permissive...

  3. A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues

    E-print Network

    A Cell Line Resource Derived from Honey Bee (Apis mellifera) Embryonic Tissues Michael J. Goblirsch comprised of honey bee cells. Such systems are important to determine the impact of these stress factors on the developmental and cell biology of honey bees. We have developed a method incorporating established insect cell

  4. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

  5. Induction of acetylcholinesterase activity by beta-ecdysone in a Drosophila cell line.

    PubMed

    Cherbas, P; Cherbas, L; Williams, C M

    1977-07-15

    When cells of the Drosophila Kc-H line are treated with larger than or equal to 10(-8) molar beta-ecdysone, they extend long processes and acquire acetylcholinesterase activity. Thus, this permanent line, derived originally from embryol cultures, may be composed of cells having some neural or glial characteristics. PMID:877552

  6. Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

    PubMed Central

    Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

    2006-01-01

    Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

  7. Defects in antigen-specific immune tolerance in continuous B cell lines from autoimmune mice.

    PubMed Central

    Brooks, M S; Aldo-Benson, M

    1986-01-01

    B cell hyperactivity and resistance to tolerance induction are well-recognized immunologic abnormalities associated with both human and murine models of systemic lupus erythematosus. Studies evaluating the role of B cells in these defects have been complicated by the difficulties of consistently isolating large numbers of B cells from T cells and other host-derived regulatory factors. We have recently developed continuous cell lines of B lymphocytes with a high degree of specificity for the antigen dinitrophenyl (DNP) from both New Zealand black times New Zealand white F1 hybrid (BWF1) and BALB/c mice, and we used them to study intrinsic B cell defects in autoimmunity. We found that the kinetics of the immune response to the antigen DNP-Ficoll of both the BWF1 and BALB/c B cell lines are not different. In addition, the BWF1 cell lines, like the BALB/c cell lines and normal B cells, require nonspecific T cell-derived factors as well as antigen to produce an immune response. Tolerance was tested in the BWF1 B cells by preincubating them with DNP-murine IgG2a (MGG), which can induce tolerance in BALB/c cell line lymphocytes. The BWF1 B cell lines were resistant to tolerance induction by DNP-MGG and required 50-fold higher dose of DNP-MGG than BALB/c cell lines for suppression. They were also relatively resistant to tolerance with trinitrophenyl-d-glutamyl lysine. Thus, DNP-specific B cells from autoimmune mice have an inherent defect in tolerance induction. PMID:2427544

  8. Vanadium mediated apoptosis and cell cycle arrest in MCF7 cell line.

    PubMed

    Ray, Rajarshi Sankar; Rana, Basabi; Swami, Biswanath; Venu, Venkatappan; Chatterjee, Malay

    2006-11-01

    Vanadium is a metal widely distributed in the environment. It is also a dietary micronutrient. It has shown insulin mimetic and chemopreventive properties and has been considered as an important pharmacological agent. In this study, we evaluated the apoptogenic role of vanadium on human breast cancer cell line MCF7. Exposure of MCF7 cells to vanadium led to the induction of apoptosis in a dose-dependent manner. Percentage of apoptosis was maximum (42.5%) at the highest non-toxic dose (250 microM). It was found that vanadium treatment brought about a prominent chromatin condensation, cell cycle arrest leading to apoptosis. These apoptosis based assays demonstrate that vanadium has the potential to be developed into an anti-cancer drug in the near future. PMID:16970931

  9. An eIF4E-interacting peptide induces cell death in cancer cell lines

    PubMed Central

    Masse, M; Glippa, V; Saad, H; Le Bloas, R; Gauffeny, I; Berthou, C; Czjzek, M; Cormier, P; Cosson, B

    2014-01-01

    The eukaryotic initiation factor eIF4E is essential for cap-dependent initiation of translation in eukaryotes. Abnormal regulation of eIF4E has been implicated in oncogenic transformation. We developed an eIF4E-binding peptide derived from Angel1, a partner of eIF4E that we recently identified. We show here that this peptide fused to a penetratin motif causes drastic and rapid cell death in several epithelial cancer cell lines. This necrotic cell death was characterized by a drop in ATP levels with F-actin network injury being a key step in extensive plasma membrane blebbing and membrane permeabilization. This synthetic eIF4E-binding peptide provides a candidate pharmacophore for a promising new cancer therapy strategy. PMID:25356869

  10. Establishment and genetic characterization of six unique tumor cell lines as preclinical models for sinonasal squamous cell carcinoma.

    PubMed

    García-Inclán, Cristina; López-Hernández, Alejandro; Alonso-Guervós, Marta; Allonca, Eva; Potes, Sira; Melón, Santiago; López, Fernando; Llorente, José Luis; Hermsen, Mario

    2014-01-01

    Sinonasal squamous cell carcinomas (SCC) are rare tumors, etiologically related to occupational exposure to wood and leather dust. In spite of surgical and radiotherapeutic advances, the 5 year survival is still 30-50%. Therefore, alternative treatment options are needed. We report the establishment and characterization of six unique human sinonasal SCC cell lines, named SCCNC1, 2, 4, 5, 6 and 7. In vitro growth and invasion characteristics were evaluated and genetic profiles were compared to those of the original primary tumors. The population doubling times ranged from 21 to 34?hours. Cell lines SCCNC2 and 7 were highly invasive in matrigel. Five cell lines carried a high number of copy number alterations, including amplifications and homozygous deletions, while one showed only three abnormalities. Sequence analysis revealed three cell lines with TP53 mutation and none with KRAS or BRAF. Overexpression of p53 was observed in five, and of EGFR in four cell lines. None of the cell lines showed strong immunopositivity of p16 or presence of human papilloma virus. In conclusion, we have created six new cell lines that are clinically and genetically representative of sinonasal SCC and that will be a useful tool for the preclinical testing of new therapeutic agents. PMID:24816148

  11. Hematopoietic cancer cell lines can support replication of Sabin poliovirus type 1.

    PubMed

    Oosterhoff, Dinja; van de Weerd, Gerard; van Eikenhorst, Gerco; de Gruijl, Tanja D; van der Pol, Leo A; Bakker, Wilfried A M

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  12. Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

    PubMed Central

    van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

    2015-01-01

    Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

  13. Authentication of scientific human cell lines: easy-to-use DNA fingerprinting.

    PubMed

    Dirks, Wilhelm G; Drexler, Hans G

    2005-01-01

    Human cell lines are an important resource for research and most often used in reverse genetic approaches or as in vitro model systems of human diseases. In this regard, it is crucial that the cells faithfully correspond to the purported objects of study. A number of recent publications have shown an unacceptable level of cell lines to be false, in part as a result of the nonavailability of a simple and easy DNA profiling technique. We have validated different single- and multiple-locus variable numbers of tandem repeats (VNTRs) enabling the establishment of a noncommercial, but good laboratory practice, method for authentication of cell lines by DNA fingerprinting. Polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of six prominent and highly polymorphic minisatellite VNTR loci, requiring only a thermal cycler and an electrophoretic system, was proven as the most reliable tool. Furthermore, the generated banding pattern and the determination of gender allows for verifying the authenticity of a given human cell line by simple agarose gel electrophoresis. The combination of rapidly generated DNA profiles based on single-locus VNTR loci and information on banding patterns of cell lines of interest by official cell banks (detailed information at the website www.dsmz.de) constitute a low-cost but highly reliable and robust method, enabling every researcher using human cell lines to easily verify cell line identity. PMID:15361654

  14. Poliovirus infection of established human blood cell lines: relationship between the differentiation stage and susceptibility of cell killing.

    PubMed

    Okada, Y; Toda, G; Oka, H; Nomoto, A; Yoshikura, H

    1987-02-01

    The replication of type 1 poliovirus in 13 established human blood cell lines differing in the differentiation stage and cell lineage was investigated. Three T (CCRF-CEM, CCRF-HSB-2, and Molt-3) and three B (Raji, CCRF-SB, and RPMI 8226) cell lines showed no cytopathic effects (CPE) or virus production. CPE associated with virus production were detected in the other seven cell lines: HL-60, ML-1, and KG-1 (granulocytic lineage), U-937 and THP-1 (monocytic lineage), K-562 (erythroid lineage), and Molt-4 (T cell lineage). These susceptible cell lines greatly differed in the speed at which the CPE progressed. The progression of CPE was faster in relatively well-differentiated cell lines such as HL-60 and U-937, independently of the multiplicity of infection, than in less differentiated cell lines such as K-562, KG-1, and THP-1. Thus, for the same lineage, the speed at which CPE progressed became proportionally higher with subsequent differentiation stages. In the K-562 cell culture, CPE were not observed until at least 5 days postinfection (p.i.), while more than 80% of HL-60 cells were killed within 3 days p.i. There were no significant differences between infected HL-60 and K-562 cells in the efficiency of infection determined at 8 hr p.i. by the indirect immunofluorescent technique, the rate of virus growth, or the amount of viral capsid protein synthesized. This indicated that there were similar viral replication cycles in the two cell lines. These observations suggest that the killing function of the virus is expressed more slowly in K-562 cells than in HL-60 cells. PMID:3027975

  15. Identification of Insect Cell Lines from 8 Lepidopteran species by DNA Amplification Fingerprinting

    NASA Astrophysics Data System (ADS)

    Khalid Nessr Alhag, Sadeq; Chao, Yao Han; Xin, Peng Jian

    DNA Amplification Fingerprinting (DAF) with arbitrarily selected primers was used to obtain DNA fingerprint profiles to distinguish among 8 lepidopteran insect cell lines. The fingerprinting pattern is a stable characteristic of the cell line because high and low passages generated the same profile. The DNA from each cell line was amplified and PCR products were analyzed by agarose gel electrophoresis. All cell lines could be distinguished from each other with following exception: Bombyx mori (Bm-e-HNU5) produced the same profile as Laphygma exigua (Le-H-HNU7) also Spodoptera exigua (UCR-SE-1C) produced identical patterns to Spodoptera litura (SL-ZSU-1). DAF will serve as an additional, valuable and reliable technique for the identification of insect cell lines.

  16. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    SciTech Connect

    Pan, Mu-Yun; Shen, Yuh-Chiang; National Research Institute of Chinese Medicine, Taipei, Taiwan ; Lu, Chien-Hsing; Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan ; Yang, Shu-Yi; Ho, Tsing-Fen; Peng, Yu-Ta; Chang, Chia-Che; Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan; Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan

    2012-12-15

    Prodigiosin is a bacterial tripyrrole pigment with potent cytotoxicity against diverse human cancer cell lines. Endoplasmic reticulum (ER) stress is initiated by accumulation of unfolded or misfolded proteins in the ER lumen and may induce cell death when irremediable. In this study, the role of ER stress in prodigiosin-induced cytotoxicity was elucidated for the first time. Comparable to the ER stress inducer thapsigargin, prodigiosin up-regulated signature ER stress markers GRP78 and CHOP in addition to activating the IRE1, PERK and ATF6 branches of the unfolded protein response (UPR) in multiple human breast carcinoma cell lines, confirming prodigiosin as an ER stress inducer. Prodigiosin transcriptionally up-regulated CHOP, as evidenced by its promoting effect on the CHOP promoter activity. Of note, knockdown of CHOP effectively lowered prodigiosin's capacity to evoke PARP cleavage, reduce cell viability and suppress colony formation, highlighting an essential role of CHOP in prodigiosin-induced cytotoxic ER stress response. In addition, prodigiosin down-regulated BCL2 in a CHOP-dependent manner. Importantly, restoration of BCL2 expression blocked prodigiosin-induced PARP cleavage and greatly enhanced the survival of prodigiosin-treated cells, suggesting that CHOP-dependent BCL2 suppression mediates prodigiosin-elicited cell death. Moreover, pharmacological inhibition of JNK by SP600125 or dominant-negative blockade of PERK-mediated eIF2? phosphorylation impaired prodigiosin-induced CHOP up-regulation and PARP cleavage. Collectively, these results identified ER stress-mediated cell death as a mode-of-action of prodigiosin's tumoricidal effect. Mechanistically, prodigiosin engages the IRE1–JNK and PERK–eIF2? branches of the UPR signaling to up-regulate CHOP, which in turn mediates BCL2 suppression to induce cell death. Highlights: ? Prodigiosin is a bacterial tripyrrole pigment with potent anticancer effect. ? Prodigiosin is herein identified as an endoplasmic reticulum (ER) stress inducer. ? Prodigiosin-induced cytotoxicity involves ER stress-mediated cell death. ? Prodigiosin transcriptionally induces CHOP to suppress BCL2 for evoking cell death. ? Prodigiosin engages the IRE1–JNK and PERK–eIF2? pathways to up-regulate CHOP.

  17. Derivation of Human Embryonic Stem Cell Lines from Vitrified Human Blastocysts.

    PubMed

    Bradley, Cara K; Schaft, Julia; Roy, Tammie K; Dumevska, Biljana; Peura, Teija T

    2016-01-01

    Human embryonic stem cells are pluripotent cells typically derived from blastulating embryos that have become excess to clinical needs in assisted reproduction programs. They provide cellular models for embryonic development and disease, and are thought to be useful for future cell replacement therapies and regenerative medicine. Here we describe methods to derive human embryonic stem cell lines. This includes blastocyst cryopreservation using a highly efficient vitrification protocol, the production and use of fibroblast feeder cells, embryo plating and passaging of resulting cellular outgrowths, and cryopreservation of putative stem cells lines. PMID:24961221

  18. Mogoltacin enhances vincristine cytotoxicity in human transitional cell carcinoma (TCC) cell line.

    PubMed

    Behnam Rassouli, F; Matin, M M; Iranshahi, M; Bahrami, A R; Neshati, V; Mollazadeh, S; Neshati, Z

    2009-03-01

    Bladder cancer is the second common cancer of the genitourinary system throughout the world and intravesical chemotherapy is usually used to reduce tumour recurrence and progression. Human transitional cell carcinoma (TCC) is an epithelial-like adherent cell line originally established from primary bladder carcinoma. Here we report the effect of mogoltacin, a sesquiterpene coumarin from Ferula badrakema on TCC cells. Mogoltacin was isolated from the fruits of F. badrakema, using silica gel column chromatography and preparative thin layer chromatography. Mogoltacin did not have any significant cytotoxicity effect on neoplastic TCC cells at 16, 32, 64, 128, 200 and 600 microg ml(-1) concentrations. In order to analyse its combination effect, TCC cells were cultured in the presence of various combining concentrations of mogoltacin and vincristine. Cells were then observed for morphological changes (by light microscopy) and cytotoxicity using MTT assay. The effect of mogoltacin on vincristine toxicity was studied after 24, 48 and 72 h of drug administration. The results of MTT assay showed that mogoltacin can significantly enhance the cytotoxicity of vincristine and confirmed the morphological observations. Results revealed that combination of 40 microg ml(-1) vincristine with 16 microg ml(-1) mogoltacin increased the cytotoxicity of vincristine after 48 h by 32.8%. PMID:18707855

  19. Detection of anti-liver cell membrane antibody using a human hepatocellular carcinoma cell line

    SciTech Connect

    Lobo-Yeo, A.; McSorley, C.; McFarlane, B.M.; Mieli-Vergani, G.; Mowat, A.P.; Vergani, D.

    1989-02-01

    A radioimmunometric technique for the detection of autoantibodies to liver membrane antigens has been developed using Alexander cells, a human hepatocellular carcinoma cell line. After incubation of Alexander cells with serum, antimembrane antibodies were detected by addition of /sup 125/I-labeled Protein A. Binding ratios in 15 children with uncontrolled autoimmune chronic active hepatitis and in seven children with primary sclerosing cholangitis were significantly higher than in 18 age-matched normal controls. Nine patients with inactive autoimmune chronic active hepatitis, 13 with alpha 1-antitrypsin deficiency and five with fulminant hepatic failure had ratios similar to controls. In nine patients with Wilson's disease, there was a modest but significant increase in binding ratio. In four children with autoimmune chronic active hepatitis, binding ratios fell during effective immunosuppressive therapy. Sera from patients with systemic lupus erythematosus or rheumatoid arthritis gave normal results, excluding that binding derives from Fc-mediated immune complex capture. A positive correlation was found between Alexander cell binding values and anti-liver-specific protein antibody titers, suggesting that the two assays detect antibodies against shared antigenic determinants. The Alexander cell assay is a simple, rapid and sensitive technique to detect antibody to liver cell membrane antigens.

  20. Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

    PubMed Central

    Gelfand, Vladimir I.

    2013-01-01

    Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport. PMID:24300413

  1. Isolation and characterization of side population cells from the human ovarian cancer cell line SK-OV-3

    PubMed Central

    RUAN, ZHENGYI; LIU, JIANHUA; KUANG, YANPING

    2015-01-01

    Ovarian cancer (OC) is the most malignant type of gynecological tumor due to its high recurrence rate following initial treatment. Previous studies have indicated that cancer stem cells (CSCs) may be a potential cause underlying the high proportion of recurrence. Side population (SP) cells isolated from cancer cell lines have been shown to exhibit characteristics associated with CSCs, but studies on SP cells in human ovarian SK-OV-3 cell line are limited. In the present study, the SP cell fraction (4.83% of the total cell population) was isolated using flow cytometry, and analyzed by immunocytochemical analysis and reverse transcription-quantitative polymerase chain reaction. The results showed that SP cells exhibited a high mean fluorescence intensity for CD44, a CSC marker, in addition to elevated expression of the CSCs-associated genes, ATP-binding cassette sub-family G member 2 and Nestin. These findings indicated the stem cell-like features of the SP cells. Furthermore, a colony formation test showed that the isolated SP cells possessed a marked capacity for self-regeneration and proliferation. In addition, a cell cycle assay involving cisplatin indicated that the SP cells were strongly resistant to chemotherapy. In conclusion, the present results suggested that SP cells isolated from the SK-OV-3 cell line exhibited properties typically associated with CSCs. Therefore, the isolated SP cells may be used to provide novel insight into potential therapies against OC.

  2. Line of attack: NK cell specificity and integration of signals

    PubMed Central

    Bryceson, Yenan T.; Long, Eric O.

    2008-01-01

    Summary Natural killer (NK) cells possess potent cytolytic activity and secrete immune modulating cytokines. The large repertoire of NK cell receptors provides versatility for the identification of infected and transformed cells, and for their elimination by NK cells. NK cell responses also stimulate and regulate the adaptive arm of the immune system. We review current knowledge about the molecular specificity of NK cell receptors and about regulation of NK cell effector functions upon encounter with target cells. Mechanisms of recognition, interplay among receptors, signal integration, and dynamic fine-tuning of NK cell responses are discussed. New insights into molecular checkpoints for NK cell effector function are highlighted and underlying reasons for the complexity in NK cell recognition and signaling are proposed. PMID:18439809

  3. Saffold virus is able to productively infect primate and rodent cell lines and induces apoptosis in these cells

    PubMed Central

    Xu, Yishi; Victorio, Carla Bianca Luena; Ng, Qimei; Tan, Yee Joo; Chua, Kaw Bing

    2014-01-01

    Saffold virus (SAFV), a newly discovered human cardiovirus of the Picornaviridae family, causes widespread infection among children, as shown by previous seroprevalence studies. To determine the host cell range of SAFV and its cytopathogenicity, eight mammalian cell lines that were available in the laboratory were screened for productive SAFV infection by a laboratory-adapted SAFV of genotype 3. Five of the cell lines (Neuro2A, CHO-K1, NIH/3T3, Vero and HEp-2) were found to be permissible. The time required for SAFV to induce complete lysis as a cytopathic effect (CPE) in these permissibly infected cells and the resultant end point virus titer differed for each cell type. HEp-2 exhibited the shortest time frame to reach full CPE compared to the others. All infected cell lines produced a high virus titer at 72 h post-infection. In addition to causing lytic cell death, SAFV also induced apoptotic cell death in host cells through both extrinsic and intrinsic pathways, although the apoptotic events in HEp-2 cells appeared to have been blocked between the early and late stages. In conclusion, laboratory-adapted SAFV is able to productively infect a number of mammalian cell lines and induce apoptosis in the infected host cells. However, apoptosis in HEp-2 cells is blocked before the end stage. PMID:26038510

  4. Molecular characterization of 7 new established cell lines from high grade serous ovarian cancer.

    PubMed

    Kreuzinger, Caroline; Gamperl, Magdalena; Wolf, Andrea; Heinze, Georg; Geroldinger, Angelika; Lambrechts, Diether; Boeckx, Bram; Smeets, Dominiek; Horvat, Reinhard; Aust, Stefanie; Hamilton, Gerhard; Zeillinger, Robert; Cacsire Castillo-Tong, Dan

    2015-07-01

    Cancer cell lines are good in vitro models to study molecular mechanisms underlying chemoresistance and cancer recurrence. Recent works have demonstrated that most of the available ovarian cancer cell lines are most unlikely high grade serous (HGSOC), the major type of epithelial ovarian cancer. We aimed at establishing well characterized HGSOC cell lines, which can be used as optimal models for ovarian cancer research. We successfully established seven cell lines from HGSOC and provided the major genomic alterations and the transcriptomic landscapes of them. They exhibited different gene expression patterns in the key pathways involved in cancer resistance. Each cell line harbored a unique TP53 mutation as their corresponding tumors and expressed cytokeratins 8/18/19 and EpCAM. Two matched lines were established from the same patient, one at diagnosis and being sensitive to carboplatin and the other during chemotherapy and being resistant. Two cell lines presented respective BRCA1 and BRCA2 mutations. To conclude, we have established seven cell lines and well characterized them at genomic and transcriptomic levels. They are optimal models to investigate the molecular mechanisms underlying the progression, chemo resistance and recurrence of HGSOC. PMID:25862976

  5. Establishment of a New Cell Line from Lepidopteran Epidermis and Hormonal Regulation on the Genes

    PubMed Central

    Shao, Hong-Lian; Zheng, Wei-Wei; Liu, Peng-Cheng; Wang, Qian; Wang, Jin-Xing; Zhao, Xiao-Fan

    2008-01-01

    When an insect molts, old cuticle on the outside of the integument is shed by apolysis and a new cuticle is formed under the old one. This process is completed by the epidermal cells which are controlled by 20-hydroxyecdysone (20E) and juvenile hormone. To understand the molecular mechanisms of integument remolding and hormonal regulation on the gene expression, an epidermal cell line from the 5th instar larval integument of Helicoverpa armigera was established and named HaEpi. The cell line has been cultured continuously for 82 passages beginning on June 30, 2005 until now. Cell doubling time was 64 h. The chromosomes were granular and the chromosome mode was from 70 to 76. Collagenase I was used to detach the cells from the flask bottom. Non-self pathogen AcMNPV induced the cells to apoptosis. The cell line was proved to be an epidermal cell line based on its unique gene expression pattern. It responded to 20E and the non-steroidal ecdysone agonist RH-2485. Its gene expression could be knocked down using RNA interference. Various genes in the cell line were investigated based on their response to 20E. This new cell line represents a platform for investigating the 20E signaling transduction pathway, the immune response mechanism in lepidopteran epidermis and interactions of the genes. PMID:18769621

  6. Development and characterization of three new diploid cell lines from Labeo rohita (Ham.).

    PubMed

    Lakra, Wazir S; Swaminathan, T Raja; Rathore, Gaurav; Goswami, Mukunda; Yadav, Kamalendra; Kapoor, Smita

    2010-01-01

    Development of cell lines from fish for identifying the pathogenesis of viral diseases and for vaccine production against viral and bacterial diseases is imperative where they are of commercial importance. Three new diploid fish cell lines (RF, RH, and RSB) were developed from fin, heart, and swim bladder of an Indian major carp, Labeo rohita, commonly called Rohu. All the cell lines were optimally maintained at 28 degrees C in Leibovitz-15 medium supplemented with 10% FBS. The propagation of RH and RSB cells was serum dependent, with a low plating efficiency (<16%), whereas RF cells showed 20% efficiency. The cytogenetic analysis revealed a diploid count of 50 chromosomes. The cells of RF and RSB were found to be epithelial, where as the cells of RH were mostly fibroblastic. The viability of the RF, RH, and RSB cell lines was 75, 70 and 72%, respectively after 6 months of storage in liquid nitrogen. The origin of the cell lines was confirmed by the amplification of 496 and 655 bp fragments of 16S rRNA and Cytochrome Oxidase Subunit I (COI) of mtDNA. The new cell lines would facilitate viral disease diagnosis and genomic studies. PMID:20730759

  7. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Totipotent embryonic stem cell lines have not been established from ungulates, however, we have developed several somatic cell lines from the in vitro culture of pig epiblast cells. One such cell line, PICM-19, was isolated via colony-cloning and was found to spontaneously differentiate into hepati...

  8. Parasite antigen-specific human T cell lines and clones. Major histocompatibility complex restriction and B cell helper function.

    PubMed Central

    Nutman, T B; Ottesen, E A; Fauci, A S; Volkman, D J

    1984-01-01

    The development of T lymphocyte lines and clones of defined specificity has become an important method for investigating both T cell recognition of foreign antigens as well as T cell influence on B cells. In the present study, human antigen-specific T cell lines and clones have been derived from a patient with a naturally acquired filarial infection. These T cells are of the helper phenotype (Leu 1+, Leu 2-, Leu 3+) and are independent of exogenous interleukin-2. Furthermore, these T cells have been shown to require both antigen-presenting cells and antigen for optimal proliferation. Helper function mediated by these T cells as manifested by the in vitro induction of parasite-specific antibody was antigen-dose dependent, requiring much lower antigen concentrations than those necessary to induce blastogenesis. More importantly, there is an absolute requirement of the T cell line for HLA-DR histocompatible antigen-presenting cells; clones derived from this T cell line show a more specific DR-related restriction--to only one of the two parental DR haplotypes in antigen stimulated proliferative responses. Such parasite antigen specific human helper T cell lines and clones should prove useful in exploring the fine control of the host response to naturally acquired helminth infections. In addition, these long-term T cell lines and clones can provide a potent tool for examining not only the events involved in human T cell responses to parasite antigens, but also into the associated cellular and humoral factors necessary for the B cell responses which follow. PMID:6233302

  9. Direct elemental analysis of cancer cell lines by total reflection X-ray fluorescence

    NASA Astrophysics Data System (ADS)

    Szoboszlai, Norbert; Réti, Andrea; Budai, Barna; Szabó, Zsuzsa; Kralovánszky, Judit; Záray, Gyula

    2008-12-01

    The elemental content of Cu, Fe and Zn in two human adenocarcinoma cell lines was investigated by total reflection X-ray fluorescence (TXRF) spectrometry. Cancer cells were sedimented directly to the quartz plates using a modified cytospin slide holder setup. Special glass stands and caps were also constructed to hold the quartz plates with the cells during the vapour-phase microwave assisted digestion. The method was validated by analysis of certified reference materials. The signal-to-noise ratio was optimized by washing the cells with different solutions. The technique was applied to the determination of Cu, Fe and Zn content of HT-29 and HCA-7 colorectal adenocarcinoma cell lines. Dry mass of the centrifuged cells were determined and the elemental analysis data reported for the two cell lines were referred either to cell numbers, to the total protein content or to the dry mass.

  10. Establishment and Characterization of Fibroblast Cell Line Derived from Siberian Tiger (Panthera tigris altaica).

    PubMed

    Liu, Changqing; Guo, Yu; Liu, Dan; Guan, Weijun; Ma, Yuehui

    2010-06-01

    The Siberian tiger ear marginal tissue fibroblast cell line (STF34) from 34 samples was successfully established using primary explants technique and cell cryoconservation technology. STF34 cells were adherent, with a population doubling time of 24?h. Chromosome analysis showed that 90.2%-91.6% of cells were diploid (2n?=?38). Isoenzyme analyses of lactate dehydrogenase and malate dehydrogenase showed that STF34 cells had no cross-contamination with other species. Tests for cell line contamination with bacteria, fungi, viruses, and mycoplasmas were all negative. Every index of the STF34 cell line meets all the standard quality controls of American Type Culture Collection. Not only has the germline of this important Siberian tiger species been preserved at the cell level, but also valuable material had been provided for genome, postgenome, and somacloning research. PMID:24845938

  11. Mechanisms of synergism between cisplatin and gemcitabine in ovarian and non-small-cell lung cancer cell lines

    PubMed Central

    Moorsel, C J A van; Pinedo, H M; Veerman, G; Bergman, A M; Kuiper, C M; Vermorken, J B; van der Vijgh, W J F; Peters, G J

    1999-01-01

    2?,2?-Difluorodeoxycytidine (gemcitabine, dFdC) and cis-diammine-dichloroplatinum (cisplatin, CDDP) are active agents against ovarian cancer and non-small-cell lung cancer (NSCLC). CDDP acts by formation of platinum (Pt)–DNA adducts; dFdC by dFdCTP incorporation into DNA, subsequently leading to inhibition of exonuclease and DNA repair. Previously, synergism between both compounds was found in several human and murine cancer cell lines when cells were treated with these drugs in a constant ratio. In the present study we used different combinations of both drugs (one drug at its IC25 and the other in a concentration range) in the human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP, its dFdC-resistant variant AG6000 and two NSCLC cell lines, H322 (human) and Lewis lung (LL) (murine). Cells were exposed for 4, 24 and 72 h with a total culture time of 96 h, and possible synergism was evaluated by median drug effect analysis by calculating a combination index (CI; CI < 1 indicates synergism). With CDDP at its IC25, the average CIs calculated at the IC50, IC75 IC90 and IC95 after 4, 24 and 72 h of exposure were < 1 for all cell lines, indicating synergism, except for the CI after 4 h exposure in the LL cell line which showed an additive effect. With dFdC at its IC25, the CIs for the combination with CDDP after 24 h were < 1 in all cell lines, except for the Cls after 4 h exposure in the LL and H322 cell lines which showed an additive effect. At 72 h exposure all Cls were < 1. CDDP did not significantly affect dFdCTP accumulation in all cell lines. CDDP increased dFdC incorporation into both DNA and RNA of the A2780 cell lines 33- and 79-fold (P < 0.01) respectively, and tended to increase the dFdC incorporation into RNA in all cell lines. In the AG6000 and LL cell lines, CDDP and dFdC induced > 25% more DNA strand breaks (DSB) than each drug alone; however, in the other cell lines no effect, or even a decrease in DSB, was observed. dFdC increased the cellular Pt accumulation after 24 h incubation only in the ADDP cell line. However, dFdC did enhance the Pt–DNA adduct formation in the A2780, AG6000, ADDP and LL cell lines (1.6-, 1.4-, 2.9- and 1.6-fold respectively). This increase in Pt–DNA adduct formation seems to be related to the incorporation of dFdC into DNA (r = 0.91). No increase in DNA platination was found in the H322 cell line. dFdC only increased Pt–DNA adduct retention in the A2780 and LL cell lines, but decreased the Pt–DNA adduct retention in the AG6000 cell line. In conclusion, the synergism between dFdC and CDDP appears to be mainly due to an increase in Pt–DNA adduct formation possibly related to changes in DNA due to dFdC incorporation into DNA. © 1999 Cancer Research Campaign PMID:10362105

  12. Presence of dopamine D-2 receptors in human tumoral cell lines

    SciTech Connect

    Sokoloff, P.; Riou, J.F.; Martres, M.P.; Schwartz, J.C. )

    1989-07-31

    ({sup 125}I) Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, ({sup 125}I) iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

  13. Establishment and characterization of a new highly metastatic human osteosarcoma cell line derived from Saos2

    PubMed Central

    Du, Lin; Fan, Qiming; Tu, Bing; Yan, Wei; Tang, Tingting

    2014-01-01

    Osteosarcoma is the most common primary malignancy of bone in adolescents and young adults. There is a shortage of tumorigenic and highly metastatic human osteosarcoma cell lines that can be used for metastasis study. Here we establish and characterize a highly metastatic human osteosarcoma cell line that is derived from Saos2 cell line based on bioluminescence. The occasional pulmonary metastatic cells developed from Saos2 were isolated, harvested, characterized and named Saos2-l. The parental Saos2 and Saos2-l cells were further characterized both in vitro and in vivo. Results showed that Saos2-l cells demonstrated increased cell adhesion, migration and invasion compared to the parental Saos2 cells. Conversely, Saos2-l cells grew at a slightly slower rate than that of the parental cells. When injected into nude mice, Saos2-l cells had a greater increase in developing pulmonary metastases compared to the parental Saos2 cells. Further transcriptional profiling analysis revealed that some gene expression were up-regulated or down-regulated in the highly metastatic Saos2-l cells, indicating possible influencing factors of metastasis. Thus, we have established and characterized a highly metastatic human osteosarcoma cell line that should serve as a valuable tool for future investigations on the pathogenesis, metastasis and potential treatments of human osteosarcoma. PMID:25031706

  14. Persistence of betanodavirus in Barramundi brain (BB) cell line involves the induction of Interferon response.

    PubMed

    Wu, Y C; Chi, S C

    2006-11-01

    The BB cell line derived from the brain tissue of a barramundi (Lates calcarifer) that survived nervous necrosis virus (NNV) infection is persistently infected with NNV. To elucidate whether interferon (IFN) plays a role in the mechanism of NNV-persistent infection in BB cell line, a virus-negative control cell line was obtained by treating BB cells with NNV-specific rabbit antiserum for 5 subcultures. After the treatment, NNV titer or RNA or capsid protein was no longer detected in the cured BB (cBB) cells. Expression of Mx gene, encoding a type I IFN-inducible antiviral protein, was found in BB cells and cBB cells following NNV infection, but not in NNV-free cBB cells. Moreover, expression of Mx gene and antiviral activity against NNV were induced in cBB cells by the treatment with MAb-neutralized BB cell supernatant. Furthermore, NNV persistent infection was induced again in cBB cell culture if multiplicity of infection (MOI) was low (< or = 1). These experimental results indicated that IFN-like cytokines existed in the culture supernatant of BB cells, and IFN-induced response played an important role in protecting the majority of cells from virus lytic infection and regulating NNV persistence in the BB cell line. PMID:16698284

  15. Carriers for metal complexes on tumour cells: the effect of cyclodextrins vs CNTs on the model guest phenanthroline-5,6-dione trithiacyclononane ruthenium(II) chloride.

    PubMed

    Braga, Susana S; Marques, Joana; Heister, Elena; Diogo, Cátia V; Oliveira, Paulo J; Paz, Filipe A Almeida; Santos, Teresa M; Marques, Maria Paula M

    2014-06-01

    The complex [Ru[9]aneS3(pdon)Cl]Cl (pdon = 1,10-phenanthroline-5,6-dione) was readily obtained from the stoichiometric reaction of Ru[9]aneS3(dmso)Cl2 with pdon. Recrystallisation in ethanol using salicylic acid as a co-crystallisation helper afforded single-crystals suitable for the collection of X-ray diffraction data which afforded a reasonable structural description. Two different kinds of molecular carriers were tested as vehicles for this complex: carbon nanotubes (CNTs) and cyclodextrins. CNTs had an insufficient loading rate for the ruthenium complex at CNT concentrations deemed non-cytotoxic on cultured cells. The cyclodextrin (CD) carriers, ?-CD and TRIMEB (standing for permethylated ?-CD), were able to form two adducts, studied by powder X-ray diffraction, thermogravimetric analysis (TGA), (13)C{(1)H} CP/MAS NMR and FT-IR spectroscopies. The DNA thermal denaturation studies showed that the complex 1 is able to intercalate with DNA. The in vitro cytotoxicity of the free complex [Ru[9]aneS3(pdon)Cl]Cl (1) and of its two CD adducts (2 and 3) was assessed on both rodent and human cell lines. By using the mouse K1735-M2 melanoma cell line and the non-tumour rat H9c2 cardiomyoblasts, the results showed that 1 and 2 significantly inhibited the growth of the tumour cell line while displaying a good safety profile on cardiomyoblasts. Compound 3 at 100 ?M inhibited the proliferation of both cell lines, with a higher activity towards the melanoma cell line. The cytotoxicity of the compounds 1-3 was further assessed on human breast cancer cell lines. Against the MDA-MB-231 line, growth inhibition occurred only with 1 and 3 at the incubation time of 96 h, both with approximate inhibition rates of 50 %; against the MCF-7 line, mild cytotoxicity was observed at 48 h of incubation, with IC50 values calculated above 100 ?M for 1, 2 and 3. PMID:24652595

  16. Establishment and characterization of 13 cell lines from a green turtle (Chelonia mydas) with fibropapillomas

    USGS Publications Warehouse

    Lu, Y.; Nerurkar, V.R.; Aguirre, A.A.; Work, T.M.; Balazs, G.H.; Yanagihara, R.

    1999-01-01

    Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30A? C in RPMI 1640 medium supplemented with 10% fetal bovine serum. Propagation of the turtle cell lines was serum dependent, and plating efficiencies ranged from 13 to 37%. The cell lines, which have been subcultivated more than 20 times, had a doubling time of approximately 30 to 36 h. When tested for their sensitivity to several fish viruses, most of the cell lines were susceptible to a rhabdovirus, spring viremia carp virus, but refractory to channel catfish virus (a herpesvirus), infectious pancreatic necrosis virus (a birnavirus), and two other fish rhabdoviruses, infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus. During in vitro subcultivation, tumor-like cell aggregates appeared in cell lines derived from lungs, testis, and periorbital and tumor tissues, and small, naked intranuclear virus particles were detected by thin-section electron microscopy. These cell lines are currently being used in attempts to isolate the putative etiologic virus of green turtle fibropapilloma.

  17. Correlation between metastatic potential and variants from colorectal tumor cell line HT-29

    PubMed Central

    Wang, Min; Vogel, Ilka; Kalthoff, Holger

    2003-01-01

    AIM: To evaluate the relationship between uPA, PAI-1, CEA, PI3K and metastatic potential in three colorectal tumor cell lines. METHODS: Metastatic model in nude rats was established by variants HT-29c and HT-29d cell lines and the metastatic potential of two tumor cell variants was compared. Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were determined using ELISA in colorectal carcinoma WiDr, HT-29 and HT-29d cell lines with different metastatic potentials. Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) was analyzed using immunohistochemistry (IHC) in these cell lines in vitro and in vivo. CEA expression was compared using fluorescence activated cell sorter (FACS) in vitro. RESULTS: The number of HT-29d cells arrested in liver dramatically decreased within the initial 24 h after injection. The taking rate of liver metastases in the variant HT-29d increased as compared with parental HT-29 cells (70% versus 50%) and a variant HT-29b cells (70% versus 60%), and extensive organs were synchronously involved in metastases. The uPA concentration of variant HT-29d cell line was significantly higher than that of the non-metastatic WiDr and the low metastatic HT-29 cell lines. The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-metastatic WiDr cells and the low metastatic HT-29 cells in vivo. CONCLUSION: The selected variant HT-29d cell exhibited an enhanced metastatic potential. The level of uPA and PAI-1 is positively correlated with the metastatic capacity of tumor cells. The expression of PI3-kinase correlates with tumor development and metastasis. PMID:14606113

  18. Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines

    PubMed Central

    Uphoff, Cord C.; Lange, Sandra; Denkmann, Sabine A.; Garritsen, Henk S. P.; Drexler, Hans G.

    2015-01-01

    Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%. PMID:25927683

  19. Calcium signaling properties of a thyrotroph cell line, mouse T?T1 cells.

    PubMed

    Tomi?, Melanija; Bargi-Souza, Paula; Leiva-Salcedo, Elias; Nunes, Maria Tereza; Stojilkovic, Stanko S

    2015-12-01

    T?T1 cells are mouse thyrotroph cell line frequently used for studies on thyroid-stimulating hormone beta subunit gene expression and other cellular functions. Here we have characterized calcium-signaling pathways in T?T1 cells, an issue not previously addressed in these cells and incompletely described in native thyrotrophs. T?T1 cells are excitable and fire action potentials spontaneously and in response to application of thyrotropin-releasing hormone (TRH), the native hypothalamic agonist for thyrotrophs. Spontaneous electrical activity is coupled to small amplitude fluctuations in intracellular calcium, whereas TRH stimulates both calcium mobilization from intracellular pools and calcium influx. Non-receptor-mediated depletion of intracellular pool also leads to a prominent facilitation of calcium influx. Both receptor and non-receptor stimulated calcium influx is substantially attenuated but not completely abolished by inhibition of voltage-gated calcium channels, suggesting that depletion of intracellular calcium pool in these cells provides a signal for both voltage-independent and -dependent calcium influx, the latter by facilitating the pacemaking activity. These cells also express purinergic P2Y1 receptors and their activation by extracellular ATP mimics TRH action on calcium mobilization and influx. The thyroid hormone triiodothyronine prolongs duration of TRH-induced calcium spikes during 30-min exposure. These data indicate that T?T1 cells are capable of responding to natively feed-forward TRH signaling and intrapituitary ATP signaling with acute calcium mobilization and sustained calcium influx. Amplification of TRH-induced calcium signaling by triiodothyronine further suggests the existence of a pathway for positive feedback effects of thyroid hormones probably in a non-genomic manner. PMID:26453278

  20. Genomic Instability of Osteosarcoma Cell Lines in Culture: Impact on the Prediction of Metastasis Relevant Genes

    PubMed Central

    Muff, Roman; Rath, Prisni; Ram Kumar, Ram Mohan; Husmann, Knut; Born, Walter; Baudis, Michael; Fuchs, Bruno

    2015-01-01

    Background Osteosarcoma is a rare but highly malignant cancer of the bone. As a consequence, the number of established cell lines used for experimental in vitro and in vivo osteosarcoma research is limited and the value of these cell lines relies on their stability during culture. Here we investigated the stability in gene expression by microarray analysis and array genomic hybridization of three low metastatic cell lines and derivatives thereof with increased metastatic potential using cells of different passages. Principal Findings The osteosarcoma cell lines showed altered gene expression during in vitro culture, and it was more pronounced in two metastatic cell lines compared to the respective parental cells. Chromosomal instability contributed in part to the altered gene expression in SAOS and LM5 cells with low and high metastatic potential. To identify metastasis-relevant genes in a background of passage-dependent altered gene expression, genes involved in "Pathways in cancer" that were consistently regulated under all passage comparisons were evaluated. Genes belonging to "Hedgehog signaling pathway" and "Wnt signaling pathway" were significantly up-regulated, and IHH, WNT10B and TCF7 were found up-regulated in all three metastatic compared to the parental cell lines. Conclusions Considerable instability during culture in terms of gene expression and chromosomal aberrations was observed in osteosarcoma cell lines. The use of cells from different passages and a search for genes consistently regulated in early and late passages allows the analysis of metastasis-relevant genes despite the observed instability in gene expression in osteosarcoma cell lines during culture. PMID:25992885

  1. Cell line-specific oxidative stress in cellular toxicity: A toxicogenomics-based comparison between liver and colon cell models.

    PubMed

    Deferme, L; Briedé, J J; Claessen, S M H; Cavill, R; Kleinjans, J C S

    2015-08-01

    Imbalance between high reactive oxygen species formation and antioxidant capacity in the colon and liver has been linked to increased cancer risk. However, knowledge about possible cell line-specific oxidative stress-mechanisms is limited. To explore this further, gene expression data from a human liver and colon cell line (HepG2/Caco-2), both exposed to menadione and H2O2 at six time points (0.5-1-2-4-8 and 24h) were compared in association with cell cycle distribution. In total, 3164 unique- and 1827 common genes were identified between HepG2 and Caco-2 cells. Despite the higher number of unique genes, most oxidative stress-related genes such as CAT, OGG1, NRF2, NF-?B, GCLC, HMOX1 and GSR were differentially expressed in both cell lines. However, cell-specific regulation of genes such as KEAP1 and GCLM, or of the EMT pathway, which are of pathophysiological importance, indicates that oxidative stress induces different transcriptional effects and outcomes in the two selected cell lines. In addition, expression levels and/or -direction of common genes were often different in HepG2 and Caco-2 cells, and this led to very diverse downstream effects as confirmed by correlating pathways to cell cycle changes. Altogether, this work contributes to obtaining a better molecular understanding of cell line-specific toxicity upon exposure to oxidative stress-inducing compounds. PMID:25800948

  2. Isolation of Chinese hamster ovary cell lines temperature conditional for the cell-surface expression of integral membrane glycoproteins

    PubMed Central

    1989-01-01

    A procedure is described to select mutants of Chinese hamster ovary cells that are conditionally defective for the cell-surface expression of integral membrane glycoproteins, including the hemagglutinin (HA) of influenza virus. Using a combination of cell sorting and biochemical screening, seven cell lines were obtained that express more cell- surface HA at 32 degrees C than at 39 degrees C. The production of infectious vesicular stomatitis virus, whose growth requires insertion of an integral membrane protein into the plasma membrane, was also temperature conditional in the majority of these mutant cell lines. Five of the lines synthesized apparently normally core-glycosylated HA at the elevated temperature but the protein was neither displayed on the cell surface nor accumulated intracellularly. In these cell lines, little or no terminally glycosylated HA molecules were observed after synthesis at 39 degrees C. By contrast, the core glycosylation of HA and several other integral membrane proteins was abnormal in the remaining two cell lines at both permissive and restrictive temperatures, due to a lesion in a cellular gene(s) that affects the formation of and/or the addition of mannose-rich oligosaccharide chains to newly synthesized polypeptides. Although HA was transported to the plasma membrane at both 32 and 39 degrees C, it did not accumulate on the cell surface at the higher temperature, apparently because of an increased rate of degradation. PMID:2537314

  3. Metabolic Profiling Comparison of Human Pancreatic Ductal Epithelial Cells and Three Pancreatic Cancer Cell Lines using NMR Based Metabonomics

    PubMed Central

    Watanabe, Miki; Sheriff, Sulaiman; Lewis, Kenneth B.; Cho, Junho; Tinch, Stuart L.; Balasubramaniam, Ambikaipakan; Kennedy, Michael A.

    2015-01-01

    Metabolic profiles of hydrophilic and lipophilic cell extracts from three cancer cell lines, Miapaca-2, Panc-1 and AsPC-1, and a non-cancerous pancreatic ductal epithelial cell line, H6C7, were examined by proton nuclear magnetic resonance spectroscopy. Over twenty five hydrophilic metabolites were identified by principal component and statistical significance analyses as distinguishing the four cell types. Fifteen metabolites were identified with significantly altered concentrations in all cancer cells, e.g. absence of phosphatidylgrycerol and phosphatidylcholine, and increased phosphatidylethanolamine and cholesterols. Altered concentrations of metabolites involved in glycerophospholipid metabolism, lipopolysaccharide and fatty acid biosynthesis indicated differences in cellular membrane composition between non-cancerous and cancer cells. In addition to cancer specific metabolites, several metabolite changes were unique to each cancer cell line. Increased N-acetyl groups in AsPC-1, octanoic acids in Panc-1, and UDP species in Miapaca-2 indicated differences in cellular membrane composition between the cancer cell lines. Induced glutamine metabolism and protein synthesis in cancer cells were indicated by absence of glutamine other metabolites such as acetate, lactate, serine, branched amino acids, and succinate. Knowledge of the specifically altered metabolic pathways identified in these pancreatic cancer cell lines may be useful for identifying new therapeutic targets and studying the effects of potential new therapeutic drugs.

  4. Physical View on the Interactions Between Cancer Cells and the Endothelial Cell Lining During Cancer Cell Transmigration and Invasion

    NASA Astrophysics Data System (ADS)

    Mierke, Claudia T.

    2015-10-01

    There exist many reviews on the biological and biochemical interactions of cancer cells and endothelial cells during the transmigration and tissue invasion of cancer cells. For the malignant progression of cancer, the ability to metastasize is a prerequisite. In particular, this means that certain cancer cells possess the property to migrate through the endothelial lining into blood or lymph vessels, and are possibly able to transmigrate through the endothelial lining into the connective tissue and follow up their invasion path in the targeted tissue. On the molecular and biochemical level the transmigration and invasion steps are well-defined, but these signal transduction pathways are not yet clear and less understood in regards to the biophysical aspects of these processes. To functionally characterize the malignant transformation of neoplasms and subsequently reveal the underlying pathway(s) and cellular properties, which help cancer cells to facilitate cancer progression, the biomechanical properties of cancer cells and their microenvironment come into focus in the physics-of-cancer driven view on the metastasis process of cancers. Hallmarks for cancer progression have been proposed, but they still lack the inclusion of specific biomechanical properties of cancer cells and interacting surrounding endothelial cells of blood or lymph vessels. As a cancer cell is embedded in a special environment, the mechanical properties of the extracellular matrix also cannot be neglected. Therefore, in this review it is proposed that a novel hallmark of cancer that is still elusive in classical tumor biological reviews should be included, dealing with the aspect of physics in cancer disease such as the natural selection of an aggressive (highly invasive) subtype of cancer cells displaying a certain adhesion or chemokine receptor on their cell surface. Today, the physical aspects can be analyzed by using state-of-the-art biophysical methods. Thus, this review will present current cancer research in a different light from a physical point of view with respect to cancer cell mechanics and the special and unique role of the endothelium on cancer cell invasion. The physical view on cancer disease may lead to novel insights into cancer disease and will help to overcome the classical views on cancer. In addition, in this review it will be discussed how physics of cancer can help to reveal and propose the functional mechanism which cancer cells use to invade connective tissue and transmigrate through the endothelium to finally metastasize. Finally, in this review it will be demonstrated how biophysical measurements can be combined with classical analysis approaches of tumor biology. The insights into physical interactions between cancer cells, the endothelium and the microenvironment may help to answer some "old," but still important questions in cancer disease progression.

  5. Response of mechanosensory hair cells of the zebrafish lateral line to aminoglycosides reveals distinct cell death pathways

    E-print Network

    Rubel, Edwin

    t We report a series of experiments investigating the kinetics of hair cell loss in lateral line of hair cell loss and the differential effects of acute versus chronic exposure to gentamicin and neomycin. Gentamicin- induced loss of half of the hair cells within 90 min and substantial additional loss, which

  6. Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature

    PubMed Central

    Charafe-Jauffret, Emmanuelle; Ginestier, Christophe; Iovino, Flora; Wicinski, Julien; Cervera, Nathalie; Finetti, Pascal; Hur, Min-Hee; Diebel, Mark E.; Monville, Florence; Dutcher, Julie; Brown, Marthy; Viens, Patrice; Xerri, Luc; Bertucci, François; Stassi, Giorgio; Dontu, Gabriela; Birnbaum, Daniel; Wicha, Max S.

    2010-01-01

    Tumors may be initiated and maintained by a cellular subcomponent that displays stem cell properties. We have utilized the expression of aldehyde dehydrogenase as assessed by the ALDEFLUOR assay to isolate and characterize “cancer stem cell” populations in 33 cell lines derived from normal and malignant mammary tissue. Twenty-three of the 33 cell lines contained an ALDEFLUOR-positive population that displayed stem cell properties in vitro and in NOD/SCID xenografts. Gene expression profiling identified a 413-gene “cancer stem cell” profile that included genes known to play a role in stem cell function as well as genes such as CXCR1/IL8RA not previously known to play such a role. Recombinant IL8 increased mammosphere formation and the ALDEFLUOR-positive population in breast cancer cell lines. Finally, we show that ALDEFLUOR-positive cells are responsible for mediating metastasis. These studies confirm the hierarchical organization of immortalized cell lines, establish techniques that can facilitate the characterization of regulatory pathways of cancer stem cells and identify potential stem cell markers and therapeutical targets. PMID:19190339

  7. MicroRNA-126 inhibits invasion in non-small cell lung carcinoma cell lines

    SciTech Connect

    Crawford, M.; Brawner, E.; Batte, K.; Yu, L.; Hunter, M.G.; Otterson, G.A.; Nuovo, G.; Marsh, C.B.; Nana-Sinkam, S.P.

    2008-09-05

    Crk is a member of a family of adaptor proteins that are involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. Increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25 nt long) that are capable of targeting genes for either degradation of mRNA or inhibition of translation. Crk is a predicted putative target gene for miR-126. Over-expression of miR126 in a lung cancer cell line resulted in a decrease in Crk protein without any alteration in the associated mRNA. These lung cancer cells exhibit a decrease in adhesion, migration, and invasion. Decreased cancer cell invasion was also evident following targeted knockdown of Crk. MiR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion and the effects on invasion may be partially mediated through Crk regulation.

  8. Exome-level comparison of primary well-differentiated neuroendocrine tumors and their cell lines.

    PubMed

    Boora, Ganesh K; Kanwar, Rahul; Kulkarni, Amit A; Pleticha, Josef; Ames, Matthew; Schroth, Gary; Beutler, Andreas S; Banck, Michaela S

    2015-01-01

    Neuroendocrine cancer cell lines are used to investigate therapeutic targets in neuroendocrine tumors (NET) and have been instrumental in the design of clinical trials targeting the PI3K/AKT/mTOR pathways, VEGF inhibitors, and somatostatin analogues. It remains unknown, however, whether the genomic makeup of NET cell lines reflect that of primary NET since comprehensive unbiased genome sequencing has not been performed on the cell lines. Four bronchopulmonary NET (BP-NET)-NCI-H720, NCI-H727, NCI-H835, and UMC11-and two pancreatic neuroendocrine tumors (panNET)-BON-1 and QGP1-were cultured. DNA was isolated, and exome sequencing was done. GATK and EXCAVATOR were used for bioinformatic analysis. We detected a total of 1,764 nonsynonymous single nucleotide variants at a rate of 8 per Mb in BP-NET and 4.3 per Mb in panNET cell lines, including 52 mutated COSMIC cancer genes in these cell lines, such as TP53, BRCA1, RB1, TSC2, NOTCH1, EP300, GNAS, KDR, STK11, and APC but not ATRX, DAXX, nor MEN1. Our data suggest that mutation rate, the pattern of copy number variations, and the mutational spectra in the BP-NET cell lines are more similar to the changes observed in small cell lung cancer than those found in primary BP-NET. Likewise, mutation rate and pattern including the absence of mutations in ATRX/DAXX, MEN1, and YY1 in the panNET cell lines BON1 and QGP1 suggest that these cell lines do not have the genetic signatures of a primary panNET. These results suggest that results from experiments with BP-NET and panNET cell lines need to be interpreted with caution. PMID:26087898

  9. Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

    PubMed Central

    Smith, Zachary J.; Lee, Changwon; Rojalin, Tatu; Carney, Randy P.; Hazari, Sidhartha; Knudson, Alisha; Lam, Kit; Saari, Heikki; Ibańez, Elisa Lazaro; Viitala, Tapani; Laaksonen, Timo; Yliperttula, Marjo; Wachsmann-Hogiu, Sebastian

    2015-01-01

    Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level. PMID:26649679

  10. Metabolic profiling of breast cancer: Differences in central metabolism between subtypes of breast cancer cell lines.

    PubMed

    Willmann, Lucas; Schlimpert, Manuel; Halbach, Sebastian; Erbes, Thalia; Stickeler, Elmar; Kammerer, Bernd

    2015-09-01

    Although the concept of aerobic glycolysis in cancer was already reported in the 1930s by Otto Warburg, the understanding of metabolic pathways remains challenging especially due to the heterogeneity of cancer. In consideration of four different time points (1, 2, 4, and 7 days of incubation), GC-MS profiling of metabolites was performed on cell extracts and supernatants of breast cancer cell lines (MDA-MB-231, -453, BT-474) with different sub classification and the breast epithelial cell line MCF-10A. To the exclusion of trypsinization, direct methanolic extraction, cell scraping and cell disruption was executed to obtain central metabolites. Major differences in biochemical pathways have been observed in the breast cancer cell lines compared to the breast epithelial cell line, as well as between the breast cancer cell lines themselves. Characteristics of breast cancer subtypes could be correlated to their individual metabolic profiles. PLS-DA revealed the discrimination of breast cancer cell lines from MCF-10A based on elevated amino acid levels. The observed metabolic signatures have great potential as biomarker for breast cancer as well as an improved understanding of subtype specific phenomenons of breast cancer. PMID:26218769

  11. A Novel Inhibitor Of Topoisomerase I is Selectively Toxic For A Subset of Non-Small Cell Lung Cancer Cell Lines | Office of Cancer Genomics

    Cancer.gov

    SW044248, identified through a screen for chemicals that are selectively toxic for NSCLC cell lines, was found to rapidly inhibit macromolecular synthesis in sensitive, but not in insensitive cells. SW044248 killed approximately 15% of a panel of 74 NSCLC cell lines and was non-toxic to immortalized human bronchial cell lines.

  12. Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA

    PubMed Central

    Lee, Jae-Ho; Ku, Ja-Lok; Park, Young-Jin; Lee, Kuhn-Uk; Kim, Woo-Ho; Park, Jae-Gahb

    1999-01-01

    AIM: To investigate the characteristics of newly established f our hepatocellular carcinoma cell lines (SNU-739, SNU-761, SNU-878 and SNU-886) from Korean hepatocellular cancer patients. METHODS: Morphologic and genetic studies were done. RESULTS: All four lines grew as a monolayer with an adherent pat tern, and their doubling times ranged from 20 to 29 h. The viability rate was relatively high (88%-94%). Neither mycoplasmal nor bacterial contamination was present. The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx, HBc and HBs transcripts were detected by reverse transcriptase-PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2), only Hep 3B showed HBx expression, and this line was used as a HBV integrated control. The RNA of albumin was detected in three lines (SNU-761, SNU- 878 and SNU-886), that of transferrin in two lines (SNU-878, SNU-886), and that of IGF- II was detected in none of the cell lines. CONCLUSION: These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis B virus. PMID:11819450

  13. Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens).

    PubMed

    Higaki, Shogo; Koyama, Yoshie; Shirai, Emi; Yokota, Tomoya; Fujioka, Yasuhiro; Sakai, Noriyoshi; Takada, Tatsuyuki

    2013-06-01

    We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz's L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and ?-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 ? promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function. PMID:23076971

  14. An analysis of astrocytic cell lines with different abilities to promote axon growth.

    PubMed

    Fok-Seang, J; Smith-Thomas, L C; Meiners, S; Muir, E; Du, J S; Housden, E; Johnson, A R; Faissner, A; Geller, H M; Keynes, R J

    1995-08-21

    The adult mammalian central nervous system (CNS) lacks the capacity to support axonal regeneration. There is increasing evidence to suggest that astrocytes, the major glial population in the CNS, may possess both axon-growth promoting and axon-growth inhibitory properties and the latter may contribute to the poor regenerative capacity of the CNS. In order to examine the molecular differences between axon-growth permissive and axon-growth inhibitory astrocytes, a panel of astrocyte cell lines exhibiting a range of axon-growth promoting properties was generated and analysed. No clear correlation was found between the axon-growth promoting properties of these astrocyte cell lines with: (i) the expression of known neurite-outgrowth promoting molecules such as laminin, fibronectin and N-cadherin; (ii) the expression of known inhibitory molecules such tenascin and chondroitin sulphate proteoglycan; (iii) plasminogen activator and plasminogen activator inhibitor activity; and (iv) growth cone collapsing activity. EM studies on aggregates formed from astrocyte cell lines, however, revealed the presence of an abundance of extracellular matrix material associated with the more inhibitory astrocyte cell lines. When matrix deposited by astrocyte cell lines was assessed for axon-growth promoting activity, matrix from permissive lines was found to be a good substrate, whereas matrix from the inhibitory astrocyte lines was a poor substrate for neuritic growth. Our findings, taken together, suggest that the functional differences between the permissive and the inhibitory astrocyte cell lines reside largely with the ECM. PMID:7583324

  15. Lymphotoxin is an autocrine growth factor for Epstein-Barr virus- infected B cell lines

    PubMed Central

    1993-01-01

    Because human lymphotoxin (LT) was originally isolated from a lymphoblastoid cell line, we investigated the role of this molecule in three newly established Epstein-Barr virus (EBV)-infected human B cell lines. These lines were derived from acute lymphoblastic leukemia (Z- 6), myelodysplastic syndrome (Z-43), and acute myelogenous leukemia (Z- 55) patients who had a prior EBV infection. Each lymphoblastoid cell line had a karyotype that was different from that of the original parent leukemic cells, and all expressed B cell, but not T cell or myeloid surface markers. In all three lines, rearranged immunoglobulin heavy chain joining region (JH) bands were found, and the presence of EBV DNA was confirmed by Southern blotting. Z-6, Z-43, and Z-55 cell lines constitutively produced 192, 48, and 78 U/ml LT, respectively, as assessed by a cytotoxicity assay and antibody neutralization. Levels of tumor necrosis factor (TNF) were undetectable. Scatchard analysis revealed that all the cell lines expressed high-affinity TNF/LT receptors with receptor densities of 4197, 1258, and 1209 sites/cell on Z-6, Z-43, and Z-55, respectively. Furthermore, labeled TNF binding could be reversed by both unlabeled TNF, as well as by LT. Studies with p60 and p80 receptor-specific antibodies revealed that the three lines expressed primarily the p80 form of the TNF receptor. When studied in a clonogenic assay, exogenous LT stimulated proliferation of all three cell lines in a dose-dependent fashion at concentrations ranging from 25 to 500 U/ml. Similar results were obtained with [3H]TdR incorporation. Monoclonal anti-LT neutralizing antibodies at concentrations of 25-500 U/ml inhibited cellular multiplication in a dose-dependent manner. It is interesting that in spite of a common receptor, TNF (1,000 U/ml) had no direct effect on Z-55 cell growth, whereas it partially reversed the stimulatory effect of exogenous LT. In addition, TNF inhibited Z-6 and Z-43 cell proliferation, and its suppressive effect was reversed by exogenous LT. Both p80 and p60 forms of soluble TNF receptors suppressed the lymphoblastoid cell line proliferation and their inhibitory effect was partially reversed by LT. Our data suggest that (a) LT is an autocrine growth factor for EBV- transformed lymphoblastoid B cell lines; and (b) anti-LT antibodies, soluble TNF/LT receptors, and TNF itself can suppress the growth of lymphoblastoid cells, probably by modulating or competing with LT.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:8382256

  16. Irinotecan induces cell cycle arrest, but not apoptosis or necrosis, in Caco-2 and CW2 colorectal cancer cell lines.

    PubMed

    Kaku, Yoshiko; Tsuchiya, Ayako; Kanno, Takeshi; Nishizaki, Tomoyuki

    2015-01-01

    Irinotecan, a topoisomerase I inhibitor, is clinically used as an anticancer drug. The present study investigated the anticancer effect of irinotecan on p53-negative Caco-2 and p53-positive CW2 human colorectal cancer cell lines. Cell viability for both Caco-2 and CW2 cells was little affected by treatment with irinotecan at concentrations ranging from 0.3 to 30 ?mol/l for 24-48 h. Irinotecan did not increase the number of TUNEL-positive cells and did not affect the population of propidium iodide (PI)-positive and annexin V-negative cells, corresponding to primary necrosis, or that of PI-positive and annexin-positive cells, corresponding to late apoptosis/secondary necrosis, in either of the two cell lines. In the cell cycle analysis, irinotecan significantly increased the proportions at the S and G2/M phases of cell cycling in parallel with a decreased population at the G1 phase in both cell lines. Irinotecan significantly inhibited tumor growth in mice inoculated with CW2 cells. Taken together, these results indicate that irinotecan induces cell cycle arrest, but not apoptosis or necrosis, both in Caco-2 and CW2 cells, leading to suppression of cell proliferation. PMID:25833236

  17. Apoptosis and cell cycle disturbances induced by coumarin and 7-hydroxycoumarin on human lung carcinoma cell lines.

    PubMed

    Lopez-Gonzalez, Jose Sullivan; Prado-Garcia, Heriberto; Aguilar-Cazares, Dolores; Molina-Guarneros, Juan A; Morales-Fuentes, Jorge; Mandoki, Juan Jose

    2004-03-01

    Coumarin and 7-hydroxycoumarin have anti-tumour actions in vitro and in vivo. There are no previous reports on the cytostatic and apoptotic actions of coumarin and 7-hydroxycoumarin in non-small cell lung carcinoma (NSCLC) cell lines. Here we report on: (1) the inhibition of cell proliferation, (2) the phase in which cell cycle arrest occurs, and (3) the induction of apoptosis. Inhibition of cell proliferation was determined by 3H-thymidine incorporation. The effects on cell cycle phases were determined at 100 microg/ml of coumarin or 7-hydroxycoumarin using propidium iodide and flow cytometry. Higher concentrations were used to study apoptosis, detected by: (1) morphological cell changes, (2) subG1 peak detection and (3) Annexin-V assay. Peripheral blood mononuclear cells (PBMC) stimulated with phytohemagglutinin were used as controls. The actions of these compounds depended on drug concentrations and on histological cell type. Coumarin and 7-hydroxycoumarin inhibited cell growth by inducing cell cycle arrest in the G1 phase in all the lung carcinoma cell lines. Apoptosis required large concentrations of the coumarin compounds and was observed in adenocarcinomas. Apoptosis was not associated with intra-nucleosomal DNA fragmentation. Apoptosis was not observed in squamous lung carcinoma cell lines, but an increase in G1 cell cycle arrest was detected. In PBMC, only large concentrations of the coumarin compounds elicited a cystostatic action. Coumarins in combination with other anti-neoplastic drugs might increase the effectiveness of NSCLC treatments. PMID:15165085

  18. Evidence of a Genomic Biomarker in Normal Human Epithelial Mammary Cell Line, MCF-10A, That Is Absent in the Human Breast Cancer Cell Line, MCF-7

    PubMed Central

    Crawford, Brian H.; Hussain, AKM A.; Jideama, Nathan M.

    2006-01-01

    This study investigated the use of DNA amplification fingerprinting (DAF) to identify biomarkers useful in the elucidating genetic factors that lead to carcinogenesis. The DNA amplification fingerprinting (DAF) technique was used to generate fingerprint profiles of a normal human mammary epithelial cell line (MCF-10A) and a human breast cancer cell line (MCF-7). When compared with one another, a polymorphic biomarker gene (262 base pairs (bps)) was identified in MCF-10A but was not present in MCF-7. This gene was cloned from the genomic DNA of the MCF-10A cell line, and subjected to Genbank database analysis. The analysis of the nucleotide sequence polymorphic marker (Genbank account: AC079630) shows that this biomarker has 100% homology with the nucleotide sequence of human chromosome 12 BAC RP11-476D10 (bps 19612-19353). The nucleotide sequence was used for possible protein translation product and the result obtained indicated that the gene codes for hypothetical protein XF2620. In order to evaluate the effects that the 262 bps biomarker would have on the morphology of MCF-7 cells, it was transfected into MCF-7 cells. There were observable changes in the morphology of the transfected cells. These changes included an increase in cell elongation and a decrease in cell aggregation. PMID:16883051

  19. Metabolic basis for differential glutamine requirements of human leukemia cell lines.

    PubMed

    Kitoh, T; Kubota, M; Takimoto, T; Hashimoto, H; Shimizu, T; Sano, H; Akiyama, Y; Mikawa, H

    1990-04-01

    We compared the ability of human leukemia cell lines of various origins to grow in glutamine-deficient media. The growth of B lymphoblastoid cell lines, including promyelocytic HL-60, is highly dependent on glutamine, whereas T-cell lines are able to proliferate in glutamine-free media. Such glutamine dependency has a good inverse correlation with the activity of glutamine synthetase. Moreover, glutamine synthetase can be induced in glutamine-deficient media, especially in glutamine-independent cells. In HL-60 cells, glutamine deprivation results in the decrease of both ATP and dATP levels. The addition of adenine to the culture medium abolishes these changes without restoring cell growth, indicating that the effects of glutamine deprivation on cell growth cannot be fully explained by the perturbation of adenine nucleotide pools. PMID:1969419

  20. Cancer Cell Line Panels Empower Genomics-Based Discovery of Precision Cancer Medicine

    PubMed Central

    Kim, Hyun Seok; Sung, Yeo-Jin

    2015-01-01

    Since the first human cancer cell line, HeLa, was established in the early 1950s, there has been a steady increase in the number and tumor type of available cancer cell line models. Cancer cell lines have made significant contributions to the development of various chemotherapeutic agents. Recent advances in multi-omics technologies have facilitated detailed characterizations of the genomic, transcriptomic, proteomic, and epigenomic profiles of these cancer cell lines. An increasing number of studies employ the power of a cancer cell line panel to provide predictive biomarkers for targeted and cytotoxic agents, including those that are already used in clinical practice. Different types of statistical and machine learning algorithms have been developed to analyze the large-scale data sets that have been produced. However, much work remains to address the discrepancies in drug assay results from different platforms and the frequent failures to translate discoveries from cell line models to the clinic. Nevertheless, continuous expansion of cancer cell line panels should provide unprecedented opportunities to identify new candidate targeted therapies, particularly for the so-called "dark matter" group of cancers, for which pharmacologically tractable driver mutations have not been identified. PMID:26256959

  1. Cancer Cell Line Panels Empower Genomics-Based Discovery of Precision Cancer Medicine.

    PubMed

    Kim, Hyun Seok; Sung, Yeo-Jin; Paik, Soonmyung

    2015-09-01

    Since the first human cancer cell line, HeLa, was established in the early 1950s, there has been a steady increase in the number and tumor type of available cancer cell line models. Cancer cell lines have made significant contributions to the development of various chemotherapeutic agents. Recent advances in multi-omics technologies have facilitated detailed characterizations of the genomic, transcriptomic, proteomic, and epigenomic profiles of these cancer cell lines. An increasing number of studies employ the power of a cancer cell line panel to provide predictive biomarkers for targeted and cytotoxic agents, including those that are already used in clinical practice. Different types of statistical and machine learning algorithms have been developed to analyze the large-scale data sets that have been produced. However, much work remains to address the discrepancies in drug assay results from different platforms and the frequent failures to translate discoveries from cell line models to the clinic. Nevertheless, continuous expansion of cancer cell line panels should provide unprecedented opportunities to identify new candidate targeted therapies, particularly for the so-called "dark matter" group of cancers, for which pharmacologically tractable driver mutations have not been identified. PMID:26256959

  2. Systematic variation in gene expression patterns in human cancer cell lines

    SciTech Connect

    Ross, Douglas T.; Scherf, Uwe; Eisen, Michael B.; Perou, Charles M.; Rees, Christian; Spellman, Paul; Iyer, Vishwanath; Jeffrey, Stefanie S.; Van de Rijn, Matt; Waltham, Mark; Pergamenschikov, Alexander; Lee, Jeffrey C.F.; Lashkari, Deval; Shalon, Dari; Myers, Timothy G.; Weinstein, John N.; Botstein, David; Brown, Patrick O.

    2000-01-01

    We used cDNA micro arrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute s screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumors from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumor specimens revealed features of the expression patterns in the tumors that had recognizable counterparts in specific cell lines, reflecting the tumor, stromal and inflammatory components of the tumor tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumors in vivo.

  3. Culture supernatants of different colon cancer cell lines induce specific phenotype switching and functional alteration of THP-1 cells.

    PubMed

    Wu, Tsung-Han; Li, Ying-Ying; Wu, Tai-Ling; Chang, John W-C; Chou, Wen-Chi; Hsieh, Ling-Ling; Chen, Jim-Ray; Yeh, Kun-Yun

    2014-07-01

    We developed an in vitro model to evaluate the effect of products secreted from different colorectal cancer (CRC) cell lines on specific phenotypic switching and functional alterations in THP-1 cells. We co-cultured the human monocytic cell line, THP-1, or phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells, (THP-1p), with supernatants from either the HT-29 (Dukes' B), HCT-15 (Dukes' C), or Colo205 (Dukes' D) cell lines, and assessed the cells for macrophage differentiation. The surface marker and cytokine profiles suggested that secreted CRC factors differentiated THP-1 cells into a "mixed" M1/M2 phenotype, although HT-29 and Colo205 supernatants induced THP-1p cells into predominantly M1-like macrophages and M2-like macrophages, respectively. Further, all three CRC supernatants enhanced the phagocytic capacity and migration of THP-1 and THP-1p cells, altering their phenotype to a more M2-kind. Therefore, different CRC cell lines induced specific phenotype switching and functional polarization of THP-1 cells. PMID:24960291

  4. Predicting Anticancer Drug Responses Using a Dual-Layer Integrated Cell Line-Drug Network Model

    PubMed Central

    Fang, Yun; Wang, Jun; Zheng, Xiaoqi; Liu, X. Shirley

    2015-01-01

    The ability to predict the response of a cancer patient to a therapeutic agent is a major goal in modern oncology that should ultimately lead to personalized treatment. Existing approaches to predicting drug sensitivity rely primarily on profiling of cancer cell line panels that have been treated with different drugs and selecting genomic or functional genomic features to regress or classify the drug response. Here, we propose a dual-layer integrated cell line-drug network model, which uses both cell line similarity network (CSN) data and drug similarity network (DSN) data to predict the drug response of a given cell line using a weighted model. Using the Cancer Cell Line Encyclopedia (CCLE) and Cancer Genome Project (CGP) studies as benchmark datasets, our single-layer model with CSN or DSN and only a single parameter achieved a prediction performance comparable to the previously generated elastic net model. When using the dual-layer model integrating both CSN and DSN, our predicted response reached a 0.6 Pearson correlation coefficient with observed responses for most drugs, which is significantly better than the previous results using the elastic net model. We have also applied the dual-layer cell line-drug integrated network model to fill in the missing drug response values in the CGP dataset. Even though the dual-layer integrated cell line-drug network model does not specifically model mutation information, it correctly predicted that BRAF mutant cell lines would be more sensitive than BRAF wild-type cell lines to three MEK1/2 inhibitors tested. PMID:26418249

  5. Leukemia derived growth factors produced by human malignant T-lymphoid cell lines.

    PubMed

    Uittenbogaart, C H; Nishanian, P G; Anisman, D J; Erikson, T K; Fahey, J L

    1986-03-01

    An autocrine (noninterleukin 2) growth factor, which we term leukemia derived growth factor (LDGF), has previously been found in the culture supernatant of the human malignant T-lymphoid cell line MOLT-4f. We now show that two other human malignant T-lymphoid cell lines, CCRF-CEM and CCRF-HSB-2 also produce such a factor. All three factors, i.e., the LDGF from MOLT-4f, CCRF-CEM, and CCRF-HSB-2 are similar to each other both in biological activity and in physicochemical characteristics. In addition to their autocrine activity, these LDGFs stimulate the growth of other malignant T-lymphoid cell lines, but they do not stimulate B-lymphoblastoid or myeloid cell lines. The results therefore suggest that these LDGFs are T-cell specific. PMID:3080240

  6. Immortalized human hepatic cell lines for in vitro testing and research purposes

    PubMed Central

    Ramboer, Eva; Vanhaecke, Tamara; Rogiers, Vera; Vinken, Mathieu

    2015-01-01

    Summary The ubiquitous shortage of primary human hepatocytes has urged the scientific community to search for alternative cell sources, such as immortalized hepatic cell lines. Over the years, several human hepatic cell lines have been produced, whether or not using a combination of viral oncogenes and human telomerase reverse transcriptase protein. Conditional approaches for hepatocyte immortalization have also been established and allow generation of growth-controlled cell lines. A variety of immortalized human hepatocytes have already proven useful as tools for liver-based in vitro testing and fundamental research purposes. The present chapter describes currently applied immortalization strategies and provides an overview of the actually available immortalized human hepatic cell lines and their in vitro applications. PMID:26272134

  7. Selection of tobacco cell lines with high yields of cinnamoyl putrescines.

    PubMed

    Berlin, J; Kukoschke, K G; Knobloch, K H

    1981-06-01

    Cell lines of NICOTIANA TABACUM resistant to either L-phenylalanine, DL-meta-fluorophenylalanine (MFP) or L-ornithine, have been selected in order to establish by this means cell strains with higher yields of cinnamoyl putrescines. Except for three MFP-resistant cell lines, all selected strains accumulated 2 to 5 times more cinnamoyl putrescines than the non-resistant wild type cells. In some lines this increase was accompanied by increased activities of enzymes which were likewise involved in the biosynthesis of cinnamoyl putrescines and in the metabolism/degradation of the selecting agents. The qualitative pattern of phenolic compounds did not change in any of the resistant cell lines. PMID:17401957

  8. Epigenetic Modulators Enhance Constitutive and Liver-Specific Reporter Expression in Murine Liver Progenitor Cell Lines.

    PubMed

    Diepeveen, Luke Aris; Watson, Michel Elyse; McSpadden, Sarah Beth; Strauss, Robyn Patricia; Callus, Bernard Andrew; Yeoh, George Cheng

    2015-10-01

    Stem cells expressing reporter constructs are extremely useful for their tracking in vivo or for determining cell lineage fate in vivo and in vitro. We generated liver progenitor cell (LPC) lines from actin-EGFP and TAT-GRE-lacZ mice. LPCs from the actin-EGFP mouse facilitate cell tracing following transplant as the reporter is constitutively expressed. LPCs from the TAT-GRE-lacZ mouse express ?-galactosidase under the control of the tyrosine aminotransferase (TAT) promoter and are only active in mature hepatocytes. We found that the utility of such LPC lines becomes severely limited by downregulation of transgene expression following extended culture. We show that epigenetic mechanisms are responsible for suppressing expression of both transgenes. Enhancement of transgene expression in both LPC lines was achieved by treating the cell lines with either the histone acetylating agent sodium butyrate or the DNA demethylating agent 5-azacytidine. PMID:25997084

  9. SunLine Transit Agency Advanced Technology Fuel Cell Bus Evaluation: Fourth Results Report

    SciTech Connect

    Eudy, L.; Chandler, K.

    2013-01-01

    SunLine Transit Agency, which provides public transit services to the Coachella Valley area of California, has demonstrated hydrogen and fuel cell bus technologies for more than 10 years. In May 2010, SunLine began demonstrating the advanced technology (AT) fuel cell bus with a hybrid electric propulsion system, fuel cell power system, and lithium-based hybrid batteries. This report describes operations at SunLine for the AT fuel cell bus and five compressed natural gas buses. The U.S. Department of Energy's National Renewable Energy Laboratory (NREL) is working with SunLine to evaluate the bus in real-world service to document the results and help determine the progress toward technology readiness. NREL has previously published three reports documenting the operation of the fuel cell bus in service. This report provides a summary of the results with a focus on the bus operation from February 2012 through November 2012.

  10. Research paper Lateral line hair cell maturation is a determinant of

    E-print Network

    Rubel, Edwin

    that cause dose-dependent sensorineural hearing loss (Smith et al., 1977) and are known to kill hair cellsResearch paper Lateral line hair cell maturation is a determinant of aminoglycoside susceptibility online 3 February 2006 Abstract Developmental differences in hair cell susceptibility to aminoglycoside

  11. USE OF A HUMAN/MOUSE HYBRID CELL LINE TO DETECT ANEUPLOIDY INDUCED BY ENVIRONMENTAL CHEMICALS

    EPA Science Inventory

    A short-term assay utilizing a human/mouse monochromosomal hybrid cell line R3-5, to detect chemically induced aneuploidy in mammalian cells is described. A single human chromosome transferred into mouse cells was used as a cytogenetic marker to quantitate abnormal chromosome seg...

  12. Characterization of P-glycoprotein and multidrug resistance proteins in rat kidney and intestinal cell lines.

    PubMed

    van de Water, Femke M; Boleij, Johanna M; Peters, Janny G P; Russel, Frans G M; Masereeuw, Rosalinde

    2007-01-01

    The activity of P-glycoprotein (Pgp/MDR1/ABCB1) and multidrug resistance proteins (MRP/ABCC) influence the pharmacokinetics and bioavailability of many drugs. Few suitable cell lines for the study of drug transport exist. Additional non-human cell lines may help clarify species differences and contribute to the current knowledge of drug transport. The aim of the present study was to characterize three rat epithelial cell lines for transporter expression and activity. Transporter expression was assessed in intestinal IEC-6 and renal GERP and NRK-52E cells using RT-PCR and Western blot analysis. Pgp and Mrp transport activity were analyzed by measuring calcein accumulation and glutathione-S-bimane efflux, respectively. The three cell lines showed Pgp expression and Pgp-dependent transport, both decreasing with culture time after reaching confluency. Besides Pgp, cells expressed Mrp1, Mrp3, Mrp4, and Mrp5, while Mrp2 and Mrp6 were absent. In addition, they showed temperature- and Mrp-dependent efflux of glutathione-S-bimane. Exposure to a panel of different inhibitors showed that this efflux was probably mediated by Mrp4. In conclusion, the three rat epithelial cell lines investigated showed Pgp and Mrp expression and transport. Mrp dependent transport was most likely mediated by Mrp4. In future, these cell lines may be used as in vitro models to study drug transport. PMID:17088052

  13. BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light

    SciTech Connect

    Giulotto, E.; Bertoni, L.; Attolini, C.; Rainaldi, G.; Anglana, M. )

    1991-04-15

    Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification.

  14. BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light.

    PubMed

    Giulotto, E; Bertoni, L; Attolini, C; Rainaldi, G; Anglana, M

    1991-04-15

    Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification. PMID:1707543

  15. Bovine anterior pituitary progenitor cell line expresses interleukin (IL)-18 and IL-18 receptor.

    PubMed

    Nagai, Y; Ogasawara, H; Taketa, Y; Aso, H; Tanaka, S; Kanaya, T; Watanabe, K; Ohwada, S; Muneta, Y; Yamaguchi, T

    2008-11-01

    In the anterior pituitary gland, inflammatory mediators regulate cell function through an immuno-endocrine pathway. Recent studies have shown that undifferentiated stem cells act as immunomodulators. These studies prompted us to establish a progenitor cell line from the bovine anterior pituitary gland and to detail its function. First, we localised interleukin (IL)-18 by immunohistochemistry to the marginal cell layer of Rathke's pouch that is assumed to embody a stem/progenitor cell compartment of the postnatal pituitary gland. A cloned anterior pituitary-derived cell line from the bovine anterior pituitary gland was established from single cell clone by the limiting dilution method and was designated as bovine anterior pituitary-derived cell line (BAPC)-1. BAPC-1 cells constantly expressed mRNAs for IL-18 and IL-18 receptor, and grew steadily and rapidly in the medium containing epidermal growth factor and basic fibroblast growth factor. The cell line also expressed the mRNAs for the stem/progenitor cell- related factors such as Nanog, Oct-4, Ptch1, Nestin, Notch1, Hes1, Lrp and Fzd4, and the mRNAs for embryonic pituitary-related factors, such as Lhx3, PitX1 and Pit-1. The nuclei of BAPC-1 were immunostained positively for Pit-1, Hes1 and beta-catenin antibodies. Furthermore, BAPC-1 cells expressed mRNAs for cytokine such as IL-1alpha, IL-6, IL-7, IL-12 and IL-15. Stimulation of BAPC-1 cells with IL-18 increased expression of mRNAs for IL-1alpha, IL-6, IL-1beta and IL-8. At day 6 in culture, BAPC-1 cells also express growth hormone mRNA. These results strongly suggest that BAPC-1 is a stem/progenitor cell line and modulates the immuno-endocrine function of the anterior pituitary cells through its cytokine production. PMID:18761716

  16. Mycoplasma hyorhinis-Contaminated Cell Lines Activate Primary Innate Immune Cells via a Protease-Sensitive Factor

    PubMed Central

    Heidegger, Simon; Jarosch, Alexander; Schmickl, Martina; Endres, Stefan; Bourquin, Carole; Hotz, Christian

    2015-01-01

    Mycoplasma are a frequent and occult contaminant of cell cultures, whereby these prokaryotic organisms can modify many aspects of cell physiology, rendering experiments that are conducted with such contaminated cells problematic. Chronic Mycoplasma contamination in human monocytic cells lines has been associated with suppressed Toll-like receptor (TLR) function. In contrast, we show here that components derived from a Mycoplasma hyorhinis-infected cell line can activate innate immunity in non-infected primary immune cells. Release of pro-inflammatory cytokines such as IL-6 by dendritic cells in response to Mycoplasma hyorhinis-infected cell components was critically dependent on the adapter protein MyD88 but only partially on TLR2. Unlike canonical TLR2 signaling that is triggered in response to the detection of Mycoplasma infection, innate immune activation by components of Mycoplasma-infected cells was inhibited by chloroquine treatment and sensitive to protease treatment. We further show that in plasmacytoid dendritic cells, soluble factors from Mycoplasma hyorhinis-infected cells induce the production of large amounts of IFN-?. We conclude that Mycoplasma hyorhinis-infected cell lines release protein factors that can potently activate co-cultured innate immune cells via a previously unrecognized mechanism, thus limiting the validity of such co-culture experiments. PMID:26565413

  17. Mig-6 overcomes gefitinib resistance by inhibiting EGFR/ERK pathway in non-small cell lung cancer cell lines

    PubMed Central

    Li, Zi-Xuan; Qu, Lian-Yue; Wen, Hi; Zhong, Hong-Shan; Xu, Ke; Qiu, Xue-Shan; Wang, En-Hua

    2014-01-01

    Non small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and is the most common cause of lung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is widely used for patients with advanced NSCLC. However, drug resistance is a major obstacle. Mig-6 is a feedback inhibitor of EGFR and its down-stream pathway; it has been shown to play a role in gefitinib sensitivity. There is neither systematical research on the relationship between Mig-6 expression and gefitinib sensitivity, nor has the contribution of up-regulated Mig-6 on the gefitinib-resistant cell lines. In the present work, four NSCLC cell lines (H1299, A549, PC-9, and PC-9/AB11) with different sensitivities to gefitinib were subjected to analysis of the expression of Mig-6. We found that Mig-6 is over-expressed in gefitinib-sensitive NSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Further analysis revealed that over-expression of Mig-6 increased cell apoptosis and inhibited proliferation of gefitinib-resistant NSCLC cells treated with gefitinib, whereas lowering the expression of Mig-6 decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in gefitinib-sensitive NSCLC cell lines. These results suggest that Mig-6 is involved in mediating the response to gefitinib in NSCLC cell lines. Additionally we demonstrated that Mig-6 could reverse gefitinib resistance through inhibition of EGFR/ERK pathway in NSCLC cells. Our work uncovered that Mig-6 may be an effective therapeutic target in gefitinib-resistant lung cancer patients. PMID:25400829

  18. TCGA Benchmark 4: Evaluating SV and SNV Calls Using Cell Line Genomes - Adam Ewing, TCGA Scientific Symposium 2012

    Cancer.gov

    Home News and Events Multimedia Library Videos TCGA Benchmark 4: Evaluating SV and SNV Calls Using Cell Line Genomes - Adam Ewing TCGA Benchmark 4: Evaluating SV and SNV Calls Using Cell Line Genomes - Adam Ewing, TCGA Scientific Symposium 2012 You

  19. Weightlessness acts on human breast cancer cell line MCF-7

    NASA Astrophysics Data System (ADS)

    Vassy, J.; Portet, S.; Beil, M.; Millot, G.; Fauvel-Lafčve, F.; Gasset, G.; Schoevaert, D.

    2003-10-01

    Because cells are sensitive to mechanical forces, weightlessness might act on stress-dependent cell changes. Human breast cancer cells MCF-7, flown in space in a Photon capsule, were fixed after 1.5, 22 and 48 h in orbit. Cells subjected to weightlessness were compared to 1g in-flight and ground controls. Post-flight, fluorescent labeling was performed to visualize cell proliferation (Ki-67), three cytoskeleton components and chromatin structure. Confocal microscopy and image analysis were used to quantify cycling cells and mitosis, modifications of the cytokeratin network and chromatin structure. Several main phenomena were observed in weightlessness: The perinuclear cytokeratin network and chromatin structure were looser. More cells were cycling and mitosis was prolonged. Finally, cell proliferation was reduced as a consequence of a cell-cycle blockade. Microtubules were altered in many cells. The results reported in the first point are in agreement with basic predictions of cellular tensegrity. The prolongation of mitosis can be explained by an alteration of microtubules. We discuss here the different mechanisms involved in weightlessness alteration of microtubules: i) alteration of their self-organization by reaction-diffusion processes, and a mathematical model is proposed, ii) activation or desactivation of microtubules stabilizing proteins, acting on both microtubule and microfilament networks in cell cortex.

  20. Defining the Diversity of Phenotypic Respecification Using Multiple Cell Lines and Reprogramming Regimens

    PubMed Central

    Alicea, Bradly; Murthy, Shashanka; Keaton, Sarah A.; Cobbett, Peter; Cibelli, Jose B.

    2013-01-01

    To better understand the basis of variation in cellular reprogramming, we performed experiments with two primary objectives: first, to determine the degree of difference, if any, in reprogramming efficiency among cells lines of a similar type after accounting for technical variables, and second, to compare the efficiency of conversion of multiple similar cell lines to two separate reprogramming regimens–induced neurons and induced skeletal muscle. Using two reprogramming regimens, it could be determined whether converted cells are likely derived from a distinct subpopulation that is generally susceptible to reprogramming or are derived from cells with an independent capacity for respecification to a given phenotype. Our results indicated that when technical components of the reprogramming regimen were accounted for, reprogramming efficiency was reproducible within a given primary fibroblast line but varied dramatically between lines. The disparity in reprogramming efficiency between lines was of sufficient magnitude to account for some discrepancies in published results. We also found that the efficiency of conversion to one phenotype was not predictive of reprogramming to the alternate phenotype, suggesting that the capacity for reprogramming does not arise from a specific subpopulation with a generally “weak grip” on cellular identity. Our findings suggest that parallel testing of multiple cell lines from several sources may be needed to accurately assess the efficiency of direct reprogramming procedures, and that testing a larger number of fibroblast lines—even lines with similar origins—is likely the most direct means of improving reprogramming efficiency. PMID:23672680

  1. Carrier-mediated transport of valproic acid in BeWo cells, a human trophoblast cell line

    E-print Network

    Utoguchi, Naoki; Audus, Kenneth L.

    2000-01-01

    The biochemical mechanisms mediating the rapid distribution of valproic acid across placenta are not precisely known. We have characterized valproic acid transport by the human trophoblast using the human choriocarcinoma cell line, BeWo. The uptake...

  2. Characterization of Multiple Bistratified Retinal Ganglion Cells in A Purkinje Cell Protein 2-Cre Transgenic Mouse Line

    PubMed Central

    Ivanova, Elena; Lee, Patrick; Pan, Zhuo-Hua

    2012-01-01

    Retinal ganglion cells are categorized into multiple classes, including multiple types of bistratified ganglion cells (BGCs). The recent use of transgenic mouse lines with specific type(s) of ganglion cells that are labeled by fluorescent markers has facilitated the morphological and physiological studies of BGCs, particularly the directional-selective BGCs. The most important benefit from using transgenic animals is the capability to perform in vivo gene manipulation. In particular, the Cre/LoxP recombination system has become a powerful tool, allowing gene deletion, over-expression, and ectopic expression in a cell type-specific and temporally controlled fashion. The key to this tool is the availability of Cre mouse lines with cell or tissue type-specific expression of Cre recombinase. In this study, we characterized the Cre-positive retinal ganglion cells in a PCP2 (Purkinje cell protein 2)-cre mouse line. We found that all of the Cre-positive retinal ganglion cells were BGCs. Based on morphological criteria, we determined that they can be grouped into five types. The On- and Off-dendrites of three of these types stratified outside of the cholinergic bands and differed from directional selective ganglion cells (DSGCs) morphologically. These cells were negative for Brn-3b and positive for both calretinin and CART retina markers. The remaining two types were identified as putative On-Off and On-DSGCs. This Cre mouse line could be useful for further studies of the molecular and functional properties of BGCs in mice. PMID:23224947

  3. MA104 Cell line presents characteristics suitable for enterovirus A71 isolation and proliferation.

    PubMed

    Li, Zhi-Hui; Yue, Ying-Ying; Li, Peng; Song, Nan-Nan; Li, Bingqing; Zhang, Ying; Meng, Hong; Jiang, Guo-Sheng; Qin, Lizeng

    2015-08-01

    Enterovirus A71 (EV-A71), one of the most important causative agents of hand, foot and mouth disease (HFMD) in children, can lead to severe clinical outcomes, even death. However, the infection spectrum of EV-A71 in different cell lines remains unknown. Therefore, in this study, the biological characteristics of EV-A71 Subgroup C4 in different cell lines were investigated. To this end, the infectivity of EV-A71Jinan1002 isolated from children with severe HFMD was assessed in 18 different host cell lines. It was found that the MA104 cell line displayed biological characteristics suitable for EV-A71 Subgroup C4 strain isolation and proliferation; indeed, it was found that a broad spectrum of cell lines can be infected by EV-A71Jinan1002. Among the screened cells, four cell lines (HEK293, RD, MA104 and Marc145) produced high 50% tissue culture infective dose (TCID50 ) values calculated in viral proliferations (ranged from 10(7.6) to 10(7.8) ); the TCID50 being negatively associated with the time to appearance of CPE. Proliferation curves demonstrated that EV-A71Jinan1002 amplifies more efficiently in MA104, Hep-2 and RD cells. Remarkably, the virus isolation rate was much higher in MA104 cells than in RD cells. Thus this study, to our knowledge, is for the first to explore the infection spectrum of EV-A71 subgroup C4 in such a large number of different cell lines. Our data provide useful reference data for facilitating further study of EV-A71. PMID:26138857

  4. Comparative analysis of micronuclei and DNA damage induced by Ochratoxin A in two mammalian cell lines.

    PubMed

    Ali, Rahat; Mittelstaedt, Roberta A; Shaddock, Joseph G; Ding, Wei; Bhalli, Javed A; Khan, Qaiser M; Heflich, Robert H

    2011-07-14

    The fungal toxin, Ochratoxin A (OTA), is a common contaminant in human food and animal feed. The present study evaluated micronucleus (MN) induction by OTA in comparison with its ability to induce cytotoxicity and DNA damage in two mammalian cell lines, CHO-K1-BH(4) Chinese hamster ovary cells and TK6 human lymphoblastoid cells. Micronuclei were evaluated by flow cytometry, cytotoxicity was estimated by relative population doubling (RPD), while direct DNA damage and oxidative DNA damage were measured with the Comet assay, performed without and with digestion by formamidopyrimidine-DNA glycosylase (fpg). For the MN and cytotoxicity measurements, the cell lines were treated for 24h (CHO cells) or 27h (TK6 cells) with 5-25?M OTA in the absence of exogenous metabolic activation. The OTA treatments resulted in concentration-responsive increases in cytotoxicity, with higher concentrations of the agent being more cytotoxic in CHO cells than TK6 cells. 15?M OTA produced positive responses for MN induction and hypodiploid events (a measure of aneugenicity) in both cell lines; this concentration of OTA also produced cytotoxicity near to the recommended limit for the assay (45±5% RPD). A time course assay with TK6 cells indicated that at least 4h of OTA treatment were required to produce a positive MN response. For the Comet assay DNA damage assessments, the cell lines were treated with 5-50?M OTA for 4h. Direct DNA damage was detected in TK6 cells, but not CHO cells, while concentration-related increases in fpg-sensitive sites were detected for both cell lines. The consistent association of oxidative DNA damage with OTA exposure suggests its involvement in producing OTA-induced clastogenicity and aneugenicity; however, based on its detection in TK6 cells direct DNA damage could be involved in any human risk posed by OTA exposure. PMID:21554981

  5. A two-dimensional gel analysis of autologous T and B lymphoblastoid cell lines.

    PubMed

    Lester, E P; Lemkin, P; Lipkin, L

    1982-04-01

    We sought polypeptide markers of pathways of lymphoid differentiation by comparing the protein-synthesis patterns of a pair of autologous human lymphoblastoid cell lines: the B-cell line CCRF-SB and the T-cell line CCRF-HSB-2. Cells were cultured in standard medium for 24 or 120 h, then biosynthetically radiolabeled with [3H]leucine. Whole cell lysates were subjected to two-dimensional gel electrophoresis and patterns of protein synthesis were made visible by fluorography. Replicate cultures showed highly reproducible patterns, but differing culture durations produced alterations in the relative synthetic rates of some proteins. Protein synthetic patterns were analyzed visually and with the GELLAB system for computer analysis of two-dimensional gel images. Qualitative and quantitative differences were found between the T- and B-cell patterns. GELLAB detected and accurately paired most of the well-defined spots in each image and constructed a unified data base from data on our initial gels. Statistical analysis of this data revealed several marker proteins, the relative synthetic rates serving to distinguish the T-cell line from the B-cell line. Such computerized analysis of protein synthetic patterns of malignant cells appears likely ultimately to provide important diagnostic information regarding human malignancies. PMID:6978777

  6. MYC amplifications in myeloma cell lines: correlation with MYC-inhibitor efficacy

    PubMed Central

    Holien, Toril; Misund, Kristine; Olsen, Oddrun Elise; Baranowska, Katarzyna Anna; Buene, Glenn; Břrset, Magne; Waage, Anders; Sundan, Anders

    2015-01-01

    In multiple myeloma, elevated MYC expression is related to disease initiation and progression. We found that in myeloma cell lines, MYC gene amplifications were common and correlated with MYC mRNA and protein. In primary cell samples MYC mRNA levels were also relatively high; however gene copy number alterations were uncommon. Elevated levels of MYC in primary myeloma cells have been reported to arise from complex genetic aberrations and are more common than previously thought. Thus, elevated MYC expression is achieved differently in myeloma cell lines and primary cells. Sensitivity of myeloma cell lines to the MYC inhibitor 10058-F4 correlated with MYC expression, supporting that the activity of 10058-F4 was through specific inhibition of MYC. PMID:26087190

  7. Immune responses to an encapsulated allogeneic islet {beta}-cell line in diabetic NOD mice

    SciTech Connect

    Black, Sasha P. . E-mail: Sasha.Black@ca.crl.com; Constantinidis, Ioannis; Cui, Hong; Tucker-Burden, Carol; Weber, Collin J.; Safley, Susan A.

    2006-02-03

    Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic {beta}-cell line ({beta}TC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of {beta}TC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic {beta}-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is First extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes.

  8. Taxotere resistance in SUIT Taxotere resistance in pancreatic carcinoma cell line SUIT 2 and its sublines

    PubMed Central

    Liu, Bin; Staren, Edgar; Iwamura, Takeshi; Appert, Hubert; Howard, John

    2001-01-01

    AIM: To investigate the specific mechanisms of intrinsic and acquired resistance to taxotere (TXT) in pancreatic adenocarcinoma (PAC). METHODS: MTT assay was used to detect the sensitivity of PAC cell line SUIT-2 and its sublines (S-007, S-013, S-020, S-028 and TXT selected SUIT-2 cell line, S2/TXT) to TXT. Mdr1 (P-gp), multidrug resistance associated protein (MRP), lung resistance protein (LRP) and ?-tubulin isotype gene expressions were detected by RT-PCR. The functionality of P-gp and MRP was tested using their specific blocker verapamil (Ver) and indomethacin (IMC), respectively. The transporter activity of P-gp was also confirmed by Rhodamine 123 accumulation assay. RESULTS: S-020 and S2/TXT were found to be significantly resistant to TXT (19 and 9.5-fold to their parental cell line SUIT-2, respectively). RT-PCR demonstrated strong expression of Mdr1 in these two cell lines, but weaker expression or no expression in other cells lines. MRP and LRP expressions were found in most of these cell lines. The TXT-resistance in S2-020 and S2/TXT could be reversed almost completely by Ver, but not by IMC. Flow cytometry showed that Ver increased the accumulation of Rhodamine-123 in these two cell lines. Compared with S-020 and SUIT-2, the levels of ?-tubulin isotype II, III expressio ns in S-2/TXT were increased remarkably. CONCLUSION: The both intrinsic and acquired TXT-related drug resistance in these PAC cell lines is mainly mediated by P-gp, but had no relationship to MRP and LRP express ions. The increases of ?-tubulin isotype II, III might be collateral changes that occur when the SUIT-2 cells are treated with TXT. PMID:11854916

  9. Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

    PubMed Central

    Liévin-Le Moal, Vanessa

    2013-01-01

    SUMMARY Hosts are protected from attack by potentially harmful enteric microorganisms, viruses, and parasites by the polarized fully differentiated epithelial cells that make up the epithelium, providing a physical and functional barrier. Enterovirulent bacteria interact with the epithelial polarized cells lining the intestinal barrier, and some invade the cells. A better understanding of the cross talk between enterovirulent bacteria and the polarized intestinal cells has resulted in the identification of essential enterovirulent bacterial structures and virulence gene products playing pivotal roles in pathogenesis. Cultured animal cell lines and cultured human nonintestinal, undifferentiated epithelial cells have been extensively used for understanding the mechanisms by which some human enterovirulent bacteria induce intestinal disorders. Human colon carcinoma cell lines which are able to express in culture the functional and structural characteristics of mature enterocytes and goblet cells have been established, mimicking structurally and functionally an intestinal epithelial barrier. Moreover, Caco-2-derived M-like cells have been established, mimicking the bacterial capture property of M cells of Peyer's patches. This review intends to analyze the cellular and molecular mechanisms of pathogenesis of human enterovirulent bacteria observed in infected cultured human colon carcinoma enterocyte-like HT-29 subpopulations, enterocyte-like Caco-2 and clone cells, the colonic T84 cell line, HT-29 mucus-secreting cell subpopulations, and Caco-2-derived M-like cells, including cell association, cell entry, intracellular lifestyle, structural lesions at the brush border, functional lesions in enterocytes and goblet cells, functional and structural lesions at the junctional domain, and host cellular defense responses. PMID:24006470

  10. Dihydrochalcone Compounds Isolated from Crabapple Leaves Showed Anticancer Effects on Human Cancer Cell Lines.

    PubMed

    Qin, Xiaoxiao; Xing, Yun Feng; Zhou, Zhiqin; Yao, Yuncong

    2015-01-01

    Seven dihydrochalcone compounds were isolated from the leaves of Malus crabapples, cv. "Radiant", and their chemical structures were elucidated by UV, IR, ESI-MS, ąH-NMR and (13)C-NMR analyses. These compounds, which include trilobatin (A1), phloretin (A2), 3-hydroxyphloretin (A3), phloretin rutinoside (A4), phlorizin (A5), 6''-O-coumaroyl-4'-O-glucopyranosylphloretin (A6), and 3'''-methoxy-6''-O-feruloy-4'-O-glucopyranosyl-phloretin (A7), all belong to the phloretin class and its derivatives. Compounds A6 and A7 are two new rare dihydrochalcone compounds. The results of a MTT cancer cell growth inhibition assay demonstrated that phloretin and these derivatives showed significant positive anticancer activities against several human cancer cell lines, including the A549 human lung cancer cell line, Bel 7402 liver cancer cell line, HepG2 human ileocecal cancer cell line, and HT-29 human colon cancer cell line. A7 had significant effects on all cancer cell lines, suggesting potential applications for phloretin and its derivatives. Adding a methoxyl group to phloretin dramatically increases phloretin's anticancer activity. PMID:26633321

  11. Atypical nuclear localization of CD133 plasma membrane glycoprotein in rhabdomyosarcoma cell lines

    PubMed Central

    NUNUKOVA, ALENA; NERADIL, JAKUB; SKODA, JAN; JAROS, JOSEF; HAMPL, ALES; STERBA, JAROSLAV; VESELSKA, RENATA

    2015-01-01

    CD133 (also known as prominin-1) is a cell surface glycoprotein that is widely used for the identification of stem cells. Furthermore, its glycosylated epitope, AC133, has recently been discussed as a marker of cancer stem cells in various human malignancies. During our recent experiments on rhabdomyosarcomas (RMS), we unexpectedly identified an atypical nuclear localization of CD133 in a relatively stable subset of cells in five RMS cell lines established in our laboratory. To the best of our knowledge, this atypical localization of CD133 has not yet been proven or analyzed in detail in cancer cells. In the present study, we verified the nuclear localization of CD133 in RMS cells using three independent anti-CD133 antibodies, including both rabbit polyclonal and mouse monoclonal antibodies. Indirect immunofluorescence and confocal microscopy followed by software cross-section analysis, transmission electron microscopy and cell fractionation with immunoblotting were also employed, and all the results undeniably confirmed the presence of CD133 in the nuclei of stable minor subpopulations of all five RMS cell lines. The proportion of cells showing an exclusive nuclear localization of CD133 ranged from 3.4 to 7.5%, with only minor differences observed among the individual anti-CD133 antibodies. Although the role of CD133 in the cell nucleus remains unclear, these results clearly indicate that this atypical nuclear localization of CD133 in a minor subpopulation of cancer cells is a common phenomenon in RMS cell lines. PMID:25977066

  12. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-28

    ... Embryonic Stem Cell Line To Be Approved for Use in NIH Funded Research SUMMARY: In compliance with the... this publication. Proposed Collection: Request for Human Embryonic Stem Cell Line to be Approved for... form is used by applicants to request that human embryonic stem cell lines be approved for use in...

  13. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-29

    ... for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded Research SUMMARY: Under the... be requested in writing. Proposed Collection: Request for Human Embryonic Stem Cell Line to be... used by applicants to request that human embryonic stem cell lines be approved for use in...

  14. 78 FR 25091 - Submission for OMB Review; 30-Day Comment Request: Request for Human Embryonic Stem Cell Line To...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-29

    ...Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH-Funded...Collection: Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH-Funded...applicants to request that human embryonic stem cell lines be approved for use in...

  15. 78 FR 13688 - Proposed Collection; 60-Day Comment Request: Request for Human Embryonic Stem Cell Line To Be...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-28

    ...Request: Request for Human Embryonic Stem Cell Line To Be Approved for Use in NIH...Collection: Request for Human Embryonic Stem Cell Line to be Approved for Use in NIH...applicants to request that human embryonic stem cell lines be approved for use in...

  16. Kidney-specific enzyme expression by human kidney cell lines generated through oncogene transfection.

    PubMed

    Robinson, P S; Goochee, C F

    1991-07-01

    The human kidney cell line 293 was generated by transfection of adenovirus DNA into normal human embryonic kidney (HEK) cells (Graham et al., 1977), whereas the human kidney cell lines ST-1i and STt-4i were generated by transfection of HEK cells with plasmids encoding SV40 viral oncogenes (Abcouwer et al., 1989). In this study, we examined kidney-specific enzyme activity levels in 293, ST-1i, and STt-4i cells to determine their ability to exhibit kidney-specific gene expression. Enzymes examined were leucine aminopeptidase (LAP), gamma-glutamyl transpeptidase (gamma-GTP), and the disaccharidases trehalase and maltase. Enzymatic activity levels were compared to three other kidney cell lines (MDCK, OK, and LLC-PK1) as well as to normal human embryonic kidney (HEK) cells and the human hepatoma cell line, Hep G2. Modulation of kidney-specific enzyme activities was assessed in response to several differentiation-inducing agents (adenosine, n-butyric acid, hexamethylene bisacetamide (HMBA), dimethyl sulfoxide (DMSO), N,N'-dimethylformamide (DMF), isobutyl methyl xanthine (IBMX), di butyryl cAMP, and retinoic acid). ST-1i and STt-4i exhibit elevated levels of LAP, gamma-GTP, trehalase, and maltase, consistent with their kidney cell origin, whereas 293 cells exhibit elevated levels of just gamma-GTP and maltase. Maltase and gamma-GTP enzyme activities in ST-1i and STt-4i cells were very responsive to the various inducing agents; 293 cells were less responsive at the inducer concentrations examined. None of the three human cell lines formed domes under any of the experimental conditions. In summary, ST-1i and STt-4i are comparable to normal HEK cells in expression of kidney-specific enzymes and in responsiveness to differentiation-inducing agents, in spite of continued expression of SV40 oncogenes. PMID:1677645

  17. Differential expression of intermediate filament proteins distinguishes classic from variant small-cell lung cancer cell lines.

    PubMed

    Broers, J L; Carney, D N; de Ley, L; Vooijs, G P; Ramaekers, F C

    1985-07-01

    The expression of intermediate filament proteins in classic and variant-type small-cell lung carcinoma (SCLC) cell lines was studied using immunocytochemical techniques, two-dimensional gel electrophoresis and immunoblotting assays. Classic SCLC cell lines contain cytokeratin proteins but no neurofilaments. In contrast, variant cell lines do not contain detectable amounts of cytokeratins but partly express neurofilaments and vimentin. These results explain apparent discrepancies on the intermediate filament content of SCLC described in the recent literature. The application of antibodies to fresh biopsy specimens of SCLC may in the future allow the identification of the variant type of cells in clinical SCLC specimens and may have a major impact on therapeutic strategy and prognosis in these patients. PMID:2409557

  18. Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines

    SciTech Connect

    Jesudasan, R.A.; Srivatsan, E.S.; Rahman, R.A.; Chandrashekharappa, S.; Evans, G.A.

    1995-03-01

    Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular arrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene. 28 refs., 9 figs.

  19. Sensitivity of gastric adenocarcinoma and normal cell lines against combined or conjugated antimetabolites.

    PubMed

    Weinreich, Jürgen; Struller, Florian; Küper, Markus; Hack, Anita; Königsrainer, Alfred; Schott, Timm C

    2013-04-01

    The in-vitro growth inhibition of cancer and normal cell lines caused by mixed or covalently linked antimetabolites should clarify whether the conjugation of antimetabolites influences cell sensitivity and growth inhibition in a manner that differs from an equimolar mixture of the same antimetabolites or not. Growth inhibition of the human gastric adenocarcinoma cell lines 23132/87 and MKN-45 in comparison with normal gastric intestinal CCL-241 and the dermal fibroblast cell line NHDF was evaluated using CASY technology. The cell lines were incubated with an equimolar mixture of 5-fluoro-2'-deoxyuridine (5FdU)+3'-C-ethynylcytidine (ECyd) or the covalently linked duplex drug 5FdU(5'?5')ECyd. The drug and metabolites of the assays and medium were determined semiquantitatively using high-performance liquid chromatography. The sensitivity of cancer and nonmalignant cell lines was clearly different against the duplex drug. A measure of 0.65 µmol/l 5FdU(5'?5')ECyd, for example, reduced the growth of MKN-45 or 23132/87 gastric cancer cells from 100% on day 0 to about 50 or 20% on day 10, respectively. However, under the same conditions, the growth of the nonmalignant NHDF and CCL-241 cell lines was not markedly inhibited. The cytostatic activity of the duplex drug is based on the active metabolites in and outside the cell formed by the degradation of 5FdU(5'?5')ECyd. The sensitivity of cell lines against the duplex drug depended on its ability to metabolize the duplex drug. 5FdU(5'?5')ECyd should be more advantageous for specific and efficient polychemotherapy of gastric cancer than the corresponding equimolar mixture of 5FdU+ECyd or a standard combination regime of single drugs. PMID:23358120

  20. Cytotoxic activity of methanol extracts from Basidiomycete mushrooms on murine cancer cell lines.

    PubMed

    Tomasi, S; Lohézic-Le Dévéhat, F; Sauleau, P; Bézivin, C; Boustie, J

    2004-04-01

    Crude methanol extracts of 58 mushroom species were screened for their cytotoxic activities against two murine cancer cell lines, L1210 and 3LL, using the tetrazolium assay. A majority of extracts (74%) exhibited IC50 > 100 microg/ml against both cell lines. A most marked activity against one of the cell lines was noted for nine species (14% of the tested species). While Amanitales and Russulales tested were not found active, Polyporales and Boletales gave better results. Four species exhibited a significant cytotoxic activity (IC50 < or = 20 microg/ml) against at least one of the two murine cancer cell lines (Ganoderma lucidum, Meripilus giganteus, Suillus granulatus, S. luteus). The last one had never been investigated for its cytotoxic compounds before. PMID:15125575

  1. Virus-Free Human Placental Cell Lines To Study Genetic Functions

    Cancer.gov

    These immortalized virus-free human placental cell lines expressing major proteins of human trophoblasts provide efficient in vitro models to study placental functions, control of tissue-specific gene expression, and other studies.

  2. Monoclonal antibodies to human glycophorin A and cell lines for the production thereof

    DOEpatents

    Vanderlaan, Martin (San Ramon, CA); Bigbee, William L. (Livermore, CA); Jensen, Ronald H. (Livermore, CA); Fong, Stella S. N. (San Diego, CA); Langlois, Richard G. (Livermore, CA)

    1988-01-01

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A.sup.N and differentiate between the M and N forms of human glycophorin A.

  3. Gap junction intercellular communication: a microinjection investigation of fibroblast and epithelial cell lines 

    E-print Network

    Pahlka, Raymond Benton

    2002-01-01

    The objectives of this research were threefold. The first objective was to develop a protocol for unbiased microinjection of the fluorescent dye Lucifer Yellow to normal fibroblast and epithelial cell lines. I determined the optimal equipment...

  4. Monoclonal antibodies to human glycophorin a and cell lines for the production thereof

    SciTech Connect

    Vanderlaan, M.; Bigbee, W.L.; Jensen, R.H.; Fong, S.S.N.; Langlois, R.G.

    1988-06-21

    Cloned mouse hybridoma cell lines have been established which continuously produce antibodies that are highly specific to and exhibit high affinity for glycophorin A/sup N/ and differentiate between the M and N forms of human glycophorin A.

  5. Mouse fibroblast L929 cells are less permissive to infection by Nelson Bay orthoreovirus compared to other mammalian cell lines.

    PubMed

    Mok, Lawrence; Wynne, James W; Grimley, Samantha; Shiell, Brian; Green, Diane; Monaghan, Paul; Pallister, Jackie; Bacic, Antony; Michalski, Wojtek P

    2015-07-01

    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus. PMID:25748429

  6. Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines.

    PubMed

    Hillger, Julia M; Schoop, Jeffison; Boomsma, Dorret I; Slagboom, P Eline; IJzerman, Adriaan P; Heitman, Laura H

    2015-12-15

    Deciphering how genetic variation in drug targets such as G protein-coupled receptors (GPCRs) affects drug response is essential for precision medicine. GPCR signaling is traditionally investigated in artificial cell lines which do not provide sufficient physiological context. Patient-derived cell lines such as lymphoblastoid cell lines (LCLs) could represent the ideal cellular model system. Here we describe a novel label-free, whole-cell biosensor method for characterizing GPCR-mediated drug responses in LCLs. Generally, such biosensor technology is deemed only compatible with adherent cell lines. We optimized and applied the methodology to study cellular adhesion properties as well as GPCR drug responses in LCLs, which are suspension cells. Coating the detector surface with the extracellular matrix protein fibronectin resulted in cell adherence and allowed detection of cellular responses. A prototypical GPCR present on these cells, i.e. the cannabinoid receptor 2 (CB2), was selected for pharmacological characterization. Receptor activation with the agonist JWH133, blockade by antagonist AM630 as well as downstream signaling inhibition by PTX could be monitored sensitively and receptor-specifically. Potencies and effects were comparable between LCLs of two genetically unrelated individuals, providing the proof-of-principle that this biosensor technology can be applied to LCLs, despite their suspension cell nature, in order to serve as an in vitro model system for the evaluation of individual genetic influences on GPCR-mediated drug responses. PMID:26143464

  7. Reprogramming somatic cells into iPS cells activates LINE-1 retroelement mobility

    PubMed Central

    Wissing, Silke; Muńoz-Lopez, Martin; Macia, Angela; Yang, Zhiyuan; Montano, Mauricio; Collins, William; Garcia-Perez, Jose Luis; Moran, John V.; Greene, Warner C.

    2012-01-01

    Long interspersed element-1 (LINE-1 or L1) retrotransposons account for nearly 17% of human genomic DNA and represent a major evolutionary force that has reshaped the structure and function of the human genome. However, questions remain concerning both the frequency and the developmental timing of L1 retrotransposition in vivo and whether the mobility of these retroelements commonly results in insertional and post-insertional mechanisms of genomic injury. Cells exhibiting high rates of L1 retrotransposition might be especially at risk for such injury. We assessed L1 mRNA expression and L1 retrotransposition in two biologically relevant cell types, human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), as well as in control parental human dermal fibroblasts (HDFs). Full-length L1 mRNA and the L1 open reading frame 1-encoded protein (ORF1p) were readily detected in hESCs and iPSCs, but not in HDFs. Sequencing analysis proved the expression of human-specific L1 element mRNAs in iPSCs. Bisulfite sequencing revealed that the increased L1 expression observed in iPSCs correlates with an overall decrease in CpG methylation in the L1 promoter region. Finally, retrotransposition of an engineered human L1 element was ?10-fold more efficient in iPSCs than in parental HDFs. These findings indicate that somatic cell reprogramming is associated with marked increases in L1 expression and perhaps increases in endogenous L1 retrotransposition, which could potentially impact the genomic integrity of the resultant iPSCs. PMID:21989055

  8. Effects of retinol on proliferation, cell adherence and extracellular matrix synthesis in a liver myofibroblast or lipocyte cell line (GRX).

    PubMed Central

    Margis, R.; Pinheiro-Margis, M.; da Silva, L. C.; Borojevic, R.

    1992-01-01

    We have studied the effect of retinol on an established murine cell line (GRX), representative of liver connective tissue cells. This cell line has myofibroblast characteristics; under retinol treatment it is induced into the lipocyte (Ito-cell) phenotype. Retinol decreased the proliferation rate in the entire cell population. It increased cell adherence to the substrate, which was correlated with the increased secretion of fibronectin. Collagen secretion was specifically decreased, whilst the total protein secretion remained stable. Heparan sulphate was decreased in the pericellular compartment, but other glycosaminoglycans were not affected by retinol treatment. Modulations of pericellular components induced by retinol may alter the relations among liver mesenchymal cells, and may be related to vitamin-A-induced modifications of the homoeostasis of hepatic connective tissue and hepatic fibrosis. Images Fig. 6 PMID:1571273

  9. Role of transient processes in resonance line spectroscopy of caesium atoms in cells with antirelaxation coating

    SciTech Connect

    Sevost'yanov, D I; Yakovlev, V P; Kozlov, A N; Vasil'ev, V V; Zibrov, S A; Velichansky, Vladimir L

    2013-07-31

    We study the peculiarities of the absorption spectra in D{sub 1,2}-lines of Cs, caused by optical pumping in cells with antirelaxation coating. In these cells the internal state of the atom, which arose under optical pumping by a monochromatic laser field, is preserved with a high probability in a collision with the wall. As a result, the optical pumping action extends to the entire volume of the cell and to all the velocities of the atoms. This leads to the speed-dependent scanning distortions of the absorption line profile. The detected features should be considered when using laserpumped quantum magnetometers with antirelaxation-coated cells. (laser spectroscopy)

  10. The RPTEC/TERT1 Cell Line as an Improved Tool for In Vitro Nephrotoxicity Assessments.

    PubMed

    Simon-Friedt, Bridget R; Wilson, Mark J; Blake, Diane A; Yu, Haini; Eriksson, Yasmin; Wickliffe, Jeffrey K

    2015-07-01

    In earlier studies, we have characterized a newly developed cell line derived from the renal proximal tubule epithelial cells (RPTEC) of a healthy human male donor in order to provide an improved in vitro model with which to investigate human diseases, such as cancer, that may be promoted by toxicant exposure. The RPTEC/TERT1 cell line has been immortalized using the human telomerase reverse transcriptase (hTERT) catalytic subunit and does not exhibit chromosomal abnormalities (Evercyte Laboratories). We have previously conducted single-compound and binary mixture experiments with the common environmental carcinogens, cadmium (Cd), and benzo[a]pyrene (B[a]P). Cells exhibited cytotoxic and compound-specific responses to low concentrations of B[a]P and Cd. We detected responses after exposure consistent with what is known regarding these cells in a normal, healthy kidney including significant gene expression changes, BPDE-DNA adducts in the presence of B[a]P, and indications of oxidative stress in the presence of Cd. The RPTEC/TERT1 cell line was also amenable to co-exposure studies due to its sensitivity and compound-specific properties. Here, we review our earlier work, compare our findings with commonly used renal cell lines, and suggest directions for future experiments. We conclude that the RPTEC/TERT1 cell line can provide a useful tool for future toxicological and mixture studies. PMID:25893367

  11. Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

    PubMed Central

    Lai, Tingfeng; Yang, Yuansheng; Ng, Say Kong

    2013-01-01

    From 2006 to 2011, an average of 15 novel recombinant protein therapeutics have been approved by US Food and Drug Administration (FDA) annually. In addition, the expiration of blockbuster biologics has also spurred the emergence of biosimilars. The increasing numbers of innovator biologic products and biosimilars have thus fuelled the demand of production cell lines with high productivity. Currently, mammalian cell line development technologies used by most biopharmaceutical companies are based on either the methotrexate (MTX) amplification technology or the glutamine synthetase (GS) system. With both systems, the cell clones obtained are highly heterogeneous, as a result of random genome integration by the gene of interest and the gene amplification process. Consequently, large numbers of cell clones have to be screened to identify rare stable high producer cell clones. As such, the cell line development process typically requires 6 to 12 months and is a time, capital and labour intensive process. This article reviews established advances in protein expression and clone screening which are the core technologies in mammalian cell line development. Advancements in these component technologies are vital to improve the speed and efficiency of generating robust and highly productive cell line for large scale production of protein therapeutics. PMID:24276168

  12. FT-infrared spectroscopic studies of lymphoma, lymphoid, and myeloid leukemia cell lines

    NASA Astrophysics Data System (ADS)

    Babrah, Jaspreet; McCarthy, Keith P.; Lush, Richard; Rye, Adam D.; Bessant, Conrad; Stone, Nicholas

    2007-07-01

    This paper presents a novel method to characterise spectral differences that distinguish leukaemia and lymphoma cell lines. This is based on objective spectral measurements of major cellular biochemical constituents and multivariate spectral processing. Fourier transform infrared (FT-IR) maps of the lymphoma, lymphoid and myeloid leukaemia cell samples were obtained using a Perkin-Elmer Spotlight 300 FT-IR imaging spectrometer. Multivariate statistical techniques incorporating principal component analysis (PCA) and linear discriminant analysis (LDA) were used to construct a mathematical model. This model was validated for reproducibility. Multivariate statistical analysis of FTIR spectra collected for each cell sample permit a combination of unsupervised and supervised methods of distinguishing cell line types. This resulted in the clustering of cell line populations, indicating distinct bio-molecular differences. Major spectral differences were observed in the 4000 to 800 cm -1 spectral region. Bands in the averaged spectra for the cell line were assigned to the major biochemical constituents including; proteins, fatty acids, carbohydrates and nucleic acids. The combination of FT-IR spectroscopy and multivariate statistical analysis provides an important insight into the fundamental spectral differences between the cell lines, which differ according to the cellular biochemical composition. These spectral differences can serve as potential biomarkers for the differentiation of leukaemia and lymphoma cells. Consequently these differences could be used as the basis for developing a spectral method for the detection and identification of haematological malignancies.

  13. New monoterpene glycosides from sunflower seeds and their protective effects against H2O2-induced myocardial cell injury.

    PubMed

    Fei, Yonghe; Zhao, Jianping; Liu, Yanli; Li, Xiaoran; Xu, Qiongming; Wang, Taoyun; Khan, Ikhlas A; Yang, Shilin

    2015-11-15

    Three new monoterpene glycosides (1-3) and eleven known compounds (4-14) were isolated from seeds of Helianthus annuus L. (sunflower). Their structures were determined by spectroscopic and chemical methods. All the compounds were isolated from sunflower seeds for the first time. Protective effects of compounds 1-14 against H2O2-induced H9c2 cardiomyocyte injury were evaluated, and compounds 1 and 2 showed some cell-protective effects. No significant DPPH radical scavenging activity was observed for compounds 1-14. PMID:25977041

  14. In vitro cytotoxicity of Indonesian stingless bee products against human cancer cell lines

    PubMed Central

    Kustiawan, Paula M.; Puthong, Songchan; Arung, Enos T.; Chanchao, Chanpen

    2014-01-01

    Objective To screen crude extracts of propolis, bee pollen and honey from four stingless bee species [Trigona incisa (T. incisa)], Timia apicalis, Trigona fusco-balteata and Trigona fuscibasis) native to East Kalimantan, Indonesia for cytotoxic activity against five human cancer cell lines (HepG2, SW620, ChaGo-I, KATO-III and BT474). Methods All samples were extracted with methanol, and then subpartitioned with n-hexane and ethyl acetate. Each crude extract was screened at 20 µg/mL for in vitro cytotoxicity against the cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, four previously shown bioactive components from propolis (apigenin, caffeic acid phenyl ester, kaempferol and naringenin) and two chemotherapeutic drugs (doxorubicin and 5-fluorouracil) were used to evaluate the sensitivity of the cell lines. Results Overall, crude extracts from propolis and honey had higher cytotoxic activities than bee pollen, but the activity was dependent upon the extraction solvent, bee species and cell line. Propolis extracts from T. incisa and Timia apicalis showed the highest and lowest cytotoxic activity, respectively. Only the HepG2 cell line was broadly sensitive to the honey extracts. For pure compounds, doxorubicin was the most cytotoxic, the four propolis compounds the least, but the ChaGo-I cell line was sensitive to kaempferol at 10 µg/mL and KATO-III was sensitive to kaempferol and apigenin at 10 µg/mL. All pure compounds were effective against the BT474 cell line. Conclusions Propolis from T. incisa and Trigona fusco-balteata contain an in vitro cytotoxic activity against human cancer cell lines. Further study is required, including the isolation and characterization of the active antiproliferative agent(s). PMID:25183275

  15. Epigenetic reprogramming and re-differentiation of a Ewing sarcoma cell line

    PubMed Central

    Moore, Joseph B.; Loeb, David M.; Hong, Kyung U.; Sorensen, Poul H.; Triche, Timothy J.; Lee, David W.; Barbato, Michael I.; Arceci, Robert J.

    2015-01-01

    Developmental reprogramming techniques have been used to generate induced pluripotent stem (iPS) cells from both normal and malignant cells. The derivation of iPS cells from cancer has the potential to provide a unique scientific tool to overcome challenges associated with the establishment of cell lines from primary patient samples and a readily expandable source of cells that may be used to model the initial disease. In the current study we developmentally reprogrammed a metastatic Ewing sarcoma (EWS) cell line to a meta-stable embryonic stem (ES)-like state sharing molecular and phenotypic features with previously established ES and iPS cell lines. EWS-iPS cells exhibited a pronounced drug resistant phenotype despite persistent expression of the oncogenic EWS-FLI1 fusion transcript. This included resistance to compounds that specifically target downstream effector pathways of EWS-FLI1, such as MAPK/ERK and PI3K/AKT, which play an important role in EWS pathogenesis. EWS-iPS cells displayed tumor initiation abilities in vivo and formed tumors exhibiting characteristic Ewing histopathology. In parallel, EWS-iPS cells re-differentiated in vitro recovered sensitivity to molecularly targeted chemotherapeutic agents, which reiterated pathophysiological features of the cells from which they were derived. These data suggest that EWS-iPS cells may provide an expandable disease model that could be used to investigate processes modulating oncogenesis, metastasis, and chemotherapeutic resistance in EWS. PMID:25806369

  16. Proteomic analysis of human glioblastoma cell lines differently resistant to a nitric oxide releasing agent.

    PubMed

    Leone, Roberta; Giussani, Paola; De Palma, Sara; Fania, Chiara; Capitanio, Daniele; Vasso, Michele; Brioschi, Loredana; Riboni, Laura; Viani, Paola; Gelfi, Cecilia

    2015-06-01

    Glioblastoma multiforme is the most aggressive astrocytoma characterized by the development of resistant cells to various cytotoxic stimuli. Nitric oxide (NO) is able to overcome tumor resistance in PTEN mutated rat C6 glioma cells due to its ability to inhibit cell growth by influencing the intracellular distribution of ceramide. The aim of this study is to monitor the effects of NO donor PAPANONOate on ceramide trafficking in human glioma cell lines, CCF-STTG1 (PTEN-mutated, p53-wt) and T98G (PTEN-harboring, p53-mutated), together with the assessment of their differential molecular signature by 2D-DIGE and MALDI mass spectrometry. In the CCF-STTG1 cell line, the results indicate that treatment with PAPANONOate decreased cell proliferation (<50%) and intracellular trafficking of ceramide, assessed by BODIPY-C5Cer, while these events were not observed in the T98G cell line. Proteomic results suggest that CCF-STTG1 cells are characterized by an increased expression of proteins involved in NO-associated ER stress (i.e. protein disulfide-isomerase A3, calreticulin, 78 kDa glucose-regulated protein), which could compromise ceramide delivery from ER to Golgi, leading to ceramide accumulation in ER and partial growth arrest. Conversely, T98G cell lines, resistant to NO exposure, are characterized by increased levels of cytosolic antioxidant proteins (i.e. glutathione-S-transferase P, peroxiredoxin 1), which might buffer intracellular NO. By providing differential ceramide distribution after NO exposure and differential protein expression of two high grade glioma cell lines, this study highlights specific proteins as possible markers for tumor aggressiveness. This study demonstrates that, in two different high grade glioma cell lines, NO exposure results in a different ceramide distribution and protein expression. Furthermore, this study highlights specific proteins as possible markers for tumor aggressiveness. PMID:25797839

  17. Dynamic gene expression by putative hair-cell progenitors during regeneration in the zebrafish lateral line

    PubMed Central

    Kim, Taeryn; Cabot, Victoria; Hudspeth, A. J.

    2014-01-01

    Hearing loss is most commonly caused by the destruction of mechanosensory hair cells in the ear. This condition is usually permanent: Despite the presence of putative hair-cell progenitors in the cochlea, hair cells are not naturally replenished in adult mammals. Unlike those of the mammalian ear, the progenitor cells of nonmammalian vertebrates can regenerate hair cells throughout life. The basis of this difference remains largely unexplored but may lie in molecular dissimilarities that affect how progenitors respond to hair-cell death. To approach this issue, we analyzed gene expression in hair-cell progenitors of the lateral-line system. We developed a transgenic line of zebrafish that expresses a red fluorescent protein in the presumptive hair-cell progenitors known as mantle cells. Fluorescence-activated cell sorting from the skins of transgenic larvae, followed by microarray-based expression analysis, revealed a constellation of transcripts that are specifically enriched in these cells. Gene expression analysis after hair-cell ablation uncovered a cohort of genes that are differentially regulated early in regeneration, suggesting possible roles in the response of progenitors to hair-cell death. These results provide a resource for studying hair-cell regeneration and the biology of sensory progenitor cells. PMID:24706895

  18. A feeder-cell independent subpopulation of the PICM-19 pig liver stem cell line capable of long-term growth and extensive expansion

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A method for the feeder-independent culture of PICM-19 pig liver stem cell line was recently devised, but the cell line’s growth was finite and the cells essentially ceased dividing after approximately 20 passages over a one year culture period. Here we report the isolation, continuous culture, and...

  19. Production of macrophage activating factor by human leukemic T cell lines.

    PubMed

    Imai, K; Suzuki, Y; Harada, T; Morikawa, S; Tanaka, A

    1985-01-01

    Human leukemic T cell lines were tested for their ability to produce a macrophage activating factor. When mouse peritoneal macrophages were cultured for 48 hr in the presence of culture supernatants from cell lines HPB-ALL, CCRF-CEM, or MOLT-4, glucose oxidation via the hexose monophosphate pathway was enhanced by five to seven fold. Culture supernatants from cell line HPB-MLT stimulated the oxidation to a lesser extent. However, cell line CCRF-HSB-2 was essentially inactive as a producer. The active supernatants also stimulated the release of hydrogen peroxide from macrophages, whereas the inactive one did not. Since treatment of the cell lines with 12-o-tetradecanoyl phorbol acetate or phytohemagglutinin had little effect on the production of the factor except HPB-ALL, the cell lines seemed to secrete the factor constitutively. The stimulatory effect was dose-dependent and evident at a concentration as low as a 1/80 dilution. The factor was resistant to heat treatment at 100 C for 20 min, nondialysable and sensitive to protease digestion. The activating factor could be partially purified by anion exchange and gel filtration chromatographies. PMID:3906371

  20. Isolation and maintenance of Rickettsia raoultii in a Rhipicephalus sanguineus tick cell line.

    PubMed

    Santibáńez, Sonia; Portillo, Aránzazu; Palomar, Ana M; Bell-Sakyi, Lesley; Romero, Lourdes; Oteo, José A

    2015-01-01

    Rickettsia raoultii, a member of the spotted fever group rickettsiae, has been implicated in cases of DEBONEL/TIBOLA/SENLAT, and has been detected in Dermacentor spp. and Rhipicephalus pumilio ticks by PCR. R. raoultii has been isolated in mammalian and tick cell lines. This study aimed to isolate R. raoultii from Spanish Dermacentor marginatus in tick cell lines. A single adult D. marginatus collected from vegetation in La Rioja (Northen Spain) in October 2012 was surface-sterilised, triturated and aliquots of the homogenate were inoculated into a panel of tick cell lines derived from embryonic Rhipicephalus sanguineus, Rhipicephalus evertsi and Ixodes ricinus. Cultures were maintained at 28 °C with weekly medium changes and checked by Gimenez stain for Rickettsia-like intracellular organisms. After 50 days of incubation, intracellular Rickettsia-like organisms were observed in the R. sanguineus cell line RML-RSE using Gimenez stain. PCR assays and sequencing of fragments of 16S RNA, ompB and ompA genes in DNA extracted from the culture suspension showed 100% identity with R. raoultii. Growth of intracellular microorganisms was not observed in preparations of the other tick cell lines. In conclusion, the tick cell line RML-RSE is a useful system for the isolation and maintenance of R. raoultii. PMID:26428859

  1. Blood group ABO-related glycosylation of urothelial cell lines: immunocytological, enzymatic, and genetic characterization.

    PubMed

    Meldgaard, P; Holmes, E H; Bennett, E P; Clausen, H; Zeuthen, J; Wolf, H; Orntoft, T F

    1994-05-01

    Three immortalized, human urothelial cell lines were characterized with respect to their ABO-related carbohydrate phenotypes using a panel of monoclonal antibodies directed to a series of carbohydrate epitopes (Lac, sialylated Lac, Le(a), sialylated Le(a), Le(x), sialylated Le(x), H types I and II, Ley, Leb, A monofucosylated types I and II, ALey, Aleb, and A type III). The glycosyltransferases forming some of these epitopes (beta 1-3/4 galactosyltransferase, alpha 1-2 fucosyltransferase, alpha 1-3 galactosyltransferase, and alpha 1-3-N-acetyl-galactosaminyltransferase) were determined by enzyme assays. The ABO gene complex was analyzed by Southern blotting, Northern blotting, and polymerase chain reaction across the O deletion and across base differences between the A and B alleles. The immunocytochemical stainings showed marked differences between the three cell lines; the high grade (tumorigenic, metastatic) cell line showed difucosylated types I and II structures, and the low grade (nontumorigenic, nonmetastatic) cell lines showed monofucosylated types I and II structures. Polymerase chain reaction genotyping of the cell lines indicated that one was OO, one was AA, and one was A plus a mutated allele. Northern blotting showed RNA encoding the A transferase. However, even though both of the A cell lines seemed to have an intact gene, which could produce A transferase and transcribed RNA, none of them showed any activity of the A gene encoded enzyme or any A-structures at the cell surface. In contrast, the three other examined glycosyltransferases were active. The three urothelial cell lines reflect in vivo findings in humans. They represent a competent system for in vitro studies of the different carbohydrate transferase genes responsible for the carbohydrate structures expressed on the cell surface in bladder tumors. PMID:8162594

  2. Establishment of Hertwig's epithelial root sheath/epithelial rests of Malassez cell line from human periodontium.

    PubMed

    Nam, Hyun; Kim, Ji-Hye; Kim, Jae-Won; Seo, Byoung-Moo; Park, Joo-Cheol; Kim, Jung-Wook; Lee, Gene

    2014-07-01

    Human Hertwig's epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cells are epithelial remnants of teeth residing in the periodontium. Although the functional roles of HERS/ERM cells have yet to be elucidated, they are a unique epithelial cell population in adult teeth and are reported to have stem cell characteristics. Therefore, HERS/ERM cells might play a role as an epithelial component for the repair or regeneration of dental hard tissues; however, they are very rare population in periodontium and the primary isolation of them is considered to be difficult. To overcome these problems, we immortalized primary HERS/ERM cells isolated from human periodontium using SV40 large T antigen (SV40 LT) and performed a characterization of the immortalized cell line. Primary HERS/ERM cells could not be maintained for more than 6 passages; however, immortalized HERS/ERM cells were maintained for more than 20 passages. There were no differences in the morphological and immunophenotypic characteristics of HERS/ERM cells and immortalized HERS/ERM cells. The expression of epithelial stem cell and embryonic stem cell markers was maintained in immortalized HERS/ERM cells. Moreover, immortalized HERS/ERM cells could acquire mesenchymal phenotypes through the epithelial-mesenchymal transition via TGF-?1. In conclusion, we established an immortalized human HERS/ERM cell line with SV40 LT and expect this cell line to contribute to the understanding of the functional roles of HERS/ERM cells and the tissue engineering of teeth. PMID:25081036

  3. Chondrocytic differentiation of peripheral neuroectodermal tumor cell line in nude mouse xenograft.

    PubMed

    Goji, J; Sano, K; Nakamura, H; Ito, H

    1992-08-01

    We have established a cell line (KU-SN) from a peripheral neuroectodermal tumor originating in the left scapula of a 4-year-old girl. The original tumor was immunoreactive with antibodies for neurofilament proteins, neuron-specific enolase, vimentin, S100 protein, and beta 2-microglobulin. Dense core granules, 50-150 nm in diameter, were identified by electron microscopy. The cell line was established from tumor cells in metastatic lung fluid. KU-SN cells were immunoreactive with the antibodies for neurofilament proteins, vimentin, neuron-specific enolase, S100 protein, glial fibrillary acidic protein, cytokeratin, and carcinoembryonic antigen. Besides these neuronal features, KU-SN cells express type 2 collagen and insulin-like growth factor 1 receptor. The addition of insulin-like growth factor 1 (100 ng/ml) increased the growth rate of KU-SN cells 2.1-fold over control. Some cells were positive for Alcian blue and alkaline phosphatase staining. Cytogenetic analysis of KU-SN cells disclosed a reciprocal chromosomal translocation [t(11,22)]. Northern blot analysis of KU-SN cells demonstrated amplified expression of the c-myc gene but not the N-myc gene. When tumor cells were transplanted into nude mice, cartilage was formed. The cartilage was immunoreactive with the antibody for HLA-ABC, indicating that it was derived from the tumor cells, not from mouse tissue. Chondrocytic differentiation was not observed in xenografts of Ewing's sarcoma cell lines SK-ES or RD-ES or the peripheral neuroectodermal tumor cell line SK-N-MC. These results indicate that KU-SN cells represent primitive neural crest cells having the potential for chondrocytic differentiation. PMID:1379122

  4. Glycolaldehyde induces apoptosis in a human breast cancer cell line.

    PubMed

    Al-Maghrebi, May A; Al-Mulla, Fahd; Benov, Ludmil T

    2003-09-01

    Activated phagocytes employ myeloperoxidase to generate glycolaldehyde, 2-hydroxypropanal, and acrolein. Because alpha-hydroxy and alpha,beta-unsaturated aldehydes are highly reactive, phagocyte-mediated formation of these products may play a role in killing bacteria and tumor cells. Using breast cancer cells, we demonstrate that glycolaldehyde inactivates glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and Cu,Zn superoxide dismutase, suppresses cell growth, and induces apoptosis. These results suggest that glycolaldehyde might be an important mediator of neutrophil anti-tumor activity. PMID:12921788

  5. Induction of apoptosis and cell cycle arrest by imexon in human pancreatic cancer cell lines.

    PubMed

    Dorr, Robert T; Raymond, Mary Ann; Landowski, Terry H; Roman, Nicholas O; Fukushima, Shoji

    2005-01-01

    Imexon is an aziridine-containing small molecule currently in Phase I clinical trials. This agent has been shown to bind to thiols and increase intracellular oxidants, inducing apoptosis in hematologic cancer cells. Pancreatic cancers are known to be sensitive to oxidation, suggesting this disease may be an appropriate target for this agent. The current report examines the activity of imexon in pancreatic cells. Imexon induced concentration-dependent and time-dependent apoptosis in a panel of six human pancreatic carcinoma cell (PCC) lines. The mean IC50 (SD) for growth inhibition by the SRB assay was 200 (101) microM for a 48 h exposure with a range of 64-358 microM. Cell killing was schedule-dependent, favoring exposure times > or =48 h. Imexon-treated MiaPaCa-2 cells underwent non-lethal growth arrest following exposure to concentrations < or =200 microM for 48 h. When concentrations were increased to 300 microM for > or =48 h, the MiaPaCa-2 cells arrested in G2 phase and activated caspases 3, 8, and 9 were detected. After a 72 h exposure to the IC80 concentration of imexon, cells exhibited a loss of mitochondrial membrane potential detected by CMXRos staining. However, there was no loss of reduced cellular thiols unless very high concentrations of > or =400 microM were used. In contrast, reactive oxygen species (ROS) were elevated in a dose-dependent fashion, starting at very low imexon concentrations. Imexon also significantly inhibited MiaPaCa-2 tumor growth in SCID mice at 100 mg/kg/d for 9 d. The tumor growth inhibition (% T/C) was 27% of control, and the tumor growth delay was 21 d, indicating an active agent by NCI standards. The levels of imexon that are cytotoxic in human PCC's are achievable based on the preliminary results of the ongoing Phase I trial. Imexon appears to be active against PCCs in vitro and has an entirely novel mechanism of action involving G2 arrest, accumulation of ROS, and the induction of apoptosis. PMID:16227632

  6. Generation and Characterisation of Novel Pancreatic Adenocarcinoma Xenograft Models and Corresponding Primary Cell Lines

    PubMed Central

    Wennerström, Anna B.; Lothe, Inger Marie Bowitz; Sandhu, Vandana; Kure, Elin H.; Myklebost, Ola; Munthe, Else

    2014-01-01

    Pancreatic adenocarcinoma is one of the most lethal cancer types, currently lacking efficient treatment. The heterogeneous nature of these tumours are poorly represented by the classical pancreatic cell lines, which have been through strong clonal selection in vitro, and are often derived from metastases. Here, we describe the establishment of novel pancreatic adenocarcinoma models, xenografts and corresponding in vitro cell lines, from primary pancreatic tumours. The morphology, differentiation grade and gene expression pattern of the xenografts resemble the orig