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Sample records for h9c2 cell line

  1. Protective effects of clovamide against H2O2-induced stress in rat cardiomyoblasts H9c2 cell line.

    PubMed

    Zamperone, Andrea; Pietronave, Stefano; Colangelo, Donato; Antonini, Silvia; Locatelli, Monica; Travaglia, Fabiano; Coïsson, Jean Daniel; Arlorio, Marco; Prat, Maria

    2014-10-01

    Cocoa contains phenolic compounds with known antioxidant and antiradical properties beneficial in different pathologies, including cardiovascular diseases. Herein, we have evaluated the protective effects of clovamide, a minor cocoa component, against oxidative stress induced in the rat cardiomyoblast cell line, also comparing it to its bio-isosteric form, rosmarinic acid, and to the main monomeric flavan-3-ol from low-molecular-weight polyphenol in cocoa, i.e. epicatechin. At nano-micro-molar concentrations, the three compounds inhibited the production of reactive oxygen species and apoptosis, evaluated under different aspects, namely, annexin V positivity, DNA fragmentation, caspase release and activation. These molecules can, thus, be considered for their bioactive beneficial activity in the context of cardiovascular pathologies and, particularly, in the protection towards oxidative stress that follows ischemic injury. Clovamide may, thus, be the primary compound for the development of innovative nutraceutical strategies towards cardiovascular diseases. PMID:25133994

  2. Mir-351-5p contributes to the establishment of a pro-inflammatory environment in the H9c2 cell line by repressing PTEN expression.

    PubMed

    da Silva, Walmir; dos Santos, Robson Augusto Souza; Moraes, Karen C M

    2016-01-01

    The activated renin-angiotensin-aldosterone system modulates several metabolic pathways that contribute to left ventricular hypertrophy and heart failure. In this metabolic system, angiotensin II modulates heart morphophysiological changes triggered by a series of inflammatory and pro-inflammatory responses; however, the fine tuning associated with the control of this biochemical pathway remains unknown. Here, we investigated elements involved in the post-transcriptional regulation of the pro-inflammatory environment in the H9c2 cardiac cell line, focusing on miRNA elements that modulate PTEN expression. A cellular model of investigation was established and the miR-315-5p was identified as a novel element targeting PTEN in this cardiac cell line, thereby controlling the protein level. This interconnected pathway contributes to the control of the pro-inflammatory environment in Ang II-treated cells. PMID:26541756

  3. 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.

  4. Safflower extract inhibiting apoptosis by inducing autophagy in myocardium derived H9C2 cell.

    PubMed

    Jia, Zhisheng; Liu, Yancai; Su, Huailing; Li, Ming; Zhang, Min; Zhu, Ye; Li, Tenjiao; Fang, Youbo; Liang, Shimin

    2015-01-01

    The Heart failure (HF) is considered as the end-stage of various heart disease and associated with high mortality globally. Progressive loss of cardiac myocytes via apoptosis is considered as the most important factor for HF pathology. In this study, we demonstrated that Safflower extract was able to inhibitthe apoptosis inducted by Angiotensin II (AngII) in a ratmyocardium derived cell line H9C2. Further examination of LC-3II conversion and autophagosome formation suggested Safflower extract induced autophagy in treated cell. Inhibition of Safflower extract induced autophagy by 3-methyladenine (3MA) abolished anti-apoptotic function of Safflower extract, while application of autophagy stimulator Rapamycin in H9C2 inhibited apoptosis as well. Moreover, treatment of H9C2 cell with Safflower extract also inhibited expression of pro-apoptotic genes BAD and Bax. In conclusion, our data indicated that Safflower extract inhibit apoptosis via inducing autophagy in myocardium cell and demonstrated the potential as novel therapeutic drug for Heart failure. PMID:26884938

  5. Naoxintong/PPARα Signaling Inhibits H9c2 Cell Apoptosis and Autophagy in Response to Oxidative Stress.

    PubMed

    Xu, Huimin; Jin, Jianhua; Chen, Lu; Li, Chunxiao; Xu, Qinggang; Shi, Juanjuan; Zhao, Buchang; Hou, Yongzhong; Wang, Hong

    2016-01-01

    Naoxintong (NXT) is an empirical formula based on the principle of traditional Chinese medicine, which has been approved by China Food and Drug Administration (CFDA) and is widely used for treatment of patients with cerebrovascular and cardiovascular diseases in China. The aim of this study is to investigate the protective mechanism of NXT on H9c2 cells (cardiogenic cell line) in response to H2O2. MTT, Western blot, and flow cytometry (FCM) methods were used to identify the protective effect of NXT extract on H2O2-induced H9c2 cells. Here we found that NXT extract significantly increased H9c2 cell viability and reduced H2O2-induced cell apoptosis and autophagy. More importantly, NXT inhibited H2O2-induced H9c2 cell apoptosis and autophagy by increasing PPARα protein levels. In contrast, silenced PPARα terminated NXT protective effect on H2O2-induced H9c2 cells. These findings suggest that NXT/PPARα signaling suppressed H2O2-induced H9c2 cell apoptosis and autophagy. PMID:27668004

  6. Naoxintong/PPARα Signaling Inhibits H9c2 Cell Apoptosis and Autophagy in Response to Oxidative Stress

    PubMed Central

    Xu, Huimin; Jin, Jianhua; Chen, Lu; Li, Chunxiao; Xu, Qinggang; Shi, Juanjuan; Zhao, Buchang

    2016-01-01

    Naoxintong (NXT) is an empirical formula based on the principle of traditional Chinese medicine, which has been approved by China Food and Drug Administration (CFDA) and is widely used for treatment of patients with cerebrovascular and cardiovascular diseases in China. The aim of this study is to investigate the protective mechanism of NXT on H9c2 cells (cardiogenic cell line) in response to H2O2. MTT, Western blot, and flow cytometry (FCM) methods were used to identify the protective effect of NXT extract on H2O2-induced H9c2 cells. Here we found that NXT extract significantly increased H9c2 cell viability and reduced H2O2-induced cell apoptosis and autophagy. More importantly, NXT inhibited H2O2-induced H9c2 cell apoptosis and autophagy by increasing PPARα protein levels. In contrast, silenced PPARα terminated NXT protective effect on H2O2-induced H9c2 cells. These findings suggest that NXT/PPARα signaling suppressed H2O2-induced H9c2 cell apoptosis and autophagy. PMID:27668004

  7. Naoxintong/PPARα Signaling Inhibits H9c2 Cell Apoptosis and Autophagy in Response to Oxidative Stress

    PubMed Central

    Xu, Huimin; Jin, Jianhua; Chen, Lu; Li, Chunxiao; Xu, Qinggang; Shi, Juanjuan; Zhao, Buchang

    2016-01-01

    Naoxintong (NXT) is an empirical formula based on the principle of traditional Chinese medicine, which has been approved by China Food and Drug Administration (CFDA) and is widely used for treatment of patients with cerebrovascular and cardiovascular diseases in China. The aim of this study is to investigate the protective mechanism of NXT on H9c2 cells (cardiogenic cell line) in response to H2O2. MTT, Western blot, and flow cytometry (FCM) methods were used to identify the protective effect of NXT extract on H2O2-induced H9c2 cells. Here we found that NXT extract significantly increased H9c2 cell viability and reduced H2O2-induced cell apoptosis and autophagy. More importantly, NXT inhibited H2O2-induced H9c2 cell apoptosis and autophagy by increasing PPARα protein levels. In contrast, silenced PPARα terminated NXT protective effect on H2O2-induced H9c2 cells. These findings suggest that NXT/PPARα signaling suppressed H2O2-induced H9c2 cell apoptosis and autophagy.

  8. 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

  9. Piperazine designer drugs induce toxicity in cardiomyoblast h9c2 cells through mitochondrial impairment.

    PubMed

    Arbo, Marcelo Dutra; Silva, Renata; Barbosa, Daniel José; da Silva, Diana Dias; Rossato, Luciana Grazziotin; Bastos, Maria de Lourdes; Carmo, Helena

    2014-08-17

    Abuse of synthetic drugs is widespread among young people worldwide. In this context, piperazine derived drugs recently appeared in the recreational drug market. Clinical studies and case-reports describe sympathomimetic effects including hypertension, tachycardia, and increased heart rate. Our aim was to investigate the cytotoxicity of N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(4-methoxyphenyl) piperazine (MeOPP), and 1-(3,4-methylenedioxybenzyl) piperazine (MDBP) in the H9c2 rat cardiac cell line. Complete cytotoxicity curves were obtained at a 0-20 mM concentration range after 24 h incubations with each drug. The EC50 values (μM) were 343.9, 59.6, 570.1, and 702.5 for BZP, TFMPP, MeOPP, and MDBP, respectively. There was no change in oxidative stress markers. However, a decrease in total GSH content was noted for MDBP, probably due to metabolic conjugation reactions. All drugs caused significant decreases in intracellular ATP, accompanied by increased intracellular calcium levels and a decrease in mitochondrial membrane potential that seems to involve the mitochondrial permeability transition pore. The cell death mode revealed early apoptotic cells and high number of cells undergoing secondary necrosis. Among the tested drugs, TFMPP seems to be the most potent cytotoxic compound. Overall, piperazine designer drugs are potentially cardiotoxic and support concerns on risks associated with the intake of these drugs.

  10. 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

  11. PROTECTIVE EFFECT OF LACTUCA SERRIOLA ON DOXORUBICIN-INDUCED TOXICITY IN H9C2 CELLS.

    PubMed

    Hosseini, Azar; Mahdian, Davood

    2016-01-01

    The use of doxorubicin (DOX) is limited by its dose-dependency because of its cardiotoxicity. Reactive oxygen species (ROS) play an important role in the pathological process. The aim of this study is to evaluate the protective effect of Lactuca seniola against DOX-induced apoptosis and death in H9C2 cells. The cells were incubated with different concentrations of extract for 4 h which continued in the presence or absence of 5 µM doxorubicin for 24 h. Cell viability, apoptotic induction and the level of apoptotic proteins were determined by using MTT, PI and immunoblotting assays, respectively. The level of lipid peroxidation was measured by fluorimetric method. DOX significantly decreased cell viability which was accompanied by an increase in ROS production and lipid peroxidation. Pretreatment with Lactuca seniola increased the viability of cardiomyocytes and could decrease lipid peroxidation. Also, Lactuca seriola inhibited the reduction of anti-apoptotic Bcl-2 protein and elevation of apoptotic Bax and caspase-3 proteins. In conclusion, Lactuca seniola exerts protective effect against oxidative stress-induced cardiomyocytes damage. Therefore, it has the potential to be used as cardioprotective agent by the patients with cardiovascular diseases. PMID:27476284

  12. Proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in H9c2 cells.

    PubMed

    Lee, Hyunjung; Park, Jinyoung; Kim, Eunice EunKyeong; Yoo, Young Sook; Song, Eun Joo

    2016-05-01

    The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol- induced cardiac hypertrophy. We demonstrated that cholesterol- induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol- induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure. [BMB Reports 2016; 49(5): 270-275]. PMID:26592933

  13. Proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in H9c2 cells

    PubMed Central

    Lee, Hyunjung; Park, Jinyoung; Kim, Eunice EunKyeong; Yoo, Young Sook; Song, Eun Joo

    2016-01-01

    The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol-induced cardiac hypertrophy. We demonstrated that cholesterol-induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol-induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure. [BMB Reports 2016; 49(5): 270-275] PMID:26592933

  14. H2O 2 induces myocardial hypertrophy in H9c2 cells: a potential role of Ube3a.

    PubMed

    Song, Rui; Zhang, Jie; Zhang, Lijuan; Wang, Guanghua; Wo, Da; Feng, Jian; Li, Xucheng; Li, Jue

    2015-01-01

    Myocardial hypertrophy that often leads to eventual heart failure is a leading cause of mortality worldwide. While both apoptosis and cell proliferation have been reported to play an important part in heart failure, its exact triggering mechanism is still unclear. Reports have shown that low concentrations of H2O2 (10-30 µM) can induce myocardial hypertrophy without affecting survival. The ubiquitin ligase Ube3a has been reported to have a close affiliation with Angelman syndrome; but many ubiquitin ligases have been reported in a variety of cardiovascular conditions including myocardial hypertrophy. However, the relationship between Ube3a and myocardial hypertrophy has never been reported in literature. The rat cardiac myoblast cell line H9c2 and primary neonatal cardiomyocytes showed similar hypertrophic responses in vitro. In this report, we utilized H2O2 treatment on H9c2 cells to induce myocardial hypertrophy and determined the relationship between Ube3a and myocardial hypertrophy. Our results showed that 10-20 μM H2O2 can induce myocardial hypertrophy without affecting cell viability and inducing cell apoptosis, while the corresponding transcription and translation levels of Ube3a are significantly increased during the process. Therefore, these findings underline that Ube3a may play an important role in myocardial hypertrophy. PMID:24917194

  15. 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.

  16. N-acetylcysteine amide decreases oxidative stress but not cell death induced by doxorubicin in H9c2 cardiomyocytes

    PubMed Central

    Shi, Rong; Huang, Chuan-Chin; Aronstam, Robert S; Ercal, Nuran; Martin, Adam; Huang, Yue-Wern

    2009-01-01

    Background While doxorubicin (DOX) is widely used in cancer chemotherapy, long-term severe cardiotoxicity limits its use. This is the first report of the chemoprotective efficacy of a relatively new thiol antioxidant, N-acetylcysteine amide (NACA), on DOX-induced cell death in cardiomyocytes. We hypothesized that NACA would protect H9c2 cardiomyocytes from DOX-induced toxicity by reducing oxidative stress. Accordingly, we determined the ability of NACA to mitigate the cytotoxicity of DOX in H9c2 cells and correlated these effects with the production of indicators of oxidative stress. Results DOX at 5 μM induced cardiotoxicity while 1) increasing the generation of reactive oxygen species (ROS), 2) decreasing levels and activities of antioxidants and antioxidant enzymes (catalase, glutathione peroxidase, glutathione reductase) and 3) increasing lipid peroxidation. NACA at 750 μM substantially reduced the levels of ROS and lipid peroxidation, as well as increased both GSH level and GSH/GSSG ratio. However, treating H9c2 cells with NACA did little to protect H9c2 cells from DOX-induced cell death. Conclusion Although NACA effectively reduced oxidative stress in DOX-treated H9c2 cells, it had minimal effects on DOX-induced cell death. NACA prevented oxidative stress by elevation of GSH and CYS, reduction of ROS and lipid peroxidation, and restoration of antioxidant enzyme activities. Further studies to identify oxidative stress-independent pathways that lead to DOX-induced cell death in H9c2 are warranted. PMID:19368719

  17. Hydrogen sulfide attenuates doxorubicin-induced cardiotoxicity by inhibiting the expression of peroxiredoxin III in H9c2 cells.

    PubMed

    Liu, Mi-Hua; Lin, Xiao-Long; Yuan, Cong; He, Jun; Tan, Tian-Ping; Wu, Shao-Jian; Yu, Shan; Chen, Li; Liu, Jun; Tian, Wei; Chen, Yu-Dan; Fu, Hong-Yun; Li, Jian; Zhang, Yuan

    2016-01-01

    Doxorubicin (DOX) is a widely used chemotherapeutic agent, which can give rise to severe cardiotoxicity, limiting its clinical use. Preliminary evidence suggests that hydrogen sulfide (H2S) may exert protective effects on DOX‑induced cardiotoxicity. Therefore, the aim of the present study was to investigate whether peroxiredoxin III is involved in the cardioprotection of H2S against DOX‑induced cardiotoxicity. The results demonstrated that DOX not only markedly induced injuries, including cytotoxicity and apoptosis, it also increased the expression levels of peroxiredoxin III. Notably, pretreatment with sodium hydrosulfide significantly attenuated the DOX‑induced decrease in cell viability and increase in apoptosis, and also reversed the increased expression levels of peroxiredoxin III in H9c2 cardiomyocytes. In addition, pretreatment of the H9c2 cells with N‑acetyl‑L‑cysteine, a scavenger of reactive oxygen species, prior to exposure to DOX markedly decreased the expression levels of peroxiredoxin III. In conclusion, the results of the present study suggested that exogenous H2S attenuates DOX‑induced cardiotoxicity by inhibiting the expression of peroxiredoxin III in H9c2 cells. In the present study, the apoptosis of H9c2 cardiomyocytes was assessed using an methyl thiazolyl tetrazolium assay and Hoechst staining. The levels of Prx III and cystathionine-γ-lyase were examined by western blotting.

  18. 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 μM 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.

  19. 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 μM 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

  20. Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells

    PubMed Central

    Hwang, Jin-Taek; Kwon, Dae Young; Park, Ock Jin

    2007-01-01

    Resveratrol, one of polyphenols derived from red wine, has been shown to protect against cell death, possibly through the association with several signaling pathways. Currently numerous studies indicate that cardiovascular diseases are linked to the release of intracellular reactive oxygen species (ROS) often generated in states such as ischemia/reperfusion injury. In the present study, we investigated whether resveratrol has the capability to control intracellular survival signaling cascades involving AMP-activated kinase (AMPK) in the inhibitory process of cardiac injury. We hypothesized that resveratrol may exert a protective effect on damage to heart muscle through modulating of the AMPK signaling pathway. We mimicked ischemic conditions by inducing cell death with H2O2 in H9c2 muscle cells. In this experiment, resveratrol induced strong activation of AMPK and inhibited the occurrence of cell death caused by treatment with H2O2. Under the same conditions, inhibition of AMPK using dominant negative AMPK constructs dramatically abolished the effect of resveratrol on cell survival in H2O2-treated cardiac muscle cells. These results indicate that resveratrol-induced cell survival is mediated by AMPK in H9c2 cells and may exert a novel therapeutic effect on oxidative stress induced in cardiac disorders. PMID:18850225

  1. 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.

  2. Expression of human myoglobin in H9c2 cells enhances toxicity to added hydrogen peroxide.

    PubMed

    Witting, Paul K; Liao, Wen-Qiang; Matthew J Harris; Neuzil, Jiri

    2006-09-22

    Hydrogen peroxide (H2O2) is implicated in cardiac myocyte (CM) damage during myocardial ischemia-reperfusion (IR) injury. Myoglobin (Mb) is present in CM at significant concentrations and reacts with H2O2 to yield one- and two-electron oxidants that may promote myocardial injury. Paradoxically, hearts from mice lacking Mb are more susceptible to H2O2-induced dysfunction than the corresponding controls [U. Flogel, A. Godecke, L.O. Klotz, J. Schrader, Role of myoglobin in the anti-oxidant defense of the heart, FASEB J. 18 (2004) 1156-1158]. We have overexpressed wild-type or Y103F variant of human Mb in cultured CMs to test whether Mb protects against H2O2 insult. Contrary to expectation, cells expressing WT or the Y103F Mb show increased mitochondrial dysfunction and apoptosis, and decreased ATP in response to H2O2 that follows the order native < Y103F Mb < WT human Mb consistent with the increasing pro-oxidant activity for these proteins. These data indicate that (i) Mb promotes oxidative damage to cultured CM and (ii) Mb may be a useful target for the design of inhibitors of myocardial IR injury. PMID:16887098

  3. Expression of human myoglobin in H9c2 cells enhances toxicity to added hydrogen peroxide

    SciTech Connect

    Witting, Paul K. . E-mail: pwitting@anzac.edu.au; Liao Wenqiang; Harris, Matthew J.; Neuzil, Jiri

    2006-09-22

    Hydrogen peroxide (H{sub 2}O{sub 2}) is implicated in cardiac myocyte (CM) damage during myocardial ischemia-reperfusion (IR) injury. Myoglobin (Mb) is present in CM at significant concentrations and reacts with H{sub 2}O{sub 2} to yield one- and two-electron oxidants that may promote myocardial injury. Paradoxically, hearts from mice lacking Mb are more susceptible to H{sub 2}O{sub 2}-induced dysfunction than the corresponding controls [U. Flogel, A. Godecke, L.O. Klotz, J. Schrader, Role of myoglobin in the anti-oxidant defense of the heart, FASEB J. 18 (2004) 1156-1158]. We have overexpressed wild-type or Y103F variant of human Mb in cultured CMs to test whether Mb protects against H{sub 2}O{sub 2} insult. Contrary to expectation, cells expressing WT or the Y103F Mb show increased mitochondrial dysfunction and apoptosis, and decreased ATP in response to H{sub 2}O{sub 2} that follows the order native < Y103F Mb < WT human Mb consistent with the increasing pro-oxidant activity for these proteins. These data indicate that (i) Mb promotes oxidative damage to cultured CM and (ii) Mb may be a useful target for the design of inhibitors of myocardial IR injury.

  4. Mitoprotective antioxidant EUK-134 stimulates fatty acid oxidation and prevents hypertrophy in H9C2 cells.

    PubMed

    Purushothaman, Sreeja; Nair, R Renuka

    2016-09-01

    Oxidative stress is an important contributory factor for the development of cardiovascular diseases like hypertension-induced hypertrophy. Mitochondrion is the major source of reactive oxygen species. Hence, protecting mitochondria from oxidative damage can be an effective therapeutic strategy for the prevention of hypertensive heart disease. Conventional antioxidants are not likely to be cardioprotective, as they cannot protect mitochondria from oxidative damage. EUK-134 is a salen-manganese complex with superoxide dismutase and catalase activity. The possible role of EUK-134, a mitoprotective antioxidant, in the prevention of hypertrophy of H9C2 cells was examined. The cells were stimulated with phenylephrine (50 μM), and hypertrophy was assessed based on cell volume and expression of brain natriuretic peptide and calcineurin. Enhanced myocardial lipid peroxidation and protein carbonyl content, accompanied by nuclear factor-kappa B gene expression, confirmed the presence of oxidative stress in hypertrophic cells. Metabolic shift was evident from reduction in the expression of medium-chain acyl-CoA dehydrogenase. Mitochondrial oxidative stress was confirmed by the reduced expression of mitochondria-specific antioxidant peroxiredoxin-3 and enhanced mitochondrial superoxide production. Compromised mitochondrial function was apparent from reduced mitochondrial membrane potential. Pretreatment with EUK-134 (10 μM) was effective in the prevention of hypertrophic changes in H9C2 cells, reduction of oxidative stress, and prevention of metabolic shift. EUK-134 treatment improved the oxidative status of mitochondria and reversed hypertrophy-induced reduction of mitochondrial membrane potential. Supplementation with EUK-134 is therefore identified as a novel approach to attenuate cardiac hypertrophy and lends scope for the development of EUK-134 as a therapeutic agent in the management of human cardiovascular disease. PMID:27514538

  5. 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

  6. 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.

  7. Polyphenol-rich apple (Malus domestica L.) peel extract attenuates arsenic trioxide induced cardiotoxicity in H9c2 cells via its antioxidant activity.

    PubMed

    Vineetha, Vadavanath Prabhakaran; Girija, Seetharaman; Soumya, Rema Sreenivasan; Raghu, Kozhiparambil Gopalan

    2014-03-01

    Evidences suggest that apple peel has a wide range of polyphenols having antioxidant activity and its consumption has been linked with improved health benefits. Arsenic trioxide (ATO) is a very effective drug for the treatment of acute promyelocytic leukemia (APL) but it leads to cardiotoxicity mediated through alterations in various cardiac ion channels and by increasing the intracellular calcium level and reactive oxygen species (ROS). The aim of the present investigation was to study the effect of methanolic extract of apple peel (APME) and aqueous extract of apple peel (APAE) on ATO (5 μM) induced toxicity in the H9c2 cardiac myoblast cell line. We estimated the cellular status of innate antioxidant enzymes, level of ROS, mitochondrial superoxide, glutathione and intracellular calcium with ATO and apple peel extracts. Prior to the cell line based study, we had evaluated the antioxidant potential of apple peel extract by 1,1-diphenyl-2-picrylhydrazyl (DPPH), total reducing power (TRP), superoxide anion and hydroxyl radical scavenging activity, in addition to quantifying total phenolic and flavonoid content. Both the extracts showed considerable antioxidant activity in cell-free chemical assays. In addition, both APME and APAE prevented the alteration in antioxidant status induced by ATO in H9c2 cells. Significant differential alterations had been observed in the activity of lactate dehydrogenase, superoxide dismutase, catalase, glutathione, glutathione peroxidase, thioredoxin reductase, xanthine oxidase, calcium overload and caspase 3 activity with ATO. The overall result revealed the protective property of polyphenol-rich apple peel extract against ATO induced cardiac toxicity via its antioxidant activity.

  8. Transforming growth factor-beta type I receptor/ALK5 contributes to doxazosin-induced apoptosis in H9C2 cells.

    PubMed

    Yang, Yi-Fan; Wu, Chau-Chung; Chen, Wen-Pin; Su, Ming-Jai

    2009-12-01

    The mechanism of doxazosin-induced apoptosis through alpha(1)-adrenoceptor-independent pathway has been reported in various types of cell models. However, the molecular events involved in this effect are still not fully discovered. In present study, we proposed that the transforming growth factor-beta type I receptor (TbetaRI/ALK5) may contribute to the doxazosin-induced apoptosis in H9C2 cardiomyoblasts. Via the detection of cell viability, apoptotic nuclei, and caspase-3 activity, we found that doxazosin induced concentration- and time-dependent apoptosis in H9C2 cells. The cell apoptosis induced by 30 muM doxazosin was exacerbated by the addition of 10 ng/ml transforming growth factor-beta1 (TGF-beta1). Doxazosin or TGF-beta1 alone respectively elevated p38 mitogen-activated protein kinases (MAPK) and Smad3 protein phosphorylation in H9C2 cells. However, the cotreatment of doxazosin and TGF-beta1 attenuated the TGF-beta1-induced Smad3 protein phosphorylation and increased doxazosin-induced p38 MAPK protein phosphorylation. Furthermore, inhibitors of TbetaRI/ALK5 (SB431542) and p38 MAPK (SB202190) or TbetaRI/ALK5 knockdown all dramatically reduced the doxazosin-induced apoptosis in H9C2 cells. In conclusion, our results demonstrated that TbetaRI/ALK5-p38 MAPK phosphorylation signaling pathway could contribute to doxazosin-induced cell apoptosis, which could be further enhanced by TGF-beta1 in association with attenuating Smad3 phosphorylation in H9C2 cells.

  9. Catha edulis Extract Induces H9c2 Cell Apoptosis by Increasing Reactive Oxygen Species Generation and Activation of Mitochondrial Proteins

    PubMed Central

    Mohan, Syam; Abdelwahab, Siddig Ibrahim; Hobani, Yahya Hasan; Syam, Suvitha; Al-Zubairi, Adel S.; Al-Sanousi, Rashad; Oraiby, Magbool Essa

    2016-01-01

    Background: Catha edulis (Khat) is an evergreen shrub or small tree, traditionally used by various peoples of the Arabian Peninsula and Africa as an integral component of the socioeconomic traditions. It is believed that the psychostimulant nature and toxic nature of khat is primarily due to the presence of cathinone and cathine respectively. Studies have shown that khat chewing is closely associated with cardiac complications, especially myocardial infarction. Hence in this study, we exposed cathine-rich khat extract in a cardiomyoblast H9c2 (2-1) cell line to check the cell death mechanism. Materials and Methods: Extraction of Catha edulis leaves was done and the presence of cathine was confirmed with LC-MS-MS. The anti-proliferative activity was assayed using MTT and apoptosis detection by acridine orange/propidium iodide assay. The expression of Bcl-2 and Bax protein and caspase-3/7 expression were analyzed. The level of reactive oxygen species generation was also evaluated. Results: The khat extract showed an IC50 value of 86.5 μg/ml at 48 hours treatment. We have observed significant early apoptosis events by intervened acridine orange within the fragmented DNA with bright green fluorescence upon treatment. The Bcl-2 expression in the treatment with IC50 concentration of khat extract for 24, 48 and 72 hours of incubation significantly decreased with increase in bax level. The increased activation of caspase-3/7 was significantly observed upon treatment together with significant increase of ROS was detected at 24 and 48 hours treatment. Conclusion: Collectively, our results provide insight into the mechanisms by which Catha edulis leaves mediate cell death in cardiomyocytes. SUMMARY Catha edulis (Khat) is an evergreen psychotropic shrub or small treeExtraction of khat leaves was done and the presence of cathine was confirmed with liquid chromatography-mass spectrometry-mass spectrometryThe khat extract showed an IC50 value of 86.5 μg/ml at 48 h treatment in

  10. Jujuboside A Protects H9C2 Cells from Isoproterenol-Induced Injury via Activating PI3K/Akt/mTOR Signaling Pathway

    PubMed Central

    Han, Dandan; Wan, Changrong; Liu, Fenghua; Xu, Xiaolong; Jiang, Linshu; Xu, Jianqin

    2016-01-01

    Jujuboside A is a kind of the saponins isolated from the seeds of Ziziphus jujuba, which possesses multiple biological effects, such as antianxiety, antioxidant, and anti-inflammatory effects; however, its mediatory effect on isoproterenol-stimulated cardiomyocytes has not been investigated yet. In this study, we tried to detect the protective effect and potential mechanism of JUA on ISO-induced cardiomyocytes injury. H9C2 cells were treated with ISO to induce cell damage. Cells were pretreated with JUA to investigate the effects on the cell viability, morphological changes, light chain 3 conversion, and the activation of PI3K/Akt/mTOR signaling pathway. Results showed that ISO significantly inhibited the cell viability in a time- and dose-dependent manner. JUA pretreatment could reverse the reduction of cell viability and better the injury of H9C2 cells induced by ISO. Western blot analysis showed that JUA could accelerate the phosphorylation of PI3K, Akt, and mTOR. Results also indicated that JUA could significantly decrease the ratio of microtubule-associated protein LC3-II/I in H9C2 cells. Taken together, our research showed that JUA could notably reduce the damage cause by ISO via promoting the phosphorylation of PI3K, Akt, and mTOR and inhibiting LC3 conversion, which may be a potential choice for the treatment of heart diseases. PMID:27293469

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

    PubMed

    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

  12. Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation

    PubMed Central

    Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

    2016-01-01

    The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H2O2-induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H2O2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H2O2-induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H2O2-induced cell death. Western blot analysis demonstrated that H2O2-induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H2O2-induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent upregulation of Bcl-2. In

  13. Polysaccharide from Fuzi likely protects against starvation-induced cytotoxicity in H9c2 cells by increasing autophagy through activation of the AMPK/mTOR pathway.

    PubMed

    Liao, Li-Zhen; Chen, Yan-Ling; Lu, Li-He; Zhao, Yong-Hua; Guo, Hua-Lei; Wu, Wei-Kang

    2013-01-01

    There is increasing evidence that starvation induces autophagy, which may be protective during starvation, in an AMPK-dependent manner. Polysaccharides from Fuzi (FPS) reportedly have protective effects on nutrition-limited livers. The present study was designed to determine whether FPS protected H9c2 cells against starvation-induced cytotoxicity using an AMPK/mTOR-dependent mechanism. H9c2 cells were incubated in serum and glucose starvation media for 12 hours to establish a cell injury model. 3-Methyladenine (3MA, an autophagy inhibitor) was used to identify the exact role of autophagy in starvation. Cells were incubated with different FPS concentrations, and the cell injury levels, autophagy activity and AMPK/mTOR phosphorylation were measured. Adenine 9-β-D-arabinofuranoside (Ara-A, an AMPK inhibitor) and 5-amino-4-imidazole-carboxamide riboside (AICAR, an AMPK activator) were used to identify whether the AMPK/mTOR pathway was involved in FPS-mediated cardioprotection. We demonstrated that starvation decreased cell viability in a time-dependent manner, and 3MA-induced autophagy inhibition aggravated the reduced cell viability. FPS treatment attenuated the cell viability decrement and the starvation-induced decline in the mitochondrial membrane potential (MMP), and autophagy; also, the AMPK/mTOR pathways were activated during treatment. Ara-A treatment abolished the protective effect of FPS, while AICAR treatment had a similar effect to FPS. We conclude that autophagy attenuates starvation-induced cardiomyocyte death, and FPS increases autophagy activity to protect against starvation-induced cytotoxicity in H9c2 cells, likely through AMPK/mTOR pathway activation.

  14. Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB 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-κB (NF-κB). The NF-κB 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-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB 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

  15. 3,3'-Diindolylmethane attenuates cardiac H9c2 cell hypertrophy through 5'-adenosine monophosphate-activated protein kinase-α.

    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 immunofluorescence to analyze cardiac α-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 β 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 α (AMPKα)/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 AMPKα and inhibited the MAPK‑mTOR signaling pathway in response to hypertrophic stimuli. PMID:25816057

  16. Testosterone protects female embryonic heart H9c2 cells against severe metabolic stress by activating estrogen receptors and up-regulating IES SUR2B.

    PubMed

    Ballantyne, Thomas; Du, Qingyou; Jovanović, Sofija; Neemo, Andrew; Holmes, Robert; Sinha, Sharabh; Jovanović, Aleksandar

    2013-02-01

    A recent clinical study demonstrated that a testosterone supplementation improves functional capacity in elderly female patients suffering from heart failure. These findings prompted us to consider possible mechanisms of testosterone-induced cardioprotection in females. To address this question we have used a pure female population of rat heart embryonic H9c2 cells. Pre-treatment of cells with testosterone for 24h significantly increased survival of H9c2 cells exposed to 2,4-dinitrophenol (DNP), an inhibitor of oxidative phosphorylation. These cells expressed low level of androgen receptors and the effect of testosterone was not modified by hydroxyflutamide, an antagonist of androgen receptor. In contrast, cyclohexamide, an inhibitor of protein biosynthesis, and tamoxifene, a partial agonist of estrogen receptors, abolished cardioprotection afforded by testosterone. In addition, finasteride, an inhibitor of 5α-reductase, and anastrazole, an inhibitor of α-aromatase, also blocked testosterone-induced cytoprotection. Real time RT-PCR revealed that testosterone did not regulate the expression of nine subunits and accessory proteins of sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. On the other hand, testosterone, as well as 17β-estradiol, up-regulated a putative mitochondrial K(ATP) channel subunit, mitochondrial sulfonylurea receptor 2B intraexonics splice variant (IES SUR2B), without affecting expression of IES SUR2A. Tamoxifene inhibited testosterone-induced up-regulation of IES SUR2B without affecting IES SUR2A. In conclusion, this study has shown that testosterone protect female embryonic heart H9c2 cells against severe metabolic stress by its conversion into metabolites that activate estrogen receptors and up-regulate IES SUR2B.

  17. Ultrasound-targeted microbubble destruction (UTMD) assisted delivery of shRNA against PHD2 into H9C2 cells.

    PubMed

    Zhang, Li; Sun, Zhenxing; 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 300μl/ml, an exposure time of 45s, and a plasmid concentration of 15μg/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-1α and downstream angiogenesis factors VEGF, TGF-β and bFGF. UTMD/PEI, combined with albumin-coated microbubbles, warrants further investigation for therapeutic gene delivery. PMID:26267649

  18. 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

  19. Luteolin Prevents H2O2-Induced Apoptosis in H9C2 Cells through Modulating Akt-P53/Mdm2 Signaling Pathway.

    PubMed

    Chang, Hong; Li, Chun; Huo, Kuiyuan; Wang, Qiyan; Lu, Linghui; Zhang, Qian; Wang, Yong; Wang, Wei

    2016-01-01

    Introduction. Luteolin, a falconoid compound in many Chinese herbs and formula, plays important roles in cardiovascular diseases. The underlying mechanism of luteolin remains to be further elaborated. Methods. A model of hydrogen peroxide- (H2O2-) induced H9C2 cells apoptosis was established. Cell viabilities were examined with an MTT assay. 2',7'-Dichlorofluorescin diacetate (DCFH-DA) and flow cytometry were used to detect ROS level and apoptosis rate, respectively. The expressions of signaling proteins related to apoptosis were analyzed by western blot and mRNA levels were detected by real-time polymerase chain reaction (PCR). Quercetin was applied as positive drug. Results. Incubation with various concentrations of H2O2 (0, 50, 100, and 200 μM) for 1 h caused dose-dependent loss of cell viability and 100 μM H2O2 reduced the cell viability to approximately 50%. Treatments with luteolin and quercetin protected cells from H2O2-induced cytotoxicity and reduced cellular ROS level and apoptosis rate. Moreover, luteolin could downregulate the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 in apoptotic signaling pathway. Further study showed that the expressions of Akt, Bcl-2, and Mdm2 were upregulated by luteolin. Conclusion. Luteolin protects H9C2 cells from H2O2-induced apoptosis. The protective and antiapoptotic effects of luteolin could be mediated by regulating the Akt-P53/Mdm2 apoptotic pathway. PMID:27525270

  20. Luteolin Prevents H2O2-Induced Apoptosis in H9C2 Cells through Modulating Akt-P53/Mdm2 Signaling Pathway

    PubMed Central

    Li, Chun; Wang, Qiyan; Lu, Linghui; Zhang, Qian

    2016-01-01

    Introduction. Luteolin, a falconoid compound in many Chinese herbs and formula, plays important roles in cardiovascular diseases. The underlying mechanism of luteolin remains to be further elaborated. Methods. A model of hydrogen peroxide- (H2O2-) induced H9C2 cells apoptosis was established. Cell viabilities were examined with an MTT assay. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and flow cytometry were used to detect ROS level and apoptosis rate, respectively. The expressions of signaling proteins related to apoptosis were analyzed by western blot and mRNA levels were detected by real-time polymerase chain reaction (PCR). Quercetin was applied as positive drug. Results. Incubation with various concentrations of H2O2 (0, 50, 100, and 200 μM) for 1 h caused dose-dependent loss of cell viability and 100 μM H2O2 reduced the cell viability to approximately 50%. Treatments with luteolin and quercetin protected cells from H2O2-induced cytotoxicity and reduced cellular ROS level and apoptosis rate. Moreover, luteolin could downregulate the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 in apoptotic signaling pathway. Further study showed that the expressions of Akt, Bcl-2, and Mdm2 were upregulated by luteolin. Conclusion. Luteolin protects H9C2 cells from H2O2-induced apoptosis. The protective and antiapoptotic effects of luteolin could be mediated by regulating the Akt-P53/Mdm2 apoptotic pathway. PMID:27525270

  1. Oxidative stress does not play a primary role in the toxicity induced with clinical doses of doxorubicin in myocardial H9c2 cells.

    PubMed

    Rharass, Tareck; Gbankoto, Adam; Canal, Christophe; Kurşunluoğlu, Gizem; Bijoux, Amandine; Panáková, Daniela; Ribou, Anne-Cécile

    2016-02-01

    The implication of oxidative stress as primary mechanism inducing doxorubicin (DOX) cardiotoxicity is still questionable as many in vitro studies implied supra-clinical drug doses or unreliable methodologies for reactive oxygen species (ROS) detection. The aim of this study was to clarify whether oxidative stress is involved in compliance with the conditions of clinical use of DOX, and using reliable tools for ROS detection. We examined the cytotoxic mechanisms of 2 μM DOX 1 day after the beginning of the treatment in differentiated H9c2 rat embryonic cardiac cells. Cells were exposed for 2 or 24 h with DOX to mimic a single chronic dosage or to favor accumulation, respectively. We found that apoptosis was prevalent in cells exposed for a short period with DOX: cells showed typical hallmarks as loss of anchorage ability, mitochondrial hyperpolarization followed by the collapse of mitochondrial activity, and nuclear condensation. Increasing the exposure period favored a shift to necrosis as the cells preferentially exhibited early DNA impairment and nuclear swelling. In either case, measuring the fluorescence lifetime of 1-pyrenebutyric acid or the intensities of dihydroethidium or amplex red showed a consistent pattern in ROS production which was a slight increased level far from representative of an oxidative stress. Moreover, pre-treatment with dexrazoxane provided a cytoprotective effect although it failed to detoxify ROS. Our data support that oxidative stress is unlikely to be the primary mechanism of DOX cardiac toxicity in vitro.

  2. Pan-histone deacetylase inhibitors regulate signaling pathways involved in proliferative and pro-inflammatory mechanisms in H9c2 cells

    PubMed Central

    2012-01-01

    Background We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA. Results We incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGFβ, TNF-α, IFNγ and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGFβ- and TNF-α-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-α and IFNγ specific networks at 6 h, followed by elicitation of IL-6 and IFNγ-centered gene networks at 24h. Conclusions Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene

  3. Suppression of antioxidant Nrf-2 and downstream pathway in H9c2 cells by advanced glycation end products (AGEs) via ERK phosphorylation.

    PubMed

    Ko, Shun-Yao; Chang, Shu-Shing; Lin, I-Hsuan; Chen, Hong-I

    2015-11-01

    Diabetic cardiomyopathy is related to oxidative stress and correlated with the presence of advanced glycation end products (AGEs). In a clinical setting, AGEs can be detected in patients presenting diabetic cardiomyopathy; however, the underlying mechanism has yet to be elucidated. In our previous study, AGEs increase cell hypertrophy via ERK phosphorylation in a process closely related to ROS production. Thus, we propose that AGEs regulate the antioxidant gene nuclear factor-erythroid 2-related factor (Nrf-2). In H9c2 cells treated with AGEs, the expression of Nrf-2 was reduced; however, ERK phosphorylation was shown to increase. Treatment with H2O2 was also shown to increase Nrf-2 and ERK phosphorylation. In cells pretreatment with ROS scavenger NAC, the effects of H2O2 were reduced; however, the effects of the AGEs remained largely unchanged. Conversely, when cells were pretreated with PD98059 (ERK inhibitor), the expression of Nrf-2 was recovered following treatment with AGEs. Our results suggest that AGEs inhibit Nrf-2 via the ERK pathway; however, this influence is partly associated with ROS. Our finding further indicated that AGEs possess both ROS-dependent and ROS-independent pathways, resulting in a reduction in Nrf-2. This report reveals an important mechanism underlying the regulation of diabetic cardiomyopathy progression by AGEs. PMID:26212730

  4. Carotenoid compositions of coloured tomato cultivars and contribution to antioxidant activities and protection against H(2)O(2)-induced cell death in H9c2.

    PubMed

    Li, Hongyan; Deng, Zeyuan; Liu, Ronghua; Loewen, Steven; Tsao, Rong

    2013-01-15

    The carotenoid compositions, antioxidant activities and the potential cardio-protective role of 13 tomato cultivars with distinct colour were studied. Colour coordinates were evaluated by colorimeter and the carotenoid compositions were analysed by UPLC. Red tomatoes had the highest total carotenoid contents (TCC) and antioxidant activities, followed by purple, orange, pink and yellow ones. The TCC were 120.5-278.0 μg/gDW, and the antioxidant activities were 21.32-40.07 μmolTE/gDW (PCL), 64.42-89.98% (DPPH) and 10.47-13.76 μmolTE/g DW (ORAC), respectively. The lipophilic extracts were also found to prevent cell death in a cell-based model system using cardiac H9c2 cells and H(2)O(2), via attenuation of the caspase-3 and matrix metalloproteinase-2 activities. The extracts of different tomatoes showed strong but different antioxidant activities. Roles of total and individual carotenoids in the antioxidant activities were studied and lycopene showed the highest correlation. Results of this study can be used to guide the development of new tomato cultivars and functional foods, and benefit the consumers. PMID:23122140

  5. Remote loading of doxorubicin into liposomes by transmembrane pH gradient to reduce toxicity toward H9c2 cells.

    PubMed

    Alyane, Mohamed; Barratt, Gillian; Lahouel, Mesbah

    2016-03-01

    The use of doxorubicin (DOX) is limited by its dose-dependent cardiotoxicity. Entrapped DOX in liposome has been shown to reduce cardiotoxicity. Results showed that about 92% of the total drug was encapsulated in liposome. The release experiments showed a weak DOX leakage in both culture medium and in PBS, more than 98% and 90% of the encapsulated DOX respectively was still retained in liposomes after 24 h of incubation. When the release experiments were carried out in phosphate buffer pH5.3, the leakage of DOX from liposomes reached 37% after 24 h of incubation. Evaluation of cellular uptake of the liposomal DOX indicated the possible endocytosis of liposomes because the majority of visible fluorescence of DOX was mainly in the cytoplasm, whereas the nuclear compartment showed a weak intensity. When using unloaded fluorescent-liposomes, the fluorescence was absent in nuclei suggests that liposomes cannot cross the nuclear membrane. MTT assay and measurement of LDH release suggest that necrosis is the form of cellular death predominates in H9c2 cells exposed to high doses of DOX, while for weak doses apoptosis could be the predominate form. Entrapped DOX reduced significantly DOX toxicity after 3 and 6 h of incubation, but after 20 h entrapped DOX is more toxic than free one.

  6. Remote loading of doxorubicin into liposomes by transmembrane pH gradient to reduce toxicity toward H9c2 cells

    PubMed Central

    Alyane, Mohamed; Barratt, Gillian; Lahouel, Mesbah

    2015-01-01

    The use of doxorubicin (DOX) is limited by its dose-dependent cardiotoxicity. Entrapped DOX in liposome has been shown to reduce cardiotoxicity. Results showed that about 92% of the total drug was encapsulated in liposome. The release experiments showed a weak DOX leakage in both culture medium and in PBS, more than 98% and 90% of the encapsulated DOX respectively was still retained in liposomes after 24 h of incubation. When the release experiments were carried out in phosphate buffer pH5.3, the leakage of DOX from liposomes reached 37% after 24 h of incubation. Evaluation of cellular uptake of the liposomal DOX indicated the possible endocytosis of liposomes because the majority of visible fluorescence of DOX was mainly in the cytoplasm, whereas the nuclear compartment showed a weak intensity. When using unloaded fluorescent-liposomes, the fluorescence was absent in nuclei suggests that liposomes cannot cross the nuclear membrane. MTT assay and measurement of LDH release suggest that necrosis is the form of cellular death predominates in H9c2 cells exposed to high doses of DOX, while for weak doses apoptosis could be the predominate form. Entrapped DOX reduced significantly DOX toxicity after 3 and 6 h of incubation, but after 20 h entrapped DOX is more toxic than free one. PMID:27013909

  7. 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

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

    PubMed

    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

  9. Bone marrow-derived mesenchymal stem cells rescue injured H9c2 cells via transferring intact mitochondria through tunneling nanotubes in an in vitro simulated ischemia/reperfusion model.

    PubMed

    Han, Hui; Hu, Jinquan; Yan, Qiang; Zhu, Jinzhou; Zhu, Zhengbin; Chen, Yanjia; Sun, Jiateng; Zhang, Ruiyan

    2016-02-01

    The transplantation of mesenchymal stem cells (MSCs) is considered to be a promising treatment for ischemic heart disease; however, the therapeutic effects and underlying mechanisms of action require further evaluation. Mitochondrial dysfunction is a key event in simulated ischemia/reperfusion (SI/R) injury. The purpose of the present study was to investigate the mechanism of mitochondrial transfer, which may be involved the antiapoptotic action of co-culture with MSCs. An in vitro model of simulated ischemia/reperfusion (SI/R) was used in the present study. The apoptotic indexes were significantly increased when H9c2 cardiomyocytes were induced in the SI/R group. Following co-culture with bone marrow-derived (BM)-MSCs, H9c2 cells exhibited marked resistance against the SI/R-induced apoptotic process. Besides, mitochondrial transfer via a tunneling nanotube (TNT) like structure was detected by confocal fluorescent microscopy. In addition, following pretreated with latrunculin-A (LatA), an inhibitor of TNT formation, the BM-MSCs were not able to rescue injured H9c2 cells from apoptosis, as previously observed. In conclusion, the anti-apoptotic ability of BM-MSCs may be partially attributed to the recovery of mitochondrial dysfunction in SI/R, and the formation of TNTs appears to be involved in this action of mitochondrial transfer between adjacent cells.

  10. 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 (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β 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

  11. 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 (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β 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.

  12. β-Sitosterol enhances cellular glutathione redox cycling by reactive oxygen species generated from mitochondrial respiration: protection against oxidant injury in H9c2 cells and rat hearts.

    PubMed

    Wong, Hoi Shan; Chen, Na; Leong, Pou Kuan; Ko, Kam Ming

    2014-07-01

    Herba Cistanches (Cistanche deserticola Y. C. Ma) is a 'Yang-invigorating' tonic herb in Chinese medicine. Preliminary chemical analysis indicated that β-sitosterol (BS) is one of the chemical constituents in an active fraction of Herba Cistanches. To investigate whether BS is an active ingredient of Herba Cistanches, the effects of BS on H9c2 cells and rat hearts were examined. The results indicated that BS stimulated the mitochondrial ATP generation capacity in H9c2 cells, which was associated with the increased production of mitochondrial reactive oxygen species. BS also stimulated mitochondrial state 3 and state 4 respiration, with the resultant decrease in coupling efficiency. BS produced an up-regulation of cellular glutathione redox cycling and protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. However, the protective effect of BS against myocardial ischemia/reperfusion injury was seen in female but not male rats ex vivo. The cardioprotection afforded by BS was likely mediated by an up-regulation of mitochondrial glutathione redox cycling in female rat hearts. In conclusion, the ensemble of results suggests that BS is an active ingredient of Herba Cistanches. The gender-dependent effect of BS on myocardial protection will further be investigated.

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

  14. 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

  15. 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 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 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.

  16. 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

  17. 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.

  18. PEDF and PEDF-derived peptide 44mer inhibit oxygen-glucose deprivation-induced oxidative stress through upregulating PPARγ via PEDF-R in H9c2 cells.

    PubMed

    Zhuang, Wei; Zhang, Hao; Pan, Jiajun; Li, Zhimin; Wei, Tengteng; Cui, Huazhu; Liu, Zhiwei; Guan, Qiuhua; Dong, Hongyan; Zhang, Zhongming

    2016-04-01

    Pigment epithelial-derived factor (PEDF) is a glycoprotein with broad biological activities including inhibiting oxygen-glucose deprivation(OGD)-induced cardiomyocytes apoptosis through its anti-oxidative properties. PEDF derived peptide-44mer shows similar cytoprotective effect to PEDF. However, the molecular mechanisms mediating cardiomyocytes apoptosis have not been fully established. Here we found that PEDF and 44mer decreased the content of ROS. This content was abolished by either PEDF-R small interfering RNA (siRNA) or PPARγ antagonist. The level of Lysophosphatidic acid (LPA) and phospholipase A2 (PLA2) was observed as drawn from the ELISA assays. PEDF and 44mer sequentially induced PPARγ expression was observed both in qPCR and Western blot assays. The level of LPA and PLA2 and PPARγ expression increased by PEDF and 44mer was significantly attenuated by PEDF-R siRNA. However, PEDF and 44mer inhibited the H9c2 cells and cultured neonatal rat myocardial cells apoptosis rate. On the other hand, TUNEL assay and cleavage of procaspase-3 showed that PEDF-R siRNA or PPARγ antagonist increased the apoptosis again. We conclude that under OGD condition, PEDF and 44mer reduce H9c2 cells apoptosis and inhibit OGD-induced oxidative stress via its receptor PEDF-R and the PPARγ signaling pathway. PMID:26966066

  19. 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 PPARγ 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 PPARγ 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.

  20. Resveratrol inhibits doxorubicin-induced cardiotoxicity via sirtuin 1 activation in H9c2 cardiomyocytes

    PubMed Central

    Liu, Mi-Hua; Shan, Jian; Li, Jian; Zhang, Yuan; Lin, Xiao-Long

    2016-01-01

    Doxorubicin (DOX) is an efficient drug used in cancer therapy; however, it can induce severe cytotoxicity, which limits its clinical application. In the present study, the effects of resveratrol (RES) on sirtuin 1 (SIRT1) activation in mediating DOX-induced cytotoxicity in H9c2 cardiac cells was investigated. H9c2 cells were exposed to 5 µM DOX for 24 h to establish a model of DOX cardiotoxicity. Apoptosis of H9c2 cardiomyocytes was assessed using the MTT assay and Hoechst nuclear staining. The results demonstrated that pretreating H9c2 cells with RES prior to the exposure of DOX resulted in increased cell viability and a decreased quantity of apoptotic cells. Western blot analysis demonstrated that DOX decreased the expression level of SIRT1. These effects were significantly alleviated by co-treatment with RES. In addition, the results demonstrated that DOX administration amplified forkhead box O1 (FoxO1) and P53 expression levels in H9c2 cells. RES was also found to protect against DOX-induced increases of FoxO1 and P53 expression levels in H9c2 cells. Furthermore, the protective effects of RES were arrested by the SIRT1 inhibitor nicotinamide. In conclusion, the results demonstrated that RES protected H9c2 cells against DOX-induced injuries via SIRT1 activation. PMID:27446329

  1. 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

  2. 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.

  3. HO-1 Protects against Hypoxia/Reoxygenation-Induced Mitochondrial Dysfunction in H9c2 Cardiomyocytes

    PubMed Central

    Chen, Dongling; Jin, Zhe; Zhang, Jingjing; Jiang, Linlin; Chen, Kai; He, Xianghu; Song, Yinwei; Ke, Jianjuan; Wang, Yanlin

    2016-01-01

    Background Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial’s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism. Methods and Results The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed. Conclusions During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. PMID

  4. Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts.

    PubMed

    Sun, Xiao; Sun, Gui-Bo; Wang, Min; Xiao, Jing; Sun, Xiao-Bo

    2011-08-01

    Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 µM H(2) O(2) for 6  h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.

  5. Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts.

    PubMed

    Sun, Xiao; Sun, Gui-Bo; Wang, Min; Xiao, Jing; Sun, Xiao-Bo

    2011-08-01

    Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 µM H(2) O(2) for 6  h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways. PMID:21445859

  6. Simvastatin induces mitochondrial dysfunction and increased atrogin-1 expression in H9c2 cardiomyocytes and mice in vivo.

    PubMed

    Bonifacio, Annalisa; Mullen, Peter J; Mityko, Ileana Scurtu; Navegantes, Luiz C; Bouitbir, Jamal; Krähenbühl, Stephan

    2016-01-01

    Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10 μmol/L simvastatin for 24 h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5 mg/kg/day simvastatin for 21 days was associated with a 5 % drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy.

  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 Central

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

    2015-01-01

    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 α-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 α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for β1 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. Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2: A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection.

    PubMed

    Jovanović, Sofija; Ballantyne, Thomas; Du, Qingyou; Blagojević, Miloš; Jovanović, Aleksandar

    2016-01-01

    ATP-sensitive K(+) (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100nM) for 24h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K(+) current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10mM), which was accompanied by increased in K(+) current in response to DNP (10mM). Cytoprotection afforded by phenylephrine (100nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K(+) conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes.

  10. Morphine Attenuated the Cytotoxicity Induced by Arsenic Trioxide in H9c2 Cardiomyocytes.

    PubMed

    Amini-Khoei, Hossein; Hosseini, Mir-Jamal; Momeny, Majid; Rahimi-Balaei, Maryam; Amiri, Shayan; Haj-Mirzaian, Arya; Khedri, Mostafa; Jahanabadi, Samane; Mohammadi-Asl, Ali; Mehr, Shahram Ejtemaie; Dehpour, Ahmad Reza

    2016-09-01

    Arsenic trioxide (ATO) is an efficient drug for the treatment of the patients with acute promyelocytic leukemia (APL). Inhibition of proliferation as well as apoptosis, attenuation of migration, and induction of differentiation in tumor cells are the main mechanisms through which ATO acts against APL. Despite advantages of ATO in treatment of some malignancies, certain harmful side effects, such as cardiotoxicity, have been reported. It has been well documented that morphine has antioxidant, anti-apoptotic, and cytoprotective properties and is able to attenuate cytotoxicity. Therefore, in this study, we aimed to investigate the protective effects of morphine against ATO toxicity in H9c2 myocytes using multi-parametric assay including thiazolyl blue tetrazolium bromide (MTT) assay, reactive oxygen species (ROS) generation, caspase 3 activity, nuclear factor kappa B (NF-κB) phosphorylation assay, and expression of apoptotic markers. Our results showed that morphine (1 μM) attenuated cytotoxicity induced by ATO in H9c2 cells. Results of this study suggest that morphine may have protective properties in management of cardiac toxicity in patients who receive ATO as an anti-cancer treatment. PMID:26815588

  11. Myricitrin Attenuates High Glucose-Induced Apoptosis through Activating Akt-Nrf2 Signaling in H9c2 Cardiomyocytes.

    PubMed

    Zhang, Bin; Chen, Yaping; Shen, Qiang; Liu, Guiyan; Ye, Jingxue; Sun, Guibo; Sun, Xiaobo

    2016-01-01

    Hyperglycemia, as well as diabetes mellitus, has been shown to trigger cardiac cell apoptosis. We have previously demonstrated that myricitrin prevents endothelial cell apoptosis. However, whether myricitrin can attenuate H9c2 cell apoptosis remains unknown. In this study, we established an experiment model in H9c2 cells exposed to high glucose. We tested the hypothesis that myricitrin may inhibit high glucose (HG)-induced cardiac cell apoptosis as determined by TUNEL staining. Furthermore, myricitrin promoted antioxidative enzyme production, suppressed high glucose-induced reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) in H9c2 cells. This agent significantly inhibited apoptotic protein expression, activated Akt and facilitated the transcription of NF-E2-related factor 2 (Nrf2)-mediated protein (heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO-1) expression as determined by Western blotting. Significantly, an Akt inhibitor (LY294002) or HO-1 inhibitor (ZnPP) not only inhibited myricitrin-induced HO-1/NQO-1 upregulation but also alleviated its anti-apoptotic effects. In summary, these observations demonstrate that myricitrin activates Nrf2-mediated anti-oxidant signaling and attenuates H9c2 cell apoptosis induced by high glucose via activation of Akt signaling. PMID:27399653

  12. H9c2 cardiomyoblasts produce thyroid hormone.

    PubMed

    Meischl, Christof; Buermans, Henk P; Hazes, Thierry; Zuidwijk, Marian J; Musters, René J P; Boer, Christa; van Lingen, Arthur; Simonides, Warner S; Blankenstein, Marinus A; Dupuy, Corrine; Paulus, Walter J; Hack, C Erik; Ris-Stalpers, Carrie; Roos, Dirk; Niessen, Hans W M

    2008-05-01

    Thyroid hormone acts on a wide range of tissues. In the cardiovascular system, thyroid hormone is an important regulator of cardiac function and cardiovascular hemodynamics. Although some early reports in the literature suggested an unknown extrathyroidal source of thyroid hormone, it is currently thought to be produced exclusively in the thyroid gland, a highly specialized organ with the sole function of generating, storing, and secreting thyroid hormone. Whereas most of the proteins necessary for thyroid hormone synthesis are thought to be expressed exclusively in the thyroid gland, we now have found evidence that all of these proteins, i.e., thyroglobulin, DUOX1, DUOX2, the sodium-iodide symporter, pendrin, thyroid peroxidase, and thyroid-stimulating hormone receptor, are also expressed in cardiomyocytes. Furthermore, we found thyroglobulin to be transiently upregulated in an in vitro model of ischemia. When performing these experiments in the presence of 125 I, we found that 125 I was integrated into thyroglobulin and that under ischemia-like conditions the radioactive signal in thyroglobulin was reduced. Concomitantly we observed an increase of intracellularly produced, 125 I-labeled thyroid hormone. In conclusion, our findings demonstrate for the first time that cardiomyocytes produce thyroid hormone in a manner adapted to the cell's environment.

  13. 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-κB, 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

  14. Beneficial properties of selenium incorporated guar gum nanoparticles against ischemia/reperfusion in cardiomyoblasts (H9c2).

    PubMed

    Soumya, R S; Vineetha, V P; Salin Raj, P; Raghu, K G

    2014-11-01

    Nanotechnology for the treatment and diagnosis has been emerging recently as a potential area of research and development. In the present study, selenium incorporated guar gum nanoparticles have been prepared by nanoprecipitation and characterized by transmission electron microscopy and particle size analysis. The nanoparticles were screened for antioxidant potential (metal chelation, total reducing power and hydroxyl radical scavenging activity) and were evaluated against the cell line based cardiac ischemia/reperfusion model with special emphasis on oxidative stress and mitochondrial parameters. The cell based cardiac ischemia model was employed using H9c2 cell lines. Investigations revealed that there was a significant alteration (P ≤ 0.05) in the innate antioxidant status (glutathione↓, glutathione peroxidase↓, thioredoxin reductase↓, superoxide dismutase↓, catalase↓, lipid peroxidation↑, protein carbonyl↑, xanthine oxidase↑ and caspase 3 activity↑), mitochondrial functions (reactive oxygen species generation, membrane potential, and pore opening) and calcium homeostasis (calcium ATPase and intracellular calcium overload) during both ischemia and reperfusion. For comparative evaluation, selenium, guar gum and selenium incorporated guar gum nanoparticles were evaluated for their protective properties against ischemia/reperfusion. The study reveals that selenium incorporated guar gum nanoparticles were better at protecting the cells from ischemia/reperfusion compared to selenium and guar gum nanoparticles. The potent antioxidant capability shown by the sample in in vitro assays may be the biochemical basis of its better biological activity. Further, the nanodimensions of the particle may be the additional factor responsible for its better effect.

  15. Nanocurcumin protects cardiomyoblasts H9c2 from hypoxia-induced hypertrophy and apoptosis by improving oxidative balance.

    PubMed

    Nehra, Sarita; Bhardwaj, Varun; Kalra, Namita; Ganju, Lilly; Bansal, Anju; Saxena, Shweta; Saraswat, Deepika

    2015-06-01

    Hypoxia-induced cardiomyocyte hypertrophy is evident; however, the distinct molecular mechanism underlying the oxidative stress-mediated damages to cardiomyocytes remains unknown. Curcumin (diferuloylmethane) is known for anti-hypertrophic effects, but low bioavailability makes it unsuitable to exploit its pharmacological properties. We assessed the efficacy of nanotized curcumin, i.e. nanocurcumin, in ameliorating hypoxia-induced hypertrophy and apoptosis in H9c2 cardiomyoblasts and compared it to curcumin. H9c2 cardiomyoblasts were challenged with 0.5 % oxygen, for 24 h to assess hypoxia-induced oxidative damage, hypertrophy and consequent apoptosis. The molecular mechanism underlying the protective efficacy of nanocurcumin was evaluated in regulating Raf-1/Erk-1/2 apoptosis by caspase-3/-7 pathway and oxidative stress. Nanocurcumin ameliorated hypoxia-induced hypertrophy and apoptosis in H9c2 cells significantly (p ≤ 0.01), by downregulating atrial natriuretic factor expression, caspase-3/-7 activation, oxidative stress and stabilizing hypoxia-inducible factor-1α (HIF-1α) better than curcumin. Nanocurcumin provides insight into its use as a potential candidate in curing hypoxia-induced cardiac pathologies by restoring oxidative balance. PMID:25846484

  16. MicroRNA-29a-3p attenuates ET-1-induced hypertrophic responses in H9c2 cardiomyocytes.

    PubMed

    Li, Man; Wang, Nan; Zhang, Jian; He, Hong-Peng; Gong, Hui-Qin; Zhang, Rui; Song, Tie-Feng; Zhang, Li-Nan; Guo, Zhi-Xia; Cao, Dong-Sun; Zhang, Tong-Cun

    2016-07-01

    Transcription factor nuclear factor of activated T cells c4 (NFATc4) is the best-characterized target for the development of cardiac hypertrophy. Aberrant microRNA-29 (miR-29) expression is involved in the development of cardiac fibrosis and congestive heart failure. However, whether miR-29 regulates hypertrophic processes is still not clear. In this study, we investigated the potential functions of miR-29a-3p in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. We showed that miR-29a-3p was down-regulated in ET-1-treated H9c2 cardiomyocytes. Overexpression of miR-29a-3p significantly reduced ET-1-induced hypertrophic responses in H9c2 cardiomyocytes, which was accompanied by a decrease in NFATc4 expression. miR-29a-3p targeted directly to the 3'-UTR of NFATc4 mRNA and silenced NFATc4 expression. Our results indicate that miR-29a-3p inhibits ET-1-induced cardiomyocyte hypertrophy via inhibiting NFATc4 expression.

  17. EGCG Blocked Phenylephrin-Induced Hypertrophy in H9C2 Cardiomyocytes, by Activating AMPK-Dependent Pathway.

    PubMed

    Cai, Yi; Zhao, Li; Qin, Yuan; Wu, Xiao-Qian

    2015-05-01

    AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism. Previous studies have shown that activation of AMPK results in suppression of cardiac myocyte hypertrophy via inhibition of the p70S6 kinase (p70S6K) and eukaryotic elongation factor-2 (eEF2) signaling pathways. Epigallocatechin-3-gallate (EGCG), the major polyphenol found in green tea, possesses multiple protective effects on the cardiovascular system including cardiac hypertrophy. However, the molecular mechanisms has not been well investigated. In this study, we found that EGCG could significantly reduce natriuretic peptides type A (Nppa), brain natriuretic polypeptide (BNP) mRNA expression and decrease cell surface area in H9C2 cardiomyocytes stimulated with phenylephrine (PE). Moreover, we showed that AMPK is activated in H9C2 cardiomyocytes by EGCG, and AMPK-dependent pathway participates in the inhibitory effects of EGCG on cardiac hypertrophy. Taken together, our findings provide the first evidence that the effect of EGCG against cardiac hypertrophy may be attributed to its activation on AMPK-dependent signaling pathway, suggesting the therapeutic potential of EGCG on the prevention of cardiac remodeling in patients with pressure overload hypertrophy. PMID:25954124

  18. MicroRNA-29a-3p attenuates ET-1-induced hypertrophic responses in H9c2 cardiomyocytes.

    PubMed

    Li, Man; Wang, Nan; Zhang, Jian; He, Hong-Peng; Gong, Hui-Qin; Zhang, Rui; Song, Tie-Feng; Zhang, Li-Nan; Guo, Zhi-Xia; Cao, Dong-Sun; Zhang, Tong-Cun

    2016-07-01

    Transcription factor nuclear factor of activated T cells c4 (NFATc4) is the best-characterized target for the development of cardiac hypertrophy. Aberrant microRNA-29 (miR-29) expression is involved in the development of cardiac fibrosis and congestive heart failure. However, whether miR-29 regulates hypertrophic processes is still not clear. In this study, we investigated the potential functions of miR-29a-3p in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. We showed that miR-29a-3p was down-regulated in ET-1-treated H9c2 cardiomyocytes. Overexpression of miR-29a-3p significantly reduced ET-1-induced hypertrophic responses in H9c2 cardiomyocytes, which was accompanied by a decrease in NFATc4 expression. miR-29a-3p targeted directly to the 3'-UTR of NFATc4 mRNA and silenced NFATc4 expression. Our results indicate that miR-29a-3p inhibits ET-1-induced cardiomyocyte hypertrophy via inhibiting NFATc4 expression. PMID:26992639

  19. 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

  20. Adipocytokine, omentin inhibits doxorubicin-induced H9c2 cardiomyoblasts apoptosis through the inhibition of mitochondrial reactive oxygen species.

    PubMed

    Kazama, Kyosuke; Okada, Muneyoshi; Yamawaki, Hideyuki

    2015-02-20

    Omentin is a relatively novel adipocyte-derived cytokine mainly expressed in visceral adipose tissues. Blood omentin level decreases in the patients with obesity, hypertension, type 2 diabetes and atherosclerosis. We have previously demonstrated that omentin inhibits key pathological processes for hypertension development, including vascular inflammatory responses, contractile reactivity and structural remodeling. In addition, there are several reports demonstrating that omentin prevents cardiac hypertrophy and myocardial ischemic injury. Doxorubicin (DOX) is an effective anti-cancer drug with cardiotoxic side effect. Here we tested the hypothesis that omentin may prevent DOX-induced cardiac cytotoxicity. H9c2 rat cardiomyoblasts were treated with DOX in the absence or presence of omentin. Omentin (300 ng/ml, 3 h pretreatment) significantly inhibited DOX (1 μM, 18 h)-induced decreases in living cell number as determined by a colorimetric cell counting assay. Omentin (300 ng/ml, 3 h) significantly inhibited DOX (1 μM, 12 h)-induced cleaved caspase-3 expression as determined by Western blotting. Omentin (300 ng/ml, 3 h) significantly inhibited DOX (1 μM, 6 h)-induced mitochondrial reactive oxygen species (ROS) production as determined by a MitoSOX Red fluorescent staining. In addition, a mitochondrial respiratory chain complex I inhibitor, rotenone (0.5 μM, 3 h pretreatment), significantly inhibited DOX (1 μM, 6-18 h)-induced decreases of living cell number, cleaved caspase-3 expression and mitochondrial ROS production. In summary, we for the first time demonstrate that omentin prevents DOX-induced H9c2 cells apoptosis through the inhibition of mitochondrial ROS production. These results indicate omentin as an attractive pharmaco-therapeautic target against DOX-induced cardiac side effect. PMID:25600813

  1. Eupatilin inhibits the apoptosis in H9c2 cardiomyocytes via the Akt/GSK-3β pathway following hypoxia/reoxygenation injury.

    PubMed

    Qiao, Zengyong; Xu, Ya-Wei; Yang, Jingyu

    2016-08-01

    Eupatilin, a pharmacologically active flavone derived from the Artemisia plant species, has been reported to have anti-oxidant, anti-inflammatory, anti-allergic, and neuroprotective activities against cerebral ischemia/reperfusion (I/R). However, the role of eupatilin in myocardial I/R injury remains unclear. In the present study, we aimed to investigate the potential molecular mechanisms against hypoxia/reoxygenation (H/R) induced cardiomyocytes apoptosis in vitro. Our results showed that eupatilin markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. Eupatilin also suppressed oxidative stress and apoptosis in H9c2 cells after myocardial I/R injury. Furthermore, eupatilin obviously increased the phosphorylation of Akt and GSK-3β in H9c2 cells. Our results suggested that eupatilin could provide significant cardioprotection against myocardial I/R injury, and the potential mechanisms might involve inhibition of cardiomyocyte apoptosis through activating the Akt/GSK-3β signaling pathway. PMID:27470375

  2. Resistin-induced cardiomyocyte hypertrophy is inhibited by apelin through the inactivation of extracellular signal-regulated kinase signaling pathway in H9c2 embryonic rat cardiomyocytes

    PubMed Central

    Luo, Jian-Wei; Zheng, Xian; Cheng, Guan-Chang; Ye, Qun-Hui; Deng, Yong-Zhi; Wu, Lin

    2016-01-01

    It has been reported that resistin induces, whereas apelin inhibits cardiac hypertrophy. However, the underlying molecular mechanisms of apelin inhibiting resistin-induced cardiac hypertrophy remain unclear. The aim of the current study is to investigate the effects of apelin on resistin-induced cardiomyocyte hypertrophy and elucidate the underlying molecular mechanism. H9c2 cells were used in the present study, and cell surface area and protein synthesis were evaluated. Reverse transcription-quantitative polymerase chain reaction was performed to analyze the expression levels of hypertrophic markers, brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC). In addition, western blotting was conducted to examine phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Following treatment of H9c2 cells with resistin, cell surface area, protein synthesis, and BNP and β-MHC mRNA expression levels were increased. Subsequent to co-treatment of H9c2 cells with apelin and resistin, lead to the inhibition of resistin-induced hypertrophic effects by apelin. In addition, treatment with resistin increased phosphorylation of ERK1/2, whereas pretreatment with apelin decreased phosphorylation of ERK1/2, which was increased by resistin. These results indicate that resistin-induced cardiac hypertrophy is inhibited by apelin via inactivation of ERK1/2 cell signaling.

  3. Resistin-induced cardiomyocyte hypertrophy is inhibited by apelin through the inactivation of extracellular signal-regulated kinase signaling pathway in H9c2 embryonic rat cardiomyocytes

    PubMed Central

    Luo, Jian-Wei; Zheng, Xian; Cheng, Guan-Chang; Ye, Qun-Hui; Deng, Yong-Zhi; Wu, Lin

    2016-01-01

    It has been reported that resistin induces, whereas apelin inhibits cardiac hypertrophy. However, the underlying molecular mechanisms of apelin inhibiting resistin-induced cardiac hypertrophy remain unclear. The aim of the current study is to investigate the effects of apelin on resistin-induced cardiomyocyte hypertrophy and elucidate the underlying molecular mechanism. H9c2 cells were used in the present study, and cell surface area and protein synthesis were evaluated. Reverse transcription-quantitative polymerase chain reaction was performed to analyze the expression levels of hypertrophic markers, brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC). In addition, western blotting was conducted to examine phosphorylation of extracellular signal-regulated kinase (ERK)1/2. Following treatment of H9c2 cells with resistin, cell surface area, protein synthesis, and BNP and β-MHC mRNA expression levels were increased. Subsequent to co-treatment of H9c2 cells with apelin and resistin, lead to the inhibition of resistin-induced hypertrophic effects by apelin. In addition, treatment with resistin increased phosphorylation of ERK1/2, whereas pretreatment with apelin decreased phosphorylation of ERK1/2, which was increased by resistin. These results indicate that resistin-induced cardiac hypertrophy is inhibited by apelin via inactivation of ERK1/2 cell signaling. PMID:27699016

  4. Discovering Antioxidant Molecules in the Archaea Domain: Peroxiredoxin Bcp1 from Sulfolobus solfataricus Protects H9c2 Cardiomyoblasts from Oxidative Stress

    PubMed Central

    Sarcinelli, Carmen; Pizzo, Elio

    2016-01-01

    Peroxiredoxins (Prxs) are ubiquitous thiol peroxidases that are involved in the reduction of peroxides. It has been reported that prokaryotic Prxs generally show greater structural robustness than their eukaryotic counterparts, making them less prone to inactivation by overoxidation. This difference has inspired the search for new antioxidants from prokaryotic sources that can be used as possible therapeutic biodrugs. Bacterioferritin comigratory proteins (Bcps) of the hyperthermophilic archaeon Sulfolobus solfataricus that belong to the Prx family have recently been characterized. One of these proteins, Bcp1, was chosen to determine its antioxidant effects in H9c2 rat cardiomyoblast cells. Bcp1 activity was measured in vitro under physiological temperature and pH conditions that are typical of mammalian cells; the yeast thioredoxin reductase (yTrxR)/thioredoxin (yTrx) reducing system was used to evaluate enzyme activity. A TAT-Bcp1 fusion protein was constructed to allow its internalization and verify the effect of Bcp1 on H9c2 rat cardiomyoblasts subjected to oxidative stress. The results reveal that TAT-Bcp1 is not cytotoxic and inhibits H2O2-induced apoptosis in H9c2 cells by reducing the H2O2 content inside these cells. PMID:27752237

  5. Attenuated SAG expression exacerbates 4-hydroxy-2-nonenal-induced apoptosis and hypertrophy of H9c2 cardiomyocytes.

    PubMed

    Park, J H; Lee, J H; Park, J-W

    2015-01-01

    Oxidative stress, associated with the accumulation of reactive oxygen species (ROS), results in numerous and detrimental effects on the myocardium such as the induction of apoptotic cell death, hypertrophy, fibrosis, dysfunction, and dilatation. The product of sensitive to apoptosis gene (SAG) is a RING finger protein that has been shown to have a protective effect against apoptosis induced by oxidative stress in various cell types. The major reactive aldehydic product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), is believed to be largely responsible for cytopathological effects observed during oxidative stress. In the present study, we showed that the transfection of H9c2 clonal myoblastic cells with small interfering RNA (siRNA) specific for SAG markedly attenuated SAG expression and exacerbates HNE-induced apoptosis and hypertrophy. The knockdown of SAG expression resulted in the modulation of cellular redox status, mitochondrial function, and cellular oxidative damage. Taken together, our results showed that the suppression of SAG expression by siRNA enhanced HNE-induced apoptosis and hypertrophy of cultured cardiomyocytes via the disruption of the cellular redox balance. Given the importance of the SAG protein in the regulation of the redox status of cardiomyocytes, we conclude that this protein may be a potential new target in the development of therapeutic agents for the prevention of cardiovascular diseases. PMID:25850934

  6. Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes

    PubMed Central

    SHI, XIAOJING; LI, YANG; HU, JUN; YU, BO

    2016-01-01

    Tert-butylhydroquinone (tBHQ), an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2), has been demonstrated to attenuate oxidative stress-induced injury and the apoptosis of human neural stem cells and other cell types. However, whether tBHQ is able to exert a protective effect against oxidative stress and the apoptosis of cardiomyocytes has not yet been determined. Thus, the objective of the present study was to determine whether tBHQ protects H9c2 cardiomyocytes against ethanol-induced apoptosis. For this purpose, four sets of experiments were performed under standard culture conditions as follows: i) untreated control cells; ii) cell treatment with 200 mM ethanol; iii) cell treatment with 5 µM tBHQ; and iv) cell pre-treatment with 5 µM tBHQ for 24 h, followed by medium change and co-culture with 200 mM ethanol containing 5 µM tBHQ for a further 24 h. The viability of the cardiomyocytes was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The levels of intracellular reactive oxygen species (ROS) and apoptosis were assessed by flow cytometry. Protein expression was measured by western blot analysis, and Nrf2 nuclear localization was observed by immunofluorescence. Exposure to ethanol led to a decrease in the protein expression of Nrf2 and its downstream antioxidant enzymes, accompanied by an increase in ROS generation and in the apoptosis of H9c2 cells. Pre-treatment with tBHQ significantly prevented the H9c2 cells from undergoing ethanol-induced apoptosis. tBHQ also increased the expression of B-cell lymphoma-2 (Bcl-2), whereas Bcl-2-associated X protein (Bax) expression was decreased. tBHQ promoted Nrf2 nuclear localization and increased the expression of Nrf2, superoxide dismutase (SOD), catalase (CAT) and heme oxygenase-1 (HO-1), and simultaneously inhibited the ethanol-induced overproduction of intracellular ROS. Therefore, tBHQ confers protection against the ethanol-induced apoptosis of and activates the

  7. Tert-butylhydroquinone attenuates the ethanol-induced apoptosis of and activates the Nrf2 antioxidant defense pathway in H9c2 cardiomyocytes.

    PubMed

    Shi, Xiaojing; Li, Yang; Hu, Jun; Yu, Bo

    2016-07-01

    Tert-butylhydroquinone (tBHQ), an inducer of nuclear factor erythroid 2-related factor 2 (Nrf2), has been demonstrated to attenuate oxidative stress-induced injury and the apoptosis of human neural stem cells and other cell types. However, whether tBHQ is able to exert a protective effect against oxidative stress and the apoptosis of cardiomyocytes has not yet been determined. Thus, the objective of the present study was to determine whether tBHQ protects H9c2 cardiomyocytes against ethanol-induced apoptosis. For this purpose, four sets of experiments were performed under standard culture conditions as follows: i) untreated control cells; ii) cell treatment with 200 mM ethanol; iii) cell treatment with 5 µM tBHQ; and iv) cell pre-treatment with 5 µM tBHQ for 24 h, followed by medium change and co-culture with 200 mM ethanol containing 5 µM tBHQ for a further 24 h. The viability of the cardiomyocytes was evaluated by 3-(4,5-dimethylthiazol‑2-yl)‑2,5-diphenyltetrazolium bromide (MTT) assay. The levels of intracellular reactive oxygen species (ROS) and apoptosis were assessed by flow cytometry. Protein expression was measured by western blot analysis, and Nrf2 nuclear localization was observed by immunofluorescence. Exposure to ethanol led to a decrease in the protein expression of Nrf2 and its downstream antioxidant enzymes, accompanied by an increase in ROS generation and in the apoptosis of H9c2 cells. Pre-treatment with tBHQ significantly prevented the H9c2 cells from undergoing ethanol‑induced apoptosis. tBHQ also increased the expression of B-cell lymphoma-2 (Bcl-2), whereas Bcl-2-associated X protein (Bax) expression was decreased. tBHQ promoted Nrf2 nuclear localization and increased the expression of Nrf2, superoxide dismutase (SOD), catalase (CAT) and heme oxygenase-1 (HO-1), and simultaneously inhibited the ethanol‑induced overproduction of intracellular ROS. Therefore, tBHQ confers protection against the ethanol

  8. Dexamethasone promotes hypertrophy of H9C2 cardiomyocytes through calcineurin B pathway, independent of NFAT activation.

    PubMed

    Sangeetha, K N; Lakshmi, B S; Niranjali Devaraj, S

    2016-01-01

    Metabolic syndrome-induced cardiac hypertrophy is a global concern leading to an increase in the morbidity and mortality of patients, with the signalling mechanism associated with them still unclear. The present study attempts to understand the metabolic syndrome-associated cardiac hypertrophy through an in vitro model using external stimuli well known for inducing metabolic disorders, i.e. dexamethasone (DEX), a synthetic glucocorticoid. DEX (0.1 and 1 μM) promoted cardiac hypertrophy in H9C2 cells at 4 days of treatment as evidenced through increased cell size and protein content. A significant induction in foetal gene reprogramming was observed, confirming the establishment of hypertrophy. Moreover, the hypertrophic response at 4 days was perceived to be physiological at 0.1 μM and pathological at 1 μM based on α-MHC and IGF1R expression, but complete inhibition in the PKB/AKT expression confirmed it to be pathological hypertrophy at both the concentrations (0.1 and 1 μM). The present study reports for the first time the mechanistic insights into DEX-mediated hypertrophy. It is hypothesized to be orchestrated through the activation of AT1R that is involved in the alteration of the cardiac isoform of SERCA2 expression perturbing the calcium homeostasis. This leads to the activation of calcineurin B, independent of NFAT involvement, which in coordination with ROS induces the activation of JNK of the MAPK signalling. PMID:26511233

  9. The antioxidant edaravone prevents cardiac dysfunction by suppressing oxidative stress in type 1 diabetic rats and in high-glucose-induced injured H9c2 cardiomyoblasts.

    PubMed

    Ji, Lei; Liu, Yingying; Zhang, Ying; Chang, Wenguang; Gong, Junli; Wei, Shengnan; Li, Xudong; Qin, Ling

    2016-09-01

    Edaravone, a radical scavenger, has been recognized as a potential protective agent for cardiovascular diseases. However, little is known about the effect of edaravone in cardiac complications associated with diabetes. Here, we have demonstrated that edaravone prevents cardiac dysfunction and apoptosis in the streptozotocin-induced type 1 diabetic rat heart. Mechanistic studies revealed that edaravone treatment improved cardiac function and restored superoxide dismutase levels. In addition, treatment of diabetic animals by edaravone increased protein expressions of sirtuin-1 (SIRT-1), peroxisome proliferator activated receptor γ coactivator α (PGC-1α), nuclear factor like-2 (NRF-2), and B cell lymphoma 2 (Bcl-2), and reduced protein expressions of Bax and Caspase-3 compared to the control group. High glucose incubation resulted in the production of reactive oxygen species (ROS) and cell death. Treatment of high-glucose-incubated H9c2 cells by edaravone reduced ROS production and cell death. In addition, the treatment of high-glucose-incubated H9c2 cells by edaravone increased the activity of antioxidative stress by increasing SIRT-1, PGC-1α, and NRF-2, and this treatment also reduced apoptosis by increasing Bcl-2 expression and reducing Bax and Caspase-3 expressions. Knockdown SIRT-1 with small interferer RNA abolished the effects of edaravone. Overall, our data demonstrated that edaravone may be an effective agent against the development of diabetic cardiomyopathy. PMID:27376621

  10. Alpinate Oxyphyllae Fructus Inhibits IGFII-Related Signaling Pathway to Attenuate Ang II-Induced Pathological Hypertrophy in H9c2 Cardiomyoblasts.

    PubMed

    Tsai, Chuan-Te; Chang, Yung-Ming; Lin, Shu-Luan; Chen, Yueh-Sheng; Yeh, Yu-Lan; Padma, Viswanadha Vijaya; Tsai, Chin-Chuan; Chen, Ray-Jade; Ho, Tsung-Jung; Huang, Chih-Yang

    2016-03-01

    Angiotensin II (Ang II) is a very important cardiovascular disease inducer and may cause cardiac pathological hypertrophy and remodeling. We evaluated a Chinese traditional medicine, alpinate oxyphyllae fructus (AOF), for therapeutic efficacy for treating Ang II-induced cardiac hypertrophy. AOF has been used to treat patients with various symptoms accompanying hypertension and cerebrovascular disorders in Korea. We investigated its protective effect against Ang II-induced cytoskeletal change and hypertrophy in H9c2 cells. The results showed that treating cells with Ang II resulted in pathological hypertrophy, such as increased expression of transcription factors NFAT-3/p-NFAT-3, hypertrophic response genes (atrial natriuretic peptide [ANP] and b-type natriuretic peptide [BNP]), and Gαq down-stream effectors (PLCβ3 and calcineurin). Pretreatment with AOF (60-100 μg/mL) led to significantly reduced hypertrophy. We also found that AOF pretreatment significantly suppressed the cardiac remodeling proteins, metalloproteinase (MMP9 and MMP2), and tissue plasminogen activator (tPA), induced by Ang II challenge. In conclusion, we provide evidence that AOF protects against Ang II-induced pathological hypertrophy by specifically inhibiting the insulin-like growth factor (IGF) II/IIR-related signaling pathway in H9c2 cells. AOF might be a candidate for cardiac hypertrophy and ventricular remodeling prevention in chronic cardiovascular diseases. PMID:26987022

  11. Elatoside C protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes through the reduction of endoplasmic reticulum stress partially depending on STAT3 activation.

    PubMed

    Wang, Min; Meng, Xiang-bao; Yu, Ying-li; Sun, Gui-bo; Xu, Xu-dong; Zhang, Xiao-po; Dong, Xi; Ye, Jing-xue; Xu, Hui-bo; Sun, Yi-fan; Sun, Xiao-bo

    2014-12-01

    Endoplasmic reticulum (ER) stress-induced apoptosis has been suggested to contribute to myocardial ischemia-reperfusion (I/R) injury. Elatoside C is one of the major triterpenoid compounds isolated from Aralia elata that is known to be cardioprotective. However, its effects on I/R injury to cardiac myocytes have not been clarified. This study aimed to investigate the possible protective effect of Elatoside C against hypoxia/reoxygenation (H/R)-induced H9c2 cardiomyocyte injury and its underlying mechanisms. H9c2 cardiomyocytes were subjected to H/R in the presence of Elatoside C. Our results showed that Elatoside C (25 μM) treatment provided significant protection against H/R-induced cell death, as evidenced by improved cell viability, maintained mitochondrial membrane potential, diminished mitochondrial ROS, and reduced apoptotic cardiomyocytes (P < 0.05). These changes were associated with the inhibition of ER stress-associated apoptosis markers (GRP78, CHOP, Caspase-12 and JNK), as well as the increased phosphorylation of STAT3 and an increased Bcl2/Bax ratio. Moreover, these effects of Elatoside C were prevented by the STAT3 inhibitor Stattic. Taken together, these results suggested that Elatoside C can alleviate H/R-induced cardiomyocyte apoptosis most likely by activating the STAT3 pathways and reducing ER stress-associated apoptosis. PMID:25326083

  12. Clematichinenoside (AR) Attenuates Hypoxia/Reoxygenation-Induced H9c2 Cardiomyocyte Apoptosis via a Mitochondria-Mediated Signaling Pathway.

    PubMed

    Ding, Haiyan; Han, Rong; Chen, Xueshan; Fang, Weirong; Liu, Meng; Wang, Xuemei; Wei, Qin; Kodithuwakku, Nandani Darshika; Li, Yunman

    2016-05-30

    Mitochondria-mediated cardiomyocyte apoptosis is involved in myocardial ischemia/reperfusion (MI/R) injury. Clematichinenoside (AR) is a triterpenoid saponin isolated from the roots of Clematis chinensis with antioxidant and anti-inflammatory cardioprotection effects against MI/R injury, yet the anti-apoptotic effect and underlying mechanisms of AR in MI/R injury remain unclear. We hypothesize that AR may improve mitochondrial function to inhibit MI/R-induced cardiomyocyte apoptosis. In this study, we replicated an in vitro H9c2 cardiomyocyte MI/R model by hypoxia/reoxygenation (H/R) treatment. The viability of H9c2 cardiomyocytes was determined by MTT assay; apoptosis was evaluated by flow cytometry and TUNEL experiments; mitochondrial permeability transition pore (mPTP) opening was analyzed by a calcein-cobalt quenching method; and mitochondrial membrane potential (ΔΨm) was detected by JC-1. Moreover, we used western blots to determine the mitochondrial cytochrome c translocation to cytosolic and the expression of caspase-3, Bcl-2, and Bax proteins. These results showed that the application of AR decreased the ratio of apoptosis and the extent of mPTP opening, but increased ΔΨm. AR also inhibited H/R-induced release of mitochondrial cytochrome c and decreased the expression of the caspase-3, Bax proteins. Conversely, it remarkably increased the expression of Bcl-2 protein. Taken together, these results revealed that AR protects H9c2 cardiomyocytes against H/R-induced apoptosis through mitochondrial-mediated apoptotic signaling pathway.

  13. Berberine treatment attenuates the palmitate-mediated inhibition of glucose uptake and consumption through increased 1,2,3-triacyl-sn-glycerol synthesis and accumulation in H9c2 cardiomyocytes.

    PubMed

    Chang, Wenguang; Chen, Li; Hatch, Grant M

    2016-04-01

    Dysfunction of lipid metabolism and accumulation of 1,2-diacyl-sn-glycerol (DAG) may be a key factor in the development of insulin resistance in type 2 diabetes. Berberine (BBR) is an isoquinoline alkaloid extract that has shown promise as a hypoglycemic agent in the management of diabetes in animal and human studies. However, its mechanism of action is not well understood. To determine the effect of BBR on lipid synthesis and its relationship to insulin resistance in H9c2 cardiomyocytes, we measured neutral lipid and phospholipid synthesis and their relationship to glucose uptake. Compared with controls, BBR treatment stimulated 2-[1,2-(3)H(N)]deoxy-D-glucose uptake and consumption in palmitate-mediated insulin resistant H9c2 cells. The mechanism was though an increase in protein kinase B (AKT) activity and GLUT-4 glucose transporter expression. DAG accumulated in palmitate-mediated insulin resistant H9c2 cells and treatment with BBR reduced this DAG accumulation and increased accumulation of 1,2,3-triacyl-sn-glycerol (TAG) compared to controls. Treatment of palmitate-mediated insulin resistant H9c2 cells with BBR increased [1,3-(3)H]glycerol and [1-(14)C]glucose incorporation into TAG and reduced their incorporation into DAG compared to control. In addition, BBR treatment of these cells increased [1-(14)C]palmitic acid incorporation into TAG and decreased its incorporation into DAG compared to controls. BBR treatment did not alter phosphatidylcholine or phosphatidylethanolamine synthesis. The mechanism for the BBR-mediated decreased precursor incorporation into DAG and increased incorporation into TAG in palmitate-incubated cells was an increase in DAG acyltransferase-2 activity and its expression and a decrease in TAG hydrolysis. Thus, BBR treatment attenuates palmitate-induced reduction in glucose uptake and consumption, in part, through reduction in cellular DAG levels and accumulation of TAG in H9c2 cells. PMID:26774040

  14. Berberine treatment attenuates the palmitate-mediated inhibition of glucose uptake and consumption through increased 1,2,3-triacyl-sn-glycerol synthesis and accumulation in H9c2 cardiomyocytes.

    PubMed

    Chang, Wenguang; Chen, Li; Hatch, Grant M

    2016-04-01

    Dysfunction of lipid metabolism and accumulation of 1,2-diacyl-sn-glycerol (DAG) may be a key factor in the development of insulin resistance in type 2 diabetes. Berberine (BBR) is an isoquinoline alkaloid extract that has shown promise as a hypoglycemic agent in the management of diabetes in animal and human studies. However, its mechanism of action is not well understood. To determine the effect of BBR on lipid synthesis and its relationship to insulin resistance in H9c2 cardiomyocytes, we measured neutral lipid and phospholipid synthesis and their relationship to glucose uptake. Compared with controls, BBR treatment stimulated 2-[1,2-(3)H(N)]deoxy-D-glucose uptake and consumption in palmitate-mediated insulin resistant H9c2 cells. The mechanism was though an increase in protein kinase B (AKT) activity and GLUT-4 glucose transporter expression. DAG accumulated in palmitate-mediated insulin resistant H9c2 cells and treatment with BBR reduced this DAG accumulation and increased accumulation of 1,2,3-triacyl-sn-glycerol (TAG) compared to controls. Treatment of palmitate-mediated insulin resistant H9c2 cells with BBR increased [1,3-(3)H]glycerol and [1-(14)C]glucose incorporation into TAG and reduced their incorporation into DAG compared to control. In addition, BBR treatment of these cells increased [1-(14)C]palmitic acid incorporation into TAG and decreased its incorporation into DAG compared to controls. BBR treatment did not alter phosphatidylcholine or phosphatidylethanolamine synthesis. The mechanism for the BBR-mediated decreased precursor incorporation into DAG and increased incorporation into TAG in palmitate-incubated cells was an increase in DAG acyltransferase-2 activity and its expression and a decrease in TAG hydrolysis. Thus, BBR treatment attenuates palmitate-induced reduction in glucose uptake and consumption, in part, through reduction in cellular DAG levels and accumulation of TAG in H9c2 cells.

  15. Curcumin induces the apoptotic intrinsic pathway via upregulation of reactive oxygen species and JNKs in H9c2 cardiac myoblasts.

    PubMed

    Zikaki, Kyriaki; Aggeli, Ioanna-Katerina; Gaitanaki, Catherine; Beis, Isidoros

    2014-06-01

    Curcumin derived from the rhizome of turmeric (Curcuma longa L.), is a well known coloring culinary agent, that has therapeutic properties against diverse pathologies such as cancer, atherosclerosis and heart failure. Given the salutary potential of curcumin, deciphering its mode of action particularly in cardiac cells, is of outstanding value. Accumulating evidence implicates curcumin in the regulation of multiple signaling pathways leading to cell survival or apoptosis. Therefore, the present study aimed at elucidating the molecular mechanisms triggered by curcumin in H9c2 cells. Curcumin was found to activate p38-mitogen-activated protein kinase (p38-MAPK) as well as c-jun NH2 terminal kinases (JNKs), in a dose- and time-dependent manner. We also observed curcumin to impair cell survival by promoting apoptosis, evidenced by chromatin condensation, poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage, as well as Bax translocation and cytochrome c release into the cytosol. Curcumin-induced apoptosis was ascribed to JNKs, since hindering their activity abolished PARP fragmentation. Furthermore, we identified curcumin to exert a pro-oxidative activity, with 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining revealing up-regulation of reactive oxygen species (ROS) levels and anti-oxidants found to abrogate PARP cleavage. In conclusion, curcumin was found to stimulate the apoptotic cell death of H9c2 cells by upregulating ROS generation and triggering activation of JNKs. With reports underscoring the capacity of curcumin to perturb the cellular redox balance ensuring survival or enhancing apoptosis, determination of its mode of action appears to be critical. Future studies should assess the appropriate administration conditions of curcumin, so as to optimize its therapeutic potential against cardiovascular pathologies.

  16. 5-AIQ inhibits H{sub 2}O{sub 2}-induced apoptosis through reactive oxygen species scavenging and Akt/GSK-3β 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-3β (GSK-3β), 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-3β activation. In addition, inhibiting the Akt/GSK-3β 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-3β 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

  17. Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes

    PubMed Central

    Tan, Xian Wen; Bhave, Mrinal; Fong, Alan Yean Yip; Matsuura, Eiji; Kobayashi, Kazuko; Shen, Lian Hua; Hwang, Siaw San

    2016-01-01

    This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: “BJLN”) and a commercial rice variety, “MR219,” on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT. PMID:27239253

  18. Cytoprotective and Cytotoxic Effects of Rice Bran Extracts in Rat H9c2(2-1) Cardiomyocytes.

    PubMed

    Tan, Xian Wen; Bhave, Mrinal; Fong, Alan Yean Yip; Matsuura, Eiji; Kobayashi, Kazuko; Shen, Lian Hua; Hwang, Siaw San

    2016-01-01

    This study was aimed at preliminarily assessing the cytoprotective and antioxidative effects of rice bran extracts (RBEs) from a Sarawak local rice variety (local name: "BJLN") and a commercial rice variety, "MR219," on oxidative stress in rat H9c2(2-1) cardiomyocytes. The cardiomyocytes were incubated with different concentrations of RBE and hydrogen peroxide (H2O2), respectively, to identify their respective IC50 values and safe dose ranges. Two nonlethal and close-to-IC50 doses of RBE were selected to evaluate their respective effects on H2O2 induced oxidative stress in cardiomyocytes. Both RBEs showed dose-dependent cytotoxicity effects on cardiomyocytes. H2O2 induction of cardiomyocytes pretreated with RBE further revealed the dose-dependent cytoprotective and antioxidative effects of RBE via an increase in IC50 values of H2O2. Preliminary analyses of induction effects of RBE and H2O2 on cellular antioxidant enzyme, catalase (CAT), also revealed their potential in regulating these activities and expression profile of related gene on oxidative stress in cardiomyocytes. Pretreated cardiomyocytes significantly upregulated the enzymatic activity and expression level of CAT under the exposure of H2O2 induced oxidative stress. This preliminary study has demonstrated the potential antioxidant effects of RBE in alleviating H2O2-mediated oxidative injuries via upregulation in enzymatic activities and expression levels of CAT. PMID:27239253

  19. Metformin Protects H9C2 Cardiomyocytes from High-Glucose and Hypoxia/Reoxygenation Injury via Inhibition of Reactive Oxygen Species Generation and Inflammatory Responses: Role of AMPK and JNK

    PubMed Central

    Hu, Mingyan; Ye, Ping; Liao, Hua; Chen, Manhua

    2016-01-01

    Metformin is a first-line drug for the management of type 2 diabetes. Recent studies suggested cardioprotective effects of metformin against ischemia/reperfusion injury. However, it remains elusive whether metformin provides direct protection against hypoxia/reoxygenation (H/R) injury in cardiomyocytes under normal or hyperglycemic conditions. This study in H9C2 rat cardiomyoblasts was designed to determine cell viability under H/R and high-glucose (HG, 33 mM) conditions and the effects of cotreatment with various concentrations of metformin (0, 1, 5, and 10 mM). We further elucidated molecular mechanisms underlying metformin-induced cytoprotection, especially the possible involvement of AMP-activated protein kinase (AMPK) and Jun NH(2)-terminal kinase (JNK). Results indicated that 5 mM metformin improved cell viability, mitochondrial integrity, and respiratory chain activity under HG and/or H/R (P < 0.05). The beneficial effects were associated with reduced levels of reactive oxygen species generation and proinflammatory cytokines (TNF-α, IL-1α, and IL-6) (P < 0.05). Metformin enhanced phosphorylation level of AMPK and suppressed HG + H/R induced JNK activation. Inhibitor of AMPK (compound C) or activator of JNK (anisomycin) abolished the cytoprotective effects of metformin. In conclusion, our study demonstrated for the first time that metformin possessed direct cytoprotective effects against HG and H/R injury in cardiac cells via signaling mechanisms involving activation of AMPK and concomitant inhibition of JNK. PMID:27294149

  20. Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

    PubMed

    Majumdar, Gipsy; Raghow, Rajendra

    2016-04-01

    The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI. Using miRCURY LNA™ Universal microRNA PCR system, we demonstrate that H9c2 cells exposed to TSA for 6 and 24 h elicited differential expression of 19 and 16 microRNAs, respectively. H9c2 cells incubated in medium-containing 100 nM of TSA elicited a rapid and robust induction of miR-129-5p. Enhanced expression of miR-129-5p was also observed in the hearts of TSA-treated mice. Induction of miR-129-5p in H9c2 cells was accompanied by reduced expression of its direct target, cyclin-dependent kinase 6 (CDK6) that is a key regulator of cell cycle. Using cell division-dependent dilution of Cell Trace™ violet measurements we showed that concomitant induction of miR-129-5p and reduced CDK6 expression were mechanistically involved in TSA-induced inhibition of proliferation of H9c2 cells. Consistent with this scenario, cells expressing an antagomiR of miR-129-5p were resistant to the anti-proliferative actions of TSA. These data indicate that although TSA treatment led to altered expression of several microRNAs, the overarching action of TSA (i.e., inhibition of cell division) in H9c2 cells was achieved via miR-129-5p.

  1. Trichostatin A induces a unique set of microRNAs including miR-129-5p that blocks cyclin-dependent kinase 6 expression and proliferation in H9c2 cardiac myocytes.

    PubMed

    Majumdar, Gipsy; Raghow, Rajendra

    2016-04-01

    The pan-histone deacetylase inhibitor (HDACI), trichostatin A (TSA), was shown to normalize interleukin-18-induced cardiac hypertrophy in vivo and in vitro; evidently, this occurred via epigenetic mechanisms that profoundly altered cardiac gene expression (Majumdar et al. in, Physiol Genom, 43: 1392, 2011; BMC Genom, 13: 709, 2012). Here, we tested the hypothesis that TSA-induced changes in chromatin architecture also led to altered expression of microRNAs that in turn, contributed to the unique transcriptome of cardiac myocytes exposed to the HDACI. Using miRCURY LNA™ Universal microRNA PCR system, we demonstrate that H9c2 cells exposed to TSA for 6 and 24 h elicited differential expression of 19 and 16 microRNAs, respectively. H9c2 cells incubated in medium-containing 100 nM of TSA elicited a rapid and robust induction of miR-129-5p. Enhanced expression of miR-129-5p was also observed in the hearts of TSA-treated mice. Induction of miR-129-5p in H9c2 cells was accompanied by reduced expression of its direct target, cyclin-dependent kinase 6 (CDK6) that is a key regulator of cell cycle. Using cell division-dependent dilution of Cell Trace™ violet measurements we showed that concomitant induction of miR-129-5p and reduced CDK6 expression were mechanistically involved in TSA-induced inhibition of proliferation of H9c2 cells. Consistent with this scenario, cells expressing an antagomiR of miR-129-5p were resistant to the anti-proliferative actions of TSA. These data indicate that although TSA treatment led to altered expression of several microRNAs, the overarching action of TSA (i.e., inhibition of cell division) in H9c2 cells was achieved via miR-129-5p. PMID:26946427

  2. Phloretin ameliorates arsenic trioxide induced mitochondrial dysfunction in H9c2 cardiomyoblasts mediated via alterations in membrane permeability and ETC complexes.

    PubMed

    Vineetha, Vadavanath Prabhakaran; Soumya, Rema Sreenivasan; Raghu, Kozhiparambil Gopalan

    2015-05-01

    Arsenic trioxide (ATO), though a very effective drug for the treatment of acute promyelocytic leukemia, leads to cardiotoxicity. As mitochondria are the center of attention of cardiac cell׳s general metabolic status, it is primarily important to see the interaction of ATO with mitochondria. Studies related exclusively to the alterations in mitochondria and its associated functions caused by ATO are very limited. The present investigation aims to explore the effect of ATO on various components of electron transport chain, oxygen consumption, ATP production, mitochondrial superoxide generation, transmembrane potential, permeability pore opening, calcium homeostasis and apoptosis. Attempts were also made to see the efficacy of phloretin, a potent antioxidant flavonoid found majorly in apple peel on cardiotoxicity. The H9c2 cells exposed to ATO (5µM) exhibited increased oxidative stress with reduced innate antioxidant status, mitochondrial dysfunctions and apoptosis. It increased the intracellular calcium content, caused alterations in the activity of transcription factor Nrf2, xanthine oxidase, aconitase and caspase 3 compared to the control group. Phloretin at 2.5 and 5µM concentrations were able to protect the cells from ATO toxicity via protecting mitochondria through its antioxidant potential. The present investigation based on mitochondria reveals the probability of cardioprotective potential of phloretin for the cancer patients on ATO chemotherapy. PMID:25746422

  3. Evaluation of a dithiocarbamate derivative as a model of thiol oxidative stress in H9c2 rat cardiomyocytes.

    PubMed

    Xie, Jiashu; Potter, Ashley; Xie, Wei; Lynch, Christophina; Seefeldt, Teresa

    2014-05-01

    Thiol redox state (TRS) refers to the balance between reduced thiols and their corresponding disulfides and is mainly reflected by the ratio of reduced and oxidized glutathione (GSH/GSSG). A decrease in GSH/GSSG, which reflects a state of thiol oxidative stress, as well as thiol modifications such as S-glutathionylation, has been shown to have important implications in a variety of cardiovascular diseases. Therefore, research models for inducing thiol oxidative stress are important tools for studying the pathophysiology of these disease states as well as examining the impact of pharmacological interventions on thiol pathways. The purpose of this study was to evaluate the use of a dithiocarbamate derivative, 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA), as a pharmacological model of thiol oxidative stress by examining the extent of thiol modifications induced in H9c2 rat cardiomyocytes and its impact on cellular functions. The extent of thiol oxidative stress produced by 2-AAPA was also compared to other models of oxidative stress including hydrogen peroxide (H2O2), diamide, buthionine sulfoximine, and N,N׳-bis(2-chloroethyl)-N-nitroso-urea. Results indicated that 2-AAPA effectively inhibited glutathione reductase and thioredoxin reductase activities and decreased the GSH/GSSG ratio by causing a significant accumulation of GSSG. 2-AAPA also increased the formation of protein disulfides as well as S-glutathionylation. The alteration in TRS led to a loss of mitochondrial membrane potential, release of cytochrome c, and increase in reactive oxygen species production. Compared to other models, 2-AAPA is more potent at creating a state of thiol oxidative stress with lower cytotoxicity, higher specificity, and more pharmacological relevance, and could be utilized as a research tool to study TRS-related normal and abnormal biochemical processes in cardiovascular diseases. PMID:24607690

  4. GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes.

    PubMed

    Chang, Fen; Liu, Jing; Fu, Hui; Wang, Jinlan; Li, Fang; Yue, Hongwei; Li, Wenjing; Zhao, Jing; Yin, Deling

    2016-09-01

    Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.

  5. GSK-3β promotes PA-induced apoptosis through changing β-arrestin 2 nucleus location in H9c2 cardiomyocytes.

    PubMed

    Chang, Fen; Liu, Jing; Fu, Hui; Wang, Jinlan; Li, Fang; Yue, Hongwei; Li, Wenjing; Zhao, Jing; Yin, Deling

    2016-09-01

    Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome. PMID:27431999

  6. High concentrations of H2O2 trigger hypertrophic cascade and phosphatase and tensin homologue (PTEN) glutathionylation in H9c2 cardiomyocytes.

    PubMed

    Panera, Nadia; Gnani, Daniela; Piermarini, Emanuela; Petrini, Stefania; Bertini, Enrico; Nobili, Valerio; Pastore, Anna; Piemonte, Fiorella; Alisi, Anna

    2016-02-01

    Cardiac hypertrophy occurs in response to different stimuli and is mainly characterized by an enlargement of cardiomyocyte size. During hypertrophy, cardiomyocytes undergo not only radical changes of the cellular architecture but also activation of signaling cascades that counteract the atrophy genes. Experimental studies highlighted that chronic low concentrations of H2O2, induce a hypertrophic phenotype, while higher levels of H2O2 promote apoptosis. In this study, we explored the early and long-term hypertrophic effects of high concentrations of H2O2 on H9c2 rat cardiomyocytes. We found that 2-h stimulation with 200μM H2O2 caused an early dramatic reduction of cell viability, accompanied, 5-days later, by increased cell size and up-regulation of atrial natriuretic peptide transcription. This hypertrophic phenotype is associated to increased Akt phosphorylation and a consequent reduction of the FOXO3a and atrogin-1 gene expression. Moreover, we observed that H2O2 caused the overexpression of miR-212/miR-132 cluster concomitantly to a down-regulation of PTEN transcript without changes in its protein expression. Noteworthy, we found that the treatment of cardiomyocytes with H2O2 further led to an increase of oxidized glutathione and glutathionylation of proteins, including PTEN. In conclusion, our results permit to reconstruct the molecular cascade triggering the cardiomyocyte hypertrophy upon high concentrations of H2O2.

  7. High concentrations of H2O2 trigger hypertrophic cascade and phosphatase and tensin homologue (PTEN) glutathionylation in H9c2 cardiomyocytes.

    PubMed

    Panera, Nadia; Gnani, Daniela; Piermarini, Emanuela; Petrini, Stefania; Bertini, Enrico; Nobili, Valerio; Pastore, Anna; Piemonte, Fiorella; Alisi, Anna

    2016-02-01

    Cardiac hypertrophy occurs in response to different stimuli and is mainly characterized by an enlargement of cardiomyocyte size. During hypertrophy, cardiomyocytes undergo not only radical changes of the cellular architecture but also activation of signaling cascades that counteract the atrophy genes. Experimental studies highlighted that chronic low concentrations of H2O2, induce a hypertrophic phenotype, while higher levels of H2O2 promote apoptosis. In this study, we explored the early and long-term hypertrophic effects of high concentrations of H2O2 on H9c2 rat cardiomyocytes. We found that 2-h stimulation with 200μM H2O2 caused an early dramatic reduction of cell viability, accompanied, 5-days later, by increased cell size and up-regulation of atrial natriuretic peptide transcription. This hypertrophic phenotype is associated to increased Akt phosphorylation and a consequent reduction of the FOXO3a and atrogin-1 gene expression. Moreover, we observed that H2O2 caused the overexpression of miR-212/miR-132 cluster concomitantly to a down-regulation of PTEN transcript without changes in its protein expression. Noteworthy, we found that the treatment of cardiomyocytes with H2O2 further led to an increase of oxidized glutathione and glutathionylation of proteins, including PTEN. In conclusion, our results permit to reconstruct the molecular cascade triggering the cardiomyocyte hypertrophy upon high concentrations of H2O2. PMID:26772165

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

    PubMed

    Thakur, Anita; Alam, Md Jahangir; Ajayakumar, M R; Ghaskadbi, Saroj; Sharma, Manish; Goswami, Shyamal K

    2015-08-01

    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 2h; 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 γ-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

  9. 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 γ-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

  10. Quercetin and hydroxytyrosol attenuates xanthine/xanthine oxidase-induced toxicity in H9c2 cardiomyocytes by regulation of oxidative stress and stress-sensitive signaling pathways.

    PubMed

    Ozbek, Namik; Bali, Elif B; Karasu, Cimen

    2015-10-01

    The increased activity of xanthine/xanthine oxidase (X/XO) has been suggested as a risk factor for heart disease and herbal polyphenols exhibits cardioprotection in vitro and in vivo. To understand the cardioprotective action mechanisms of polyphenol quercetin and hydroxytyrosol, the expression levels of stress-responsive proteins were studied in X/XO-induced toxicity model of H9c2 cardiomyocyocytes. Pretreatment with each polypenol (0.1-10 μg/ml; 24 h) enhanced viability (p < 0.01; MTT test) and inhibited reactive oxygen species (ROS) generation (p < 0.001; H2DCFDA assay) against 12 h exposure to a free radical generating system, X (0.5 mM) and XO (5 mU/ml). Western blotting experiments showed that X/XO increases the phosphorylation of downstream substrate of p38, MAPK-activated protein kinase 2 (MAPKAPK-2), p44/42-MAPK (Erk1/2) and cleaved caspase-3 (p < 0.001, vs. Control), however inhibits the levels of phosphorylated c-Jun and Hsp27 (p < 0.01, vs. Control). Pretreatment with quercetin or hydroxytyrosol attenuated the phosphorylation of MAPKAPK-2 and cleaved caspase-3 in X/XO-exposed cells (p < 0.01, vs. X/XO). Hydroxytyrosol enhanced the reduction of phosphorylation of a transcriptional target c-Jun and led to overphosphorylation in protective proteins, p44/42-MAPK and Hsp27 in X/XO-exposed cells (p < 0.01, vs. X/XO). Our data suggest that quercetin and hydroxytyrosol protects cardiomyocytes against X/XO-induced oxidative toxicity by diminishing intracellular ROS and the regulation of stress-sensitive protein kinase cascades and transcription factors. PMID:26374991

  11. Effects of bpV(pic) and bpV(phen) on H9c2 cardiomyoblasts during both hypoxia/reoxygenation and H2O2-induced injuries.

    PubMed

    Tian, Youqing; Daoud, Abdelkader; Shang, Jing

    2012-03-01

    Reactive oxygen species (ROS) are involved in myocardial injury. ROS are known to inactivate lipid phosphatase and tension homolog on chromosome 10 (PTEN), an enzyme that increases apoptosis in neonatal cardiomyocytes. BpV(pic) and bpV(phen), two bisperoxovanadium molecules and PTEN inhibitors, may be involved in limiting myocardial infarction. To compare the protective effects of bpV(pic) and bpV(phen) on ROS-induced cardiomyocyte injury and their possible mechanisms, we selected two popular models of hypoxia/reoxygenation (H/R) and H2O2-induced injury in H9c2 cardiomyoblasts to investigate their effects against injury. We found that pre-treatment with bpV(pic) and bpV(phen) increased the viability and protected the morphology of H9c2 cells under the conditions of H/R and H2O2 by inhibiting LDH release, apoptosis and caspases 3/8/9 activities. However, their respective inhibitory abilities in the two models were different, suggesting that the quantity of ROS from the two models might be different. However, the conflict between ROS and PTEN may affect the action of bpV(pic) and bpV(phen). Taken together, the results demonstrate that bpV(pic) and bpV(phen) have inhibitory effects on oxidative stress-induced cardiomyocyte injury that may be partially modulated by the action of ROS on PTEN.

  12. Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes.

    PubMed

    Dziegala, Magdalena; Kasztura, Monika; Kobak, Kamil; Bania, Jacek; Banasiak, Waldemar; Ponikowski, Piotr; Jankowska, Ewa A

    2016-10-01

    The differential availability of iron during hypoxia is presumed to affect the functioning of cardiac and skeletal myocytes. Rat H9C2 cardiomyocytes and L6G8C5 myocytes were cultured for 48 h in normoxic or hypoxic conditions at the optimal, reduced or increased iron concentration. The mRNA expression levels of markers of apoptosis [B‑cell lymphoma‑2 (Bcl2; inhibition) and Bcl‑2‑activated X protein (Bax; induction)], atrophy (Atrogin), glycolysis (pyruvate kinase 2; PKM2) and iron metabolism [transferrin receptor 1 (TfR1; iron importer), ferroportin 1 (FPN1; iron exporter), ferritin heavy chain (FTH; iron storage protein) and hepcidin (HAMP; iron regulator)] were determined using reverse transcription‑quantitative polymerase chain reaction, and cell viability was measured using an tetrazolium reduction assay. Cardiomyocytes and myocytes, when exposed to hypoxia, demonstrated an increased Bax/Bcl‑2 gene expression ratio (P<0.05). Additional deferoxamine (DFO) treatment resulted in further increases in Bax/Bcl‑2 in each cell type (P<0.001 each) and this was associated with the 15% loss in viability. The analogous alterations were observed in both cell types upon ammonium ferric citrate (AFC) treatment during hypoxia; however, the increased Bax/Bcl‑2 ratio and associated viability loss was lower compared with that in case of DFO treatment (P<0.05 each). Under hypoxic conditions, myocytes demonstrated an increased expression of PKM2 (P<0.01). Additional DFO treatment caused an increase in the mRNA expression levels of PKM2 and Atrogin‑1 (P<0.001 and P<0.05, respectively), whereas AFC treatment caused an increased mRNA expression of PKM2 (P<0.01) and accompanied decreased mRNA expression of Atrogin‑1 (P<0.05). The expression augmentation of PKM2 during hypoxia was greater upon low iron compared with that of ferric salt treatment (P<0.01). Both cell types upon DFO during hypoxia demonstrated the increased expression of TfR1

  13. Influence of the availability of iron during hypoxia on the genes associated with apoptotic activity and local iron metabolism in rat H9C2 cardiomyocytes and L6G8C5 skeletal myocytes.

    PubMed

    Dziegala, Magdalena; Kasztura, Monika; Kobak, Kamil; Bania, Jacek; Banasiak, Waldemar; Ponikowski, Piotr; Jankowska, Ewa A

    2016-10-01

    The differential availability of iron during hypoxia is presumed to affect the functioning of cardiac and skeletal myocytes. Rat H9C2 cardiomyocytes and L6G8C5 myocytes were cultured for 48 h in normoxic or hypoxic conditions at the optimal, reduced or increased iron concentration. The mRNA expression levels of markers of apoptosis [B‑cell lymphoma‑2 (Bcl2; inhibition) and Bcl‑2‑activated X protein (Bax; induction)], atrophy (Atrogin), glycolysis (pyruvate kinase 2; PKM2) and iron metabolism [transferrin receptor 1 (TfR1; iron importer), ferroportin 1 (FPN1; iron exporter), ferritin heavy chain (FTH; iron storage protein) and hepcidin (HAMP; iron regulator)] were determined using reverse transcription‑quantitative polymerase chain reaction, and cell viability was measured using an tetrazolium reduction assay. Cardiomyocytes and myocytes, when exposed to hypoxia, demonstrated an increased Bax/Bcl‑2 gene expression ratio (P<0.05). Additional deferoxamine (DFO) treatment resulted in further increases in Bax/Bcl‑2 in each cell type (P<0.001 each) and this was associated with the 15% loss in viability. The analogous alterations were observed in both cell types upon ammonium ferric citrate (AFC) treatment during hypoxia; however, the increased Bax/Bcl‑2 ratio and associated viability loss was lower compared with that in case of DFO treatment (P<0.05 each). Under hypoxic conditions, myocytes demonstrated an increased expression of PKM2 (P<0.01). Additional DFO treatment caused an increase in the mRNA expression levels of PKM2 and Atrogin‑1 (P<0.001 and P<0.05, respectively), whereas AFC treatment caused an increased mRNA expression of PKM2 (P<0.01) and accompanied decreased mRNA expression of Atrogin‑1 (P<0.05). The expression augmentation of PKM2 during hypoxia was greater upon low iron compared with that of ferric salt treatment (P<0.01). Both cell types upon DFO during hypoxia demonstrated the increased expression of TfR1

  14. miR-7a/b attenuates post-myocardial infarction remodeling and protects H9c2 cardiomyoblast against hypoxia-induced apoptosis involving Sp1 and PARP-1

    PubMed Central

    Li, Rui; Geng, Hai-hua; Xiao, Jie; Qin, Xiao-teng; Wang, Fu; Xing, Jun-hui; Xia, Yan-fei; Mao, Yang; Liang, Jing-wen; Ji, Xiao-ping

    2016-01-01

    miRs (microRNAs, miRNAs) intricately regulate physiological and pathological processes. Although miR-7a/b protects against cardiomyocyte injury in ischemia/reperfusion injury, the function of miR-7a/b in myocardial infarction (MI)-induced cardiac remodeling remains unclear. Here, we sought to investigate the function of miR-7a/b in post-MI remodeling in a mouse model and to determine the underlying mechanisms involved. miR-7a/b overexpression improved cardiac function, attenuated cardiac remodeling and reduced fibrosis and apoptosis, whereas miR-7a/b silencing caused the opposite effects. Furthermore, miR-7a/b overexpression suppressed specific protein 1 (Sp1) and poly (ADP-ribose) polymerase (PARP-1) expression both in vivo and in vitro, and a luciferase reporter activity assay showed that miR-7a/b could directly bind to Sp1. Mithramycin, an inhibitor of the DNA binding activity of Sp1, effectively repressed PARP-1 and caspase-3, whereas knocking down miR-7a/b partially counteracted these beneficial effects. Additionally, an immunoprecipitation assay indicated that hypoxia triggered activation of the binding activity of Sp1 to the promoters of PARP-1 and caspase-3, which is abrogated by miR-7a/b. In summary, these findings identified miR-7a/b as protectors of cardiac remodeling and hypoxia-induced injury in H9c2 cardiomyoblasts involving Sp1 and PARP-1. PMID:27384152

  15. 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.

  16. 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

  17. Biology of SNU Cell Lines

    PubMed Central

    Ku, Ja-Lok

    2005-01-01

    SNU (Seoul National University) cell lines have been established from Korean cancer patients since 1982. Of these 109 cell lines have been characterized and reported, i.e., 17 colorectal carcinoma, 12 hepatocellular carcinoma, 11 gastric carcinoma, 12 uterine cervical carcinoma, 17 B-lymphoblastoid cell lines derived from cancer patients, 5 ovarian carcinoma, 3 malignant mixed Mllerian tumor, 6 laryngeal squamous cell carcinoma, 7 renal cell carcinoma, 9 brain tumor, 6 biliary tract, and 4 pancreatic carcinoma cell lines. These SNU cell lines have been distributed to biomedical researchers domestic and worldwide through the KCLB (Korean Cell Line Bank), and have proven to be of value in various scientific research fields. The characteristics of these cell lines have been reported in over 180 international journals by our laboratory and by many other researchers from 1987. In this paper, the cellular and molecular characteristics of SNU human cancer cell lines are summarized according to their genetic and epigenetic alterations and functional analysis. PMID:19956504

  18. 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

  19. Low abundance of mitochondrial DNA changes mitochondrial status and renders cells resistant to serum starvation and sodium nitroprusside insult.

    PubMed

    Lee, Sung Ryul; Heo, Hye Jin; Jeong, Seung Hun; Kim, Hyoung Kyu; Song, In Sung; Ko, Kyung Soo; Rhee, Byoung Doo; Kim, Nari; Han, Jin

    2015-07-01

    Mutation or depletion of mitochondrial DNA (mtDNA) can cause severe mitochondrial malfunction, originating from the mitochondrion itself, or from the crosstalk between nuclei and mitochondria. However, the changes that would occur if the amount of mtDNA is diminished are less known. Thus, we generated rat myoblast H9c2 cells containing lower amounts of mtDNA via ethidium bromide and uridine supplementation. After confirming the depletion of mtDNA by quantitative PCR and gel electrophoresis analysis, we investigated the changes in mitochondrial physical parameters by using flow cytometry. We also evaluated the resistance of these cells to serum starvation and sodium nitroprusside. H9c2 cells with diminished mtDNA contents showed decreased mitochondrial membrane potential, mass, free calcium, and zinc ion contents as compared to naïve H9c2 cells. Furthermore, cytosolic and mitochondrial reactive oxygen species levels were significantly higher in mtDNA-lowered H9c2 cells than in the naïve cells. Although the oxygen consumption rate and cell proliferation were decreased, mtDNA-lowered H9c2 cells were more resistant to serum deprivation and nitroprusside insults than the naïve H9c2 cells. Taken together, we conclude that the low abundance of mtDNA cause changes in cellular status, such as changes in reactive oxygen species, calcium, and zinc ion levels inducing resistance to stress. PMID:25825022

  20. Differential translocation of nuclear factor-kappaB in a cardiac muscle cell line under gravitational changes.

    PubMed

    Kwon, Ohwon; Tranter, Michael; Jones, W Keith; Sankovic, John M; Banerjee, Rupak K

    2009-06-01

    Microgravity (micro-g) environments have been shown to elicit dysregulation of specific genes in a wide assay of cell types. It is known that the activation of transcription factors and molecular signaling pathways influence various physiological outcomes associated with stress and adaptive responses. Nuclear factor-kappa B (NF-kappaB) is one of the most prevailing oxidation-sensitive transcription factors. It is hypothesized that simulated microgravity would activate NF-kappaB and its downstream transcriptional networks, thus suggesting a role for NF-kappaB in microgravity induced muscle atrophy. To investigate the activation of NF-kappaB in a rat cardiac cell line (H9c2) under micro-g, rotating wall vessel bioreactors were used to simulate micro-g conditions. Western blotting revealed that mean nuclear translocation of NF-kappaB p65 subunit was 69% for micro-g and 46% for unit-g dynamic control as compared with a 30 min TNF-alpha positive control (p<0.05, n=3). The results from western blots were confirmed by enzyme-linked immunosorbent assay, which showed 66% for micro-g and 45% for dynamic control as compared with positive control (p<0.05, n=3). These results show significant differential translocation of NF-kappaB p65 under simulated micro-g. These results may be expanded upon to explain physiological changes such as muscle atrophy and further identify the regulatory pathways and effector molecules activated under exposure to micro-g.

  1. Human Adrenocortical Carcinoma Cell Lines

    PubMed Central

    Wang, Tao; Rainey, William E.

    2011-01-01

    Summary The human adrenal cortex secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids are produced from unique cell types located within the three distinct zones of the adrenal cortex. Disruption of adrenal steroid production results in a variety of diseases that can lead to hypertension, metabolic syndrome, infertility and androgen excess. The adrenal cortex is also a common site for the development of adenomas, and rarely the site for the development of carcinomas. The adenomas can lead to diseases associated with adrenal steroid excess, while the carcinomas are particularly aggressive and have a poor prognosis. In vitro cell culture models provide an important tool to examine molecular and cellular mechanisms controlling both the normal and pathologic function of the adrenal cortex. Herein we discuss the human adrenocortical cell lines and their use as model systems for adrenal studies. PMID:21924324

  2. 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

  3. Cell line: 2004-2014.

    PubMed

    2014-11-20

    2014 marks Cell's 40th anniversary, and over the year we have looked back at how discoveries of the last four decades have molded our understanding of biology. The final decade of the Cell Line features a selection of the exceptional scientific work-both landmark papers and essential reviews. Select entries can be read as an "Annotated Classic," which includes the original paper and accompanying reflections of a leading scientist, considering the work from our current vantage point. Our last installment includes a harbinger of the interplay between microbiota and mammalian hosts in 2004, revolutionary papers in 2006 and 2007 unlocking cellular reprogramming, the discovery of beige adipocytes in 2012, and the first example of CRISPR-based genome editing in a nonhuman primate in 2014. In addition to landmark publications, there were innovative developments at the journal in this decade, with the complete redesign of the print journal and the creation of Leading Edge in late 2005 and the restructuring of the online display of the article in 2010. Keeping pace with the changing nature of biological research, over the decade Cell added new article types, introduced guidelines for the organization of supplementary material, and expanded the journal's web-based content to bring editors' and authors' excitement and perspective on individual papers to the readership. An interactive version of the timeline, with links to the papers, full author lists, and Annotated Classics, is available at http://dx.doi.org/10.1016/j.cell.2014.11.004. PMID:25416957

  4. Materials Data on H9C2(NO)3 (SG:33) by Materials Project

    SciTech Connect

    Kristin Persson

    2014-07-09

    Computed materials data using density functional theory calculations. These calculations determine the electronic structure of bulk materials by solving approximations to the Schrodinger equation. For more information, see https://materialsproject.org/docs/calculations

  5. Anticancer activity of synthetic bis(indolyl)methane-ortho-biaryls against human cervical cancer (HeLa) cells.

    PubMed

    Jamsheena, Vellekkatt; Shilpa, Ganesan; Saranya, Jayaram; Harry, Nissy Ann; Lankalapalli, Ravi Shankar; Priya, Sulochana

    2016-03-01

    Bis(indolyl)methane appended biaryls were designed, synthesized and evaluated in human cervical cancer cell lines (HeLa) for their anticancer activities and compared against normal rat cardiac myoblasts (H9C2) cells. Compounds 1-12 were synthesized, with variations in one of the phenyl unit, in a single step by condensation of biaryl-2-carbaldehydes with indole in the presence of para-toluenesulfonic acid. Compound 1 exhibited a GI50 value of 11.00 ± 0.707 μM and the derivatives, compounds 4 and 11 showed a GI50 value of 8.33 ± 0.416 μM and 9.13 ± 0.177 μM respectively in HeLa cells and was found to be non-toxic to H9C2 cells up to 20 μM. Furthermore, compounds 1, 4 and 11 induced caspase dependent cellular apoptosis in a concentration-dependent manner, reduced mitochondrial membrane potential, inhibited the cell migration and downregulated the production of MMP-2 and MMP-9 in HeLa cells.

  6. HLA expression in hepatocellular carcinoma cell lines.

    PubMed

    Wadee, A A; Paterson, A; Coplan, K A; Reddy, S G

    1994-08-01

    The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines.

  7. Silencing of TBX20 gene expression in rat myocardial and human embryonic kidney cells leads to cell cycle arrest in G2 phase

    PubMed Central

    Liu, Peiyan; Sun, Yueling; Qiu, Guangbin; Jiang, Hongkun; Qiu, Guangrong

    2016-01-01

    Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T-box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Cell proliferation was assessed using a cell counting kit and proliferating cell nuclear antigen expression was determined by western blotting. Analysis of cell apoptosis was achieved by annexin V-fluorescein isothiocyanate/propidium iodide staining and a fluorometric terminal deoxynucleotidyl transferase dUTP nick-end labeling system. Cell cycle analysis was achieved using fluorescence-activated cell sorting, and, an RT-qPCR array was used to profile the expression of TBX20-related genes. Silencing of TBX20 in H9c2 and HEK293 cells significantly inhibited cell proliferation, induced cell apoptosis and led to G2/M cell cycle arrest. A reduction in cyclin B1 mRNA levels and an increase in cyclin-dependent kinase inhibitor 1B mRNA levels was observed, which indicated that cells were arrested in G2 phase. Concurrently, the mRNA levels of GATA binding protein 4 were increased in both cell lines, which may provide an explanation for the abnormal cardiac hypertrophy observed in patients with congenital heart disease. These results suggest that TBX20 is required for heart morphogenesis, and inhibition of TBX20 expression may lead to the suppression of cell proliferation and cell cycle

  8. Silencing of TBX20 gene expression in rat myocardial and human embryonic kidney cells leads to cell cycle arrest in G2 phase.

    PubMed

    Liu, Peiyan; Sun, Yueling; Qiu, Guangbin; Jiang, Hongkun; Qiu, Guangrong

    2016-10-01

    Congenital heart diseases (CHDs) are the most common birth defects due to abnormal cardiac development. The T-box 20 (TBX20) gene is a member of the T‑box family of transcription factors and encodes TBX20, which is essential for early heart development. In the present study, reduced TBX20 expression was observed in CHD tissue samples compared with normal tissues, and the function of TBX20 in Rattus norvegicus myocardial cells [H9c2(2-1)] and human embryonic kidney cells (HEK293) was investigated. TBX20 was silenced in H9c2 and HEK293 cells via transfection of small interfering RNA and short hairpin RNA duplexes, respectively, and TBX20 mRNA and protein levels were subsequently examined using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis. Cell proliferation was assessed using a cell counting kit and proliferating cell nuclear antigen expression was determined by western blotting. Analysis of cell apoptosis was achieved by annexin V‑fluorescein isothiocyanate/propidium iodide staining and a fluorometric terminal deoxynucleotidyl transferase dUTP nick‑end labeling system. Cell cycle analysis was achieved using fluorescence‑activated cell sorting, and, an RT‑qPCR array was used to profile the expression of TBX20‑related genes. Silencing of TBX20 in H9c2 and HEK293 cells significantly inhibited cell proliferation, induced cell apoptosis and led to G2/M cell cycle arrest. A reduction in cyclin B1 mRNA levels and an increase in cyclin‑dependent kinase inhibitor 1B mRNA levels was observed, which indicated that cells were arrested in G2 phase. Concurrently, the mRNA levels of GATA binding protein 4 were increased in both cell lines, which may provide an explanation for the abnormal cardiac hypertrophy observed in patients with congenital heart disease. These results suggest that TBX20 is required for heart morphogenesis, and inhibition of TBX20 expression may lead to the suppression of cell

  9. Neuroblastoma cell lines showing smooth muscle cell phenotypes.

    PubMed

    Sugimoto, T; Mine, H; Horii, Y; Takahashi, K; Nagai, R; Morishita, R; Komada, M; Asada, Y; Sawada, T

    2000-12-01

    Neuroblastoma is a tumor that is derived from the neural crest. Recent studies demonstrated that several human neuroblastoma cell lines exhibit at least three morphologic types: neuroblastic (N)-type, substrate-adhesive (S)-type and intermediate (I)-type cells. However, the origin of the S-type cells has not been clearly identified. In this study, the expressions of smooth muscle-specific proteins (desmin, alpha-smooth muscle actin, basic calponin and the smooth muscle myosin heavy-chain isoforms of SM1 and SM2) in three parent and four cloned neuroblastoma cell lines, composed of S-type cells, were examined by indirect immunofluorescence, Western blot and/or by reverse transcription-polymerase chain reaction (RT-PCR). Desmin was found in two of the seven cell lines, and alpha-smooth muscle actin and basic calponin were detected in all of seven of the cell lines. In three parent cell lines and one cloned cell line composed of N-type cells, none of three smooth muscle-specific proteins were detected. In smooth muscle myosin heavy-chain isoforms, SM1 was detected in two parent cell lines composed of S-type cells (MP-N-MS and KP-N-YS) by immunofluorescence, Western blot and/or by RT-PCR, whereas the SM2 isoform was detected in one parent cell line (MP-N-MS) by RT-PCR. These findings indicate that S-type cells have either the immature or mature smooth muscle cell phenotype, and neural crest cells very likely have the ability of to differentiate into smooth muscle cells in the human system.

  10. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  11. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks’ genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area.

  12. Virus Discovery Using Tick Cell Lines.

    PubMed

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

    While ticks have been known to harbor and transmit pathogenic arboviruses for over 80 years, the application of high-throughput sequencing technologies has revealed that ticks also appear to harbor a diverse range of endogenous tick-only viruses belonging to many different families. Almost nothing is known about these viruses; indeed, it is unclear in most cases whether the identified viral sequences are derived from actual replication-competent viruses or from endogenous virus elements incorporated into the ticks' genomes. Tick cell lines play an important role in virus discovery and isolation through the identification of novel viruses chronically infecting such cell lines and by acting as host cells to aid in determining whether or not an entire replication-competent, infective virus is present in a sample. Here, we review recent progress in tick-borne virus discovery and comment on the actual and potential applications for tick cell lines in this emerging research area. PMID:27679414

  13. Killer cell lines against Shope carcinoma cells in rabbits.

    PubMed

    Takahashi, M; Yamade, I; Seto, A

    1991-09-01

    Killer cell activity against Shope carcinoma cells was not detected in PBL nor in spleen cells from tumor-bearing B/J rabbits, but was induced by in vitro culture of these cells in the presence of IL-2 and X-irradiated carcinoma cells. HTLV-I-transformed killer cell lines were successfully obtained by the culturing of PBL from an HTLV-I-infected and tumor-bearing Chbb:HM rabbit. These killer cells included large cells with azurophilic granules in the cytoplasm and with a reniform nucleus, thus resembling large granular lymphocytes. The killer activity was similar against the Vx2K cell line from a random-bred rabbit and SCB cell lines from an B/J rabbit, suggesting the absence of MHC restriction. PMID:1655241

  14. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    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. Umbelliprenin Induces Apoptosis in CLL Cell Lines.

    PubMed

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V-FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate.

  16. Umbelliprenin Induces Apoptosis in CLL Cell Lines

    PubMed Central

    Ziai, Seyed Ali; Gholami, Omid; Iranshahi, Mehrdad; Zamani, Amir Hassan; Jeddi-Tehrani, Mahmood

    2012-01-01

    Chronic lymphocytic leukemia (CLL) remains an incurable disease that requires innovative new approaches to improve therapeutic outcome. Many Ferula species, including F. asa-foetida, synthesize terpenyloxy coumarins. One of these coumarins is umbelliprenin, which has been implicated with induction of apoptosis in some cancer cell lines. In this study induction of apoptosis by umbelliprenin on Jurkat T-CLL and Raji B-CLL cell lines was studied. In this regard, cells were incubated with various concentrations of umbelliprenin in-vitro for different times and assayed for apoptosis with annexin V–FITC/PI double staining flowcytometry method. Results showed that umbelliprenin induced apoptosis in leukemic cells in a dose- and time-dependent manner and that CLL cells were more susceptible to umbelliprenin induced cell death than normal peripheral blood mononuclear cell (PBMCs). Moreover, we study the induction of apoptosis in Jurkat cells by umbelliprenin in the presence of interleukin 4 (IL-4) as an agent that causes resistance to apoptosis in CLL cells, was also student. We showed that IL-4 can not reduce apoptotic effect of umbelliprenin. The preferential toxicity of umbelliprenin for CLL cells, supports the hypothesis that oral administration of umbelliprenin in the form of foods or folk medicines containing this coumarin, might enhance protection against the development of CLL in man with little side effects. In conclusion, umbelliprenin may be an effective therapeutic agent in the treatment of CLL, and thus clinical studies with umbelliprenin may be appropriate. PMID:24250490

  17. [Characterization of a liver metastatic cell line derived from a human gastric cancer cell line].

    PubMed

    Wakasugi, J

    1990-08-01

    This study was carried out to investigate whether there is any difference of biological characteristics between a gastric cancer cell line (KATOIII) and another cell line derived from liver metastasis of the same cell line (KATOIII-H2). The liver metastasis was produced by intrasplenic injection of the fluid containing of KATOIII in nude mouse and new cell line was established using the cells of metastatic site. The results are as follows. 1) Inoculation of KATOIII-H2 into the spleen produced liver metastases in all of the experimental animals, whereas the same procedure with KATOIII produced metastasis only in 30% of the animals. 2) KATOIII-H2 exhibited more prominent platelet-aggregating activity than KATOIII. 3) There is no difference between two cell lines on doubling time, histological findings of the xenografts and chromosomal number. 4) DNA index of KATOIII-H2 is lower than KATOIII and the trisomy in NO. 20 chromosome of KATOIII-H2 was noted. The results indicate that metastatic potential is different between two cell lines and this fact is probably in a part because of the different platelet-aggregating activity of each cell line. PMID:2233668

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

    SciTech Connect

    Felthaus, O.; 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.

  19. 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

  20. Experience with the Vero cell line.

    PubMed

    Montagnon, B J; Vincent-Falquet, J C

    1998-01-01

    The Vero cell line has been managed with the Cell Bank system to produce at the 142nd passage IPV, OPV and rabies vaccines since 1982 by Pasteur Mérieux Serums & Vaccins (PMsv). The safety of the cell line was regularly validated at the Working Cell Bank (WCB) level according to the WHO and European Pharmacopoeia requirements for absence of bacteria, fungi, mycoplasma and viruses. A special emphasis was devoted to research on the absence of simian viruses (SV40, SIV, Retro-D virus and simian CMV). All these specific researches were negative. At a low level of passage, the Vero cells are not tumorigenic. Vaccines have been prepared in low passage level Vero cells and together with the excellent downstream purification have resulted in excellent safety as attested by pharmacovigilance of more than 100 million doses of IPV during 12 years, more than 20 million doses of rabies vaccine during 10 years and more than 1 billion of OPV during eight years.

  1. Cytosine methylation profiling of cancer cell lines

    PubMed Central

    Ehrich, Mathias; Turner, Julia; Gibbs, Peter; Lipton, Lara; Giovanneti, Mara; Cantor, Charles; van den Boom, Dirk

    2008-01-01

    DNA-methylation changes in human cancer are complex and vary between the different types of cancer. Capturing this epigenetic variability in an atlas of DNA-methylation changes will be beneficial for basic research as well as translational medicine. Hypothesis-free approaches that interrogate methylation patterns genome-wide have already generated promising results. However, these methods are still limited by their quantitative accuracy and the number of CpG sites that can be assessed individually. Here, we use a unique approach to measure quantitative methylation patterns in a set of >400 candidate genes. In this high-resolution study, we employed a cell-line model consisting of 59 cancer cell lines provided by the National Cancer Institute and six healthy control tissues for discovery of methylation differences in cancer-related genes. To assess the effect of cell culturing, we validated the results from colon cancer cell lines by using clinical colon cancer specimens. Our results show that a large proportion of genes (78 of 400 genes) are epigenetically altered in cancer. Although most genes show methylation changes in only one tumor type (35 genes), we also found a set of genes that changed in many different forms of cancer (seven genes). This dataset can easily be expanded to develop a more comprehensive and ultimately complete map of quantitative methylation changes. Our methylation data also provide an ideal starting point for further translational research where the results can be combined with existing large-scale datasets to develop an approach that integrates epigenetic, transcriptional, and mutational findings. PMID:18353987

  2. CellLineNavigator: a workbench for cancer cell line analysis.

    PubMed

    Krupp, Markus; Itzel, Timo; Maass, Thorsten; Hildebrandt, Andreas; Galle, Peter R; Teufel, Andreas

    2013-01-01

    The CellLineNavigator database, freely available at http://www.medicalgenomics.org/celllinenavigator, is a web-based workbench for large scale comparisons of a large collection of diverse cell lines. It aims to support experimental design in the fields of genomics, systems biology and translational biomedical research. Currently, this compendium holds genome wide expression profiles of 317 different cancer cell lines, categorized into 57 different pathological states and 28 individual tissues. To enlarge the scope of CellLineNavigator, the database was furthermore closely linked to commonly used bioinformatics databases and knowledge repositories. To ensure easy data access and search ability, a simple data and an intuitive querying interface were implemented. It allows the user to explore and filter gene expression, focusing on pathological or physiological conditions. For a more complex search, the advanced query interface may be used to query for (i) differentially expressed genes; (ii) pathological or physiological conditions; or (iii) gene names or functional attributes, such as Kyoto Encyclopaedia of Genes and Genomes pathway maps. These queries may also be combined. Finally, CellLineNavigator allows additional advanced analysis of differentially regulated genes by a direct link to the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources.

  3. High prevalence of side population in human cancer cell lines

    PubMed Central

    Boesch, Maximilian; Zeimet, Alain G.; Fiegl, Heidi; Wolf, Barbara; Huber, Julia; Klocker, Helmut; Gastl, Guenther

    2016-01-01

    Cancer cell lines are essential platforms for performing cancer research on human cells. We here demonstrate that, across tumor entities, human cancer cell lines harbor minority populations of putative stem-like cells, molecularly defined by dye extrusion resulting in the side population phenotype. These findings establish a heterogeneous nature of human cancer cell lines and argue for their stem cell origin. This should be considered when interpreting research involving these model systems. PMID:27226981

  4. On the Ontology Based Representation of Cell Lines

    PubMed Central

    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

  5. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National... tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and corresponding information will be posted in a publically held...

  6. EXAFS studies of prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

    Czapla, J.; Kwiatek, W. M.; Lekki, J.; Kisiel, A.; Steininger, R.; Goettlicher, J.

    2013-04-01

    Sulphur plays a vital role in every human organism. It is known, that sulphur-bearing compounds, such as for example cysteine and glutathione, play critical roles in development and progression of many diseases. Any alteration in sulphur's biochemistry could become a precursor of serious pathological conditions. One of such condition is prostate cancer, the most frequently diagnosed malignancy in the western world and the second leading cause of cancer related death in men. The purpose of presented studies was to examine what changes occur in the nearest chemical environment of sulphur in prostate cancer cell lines in comparison to healthy cells. The Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy was used, followed by theoretical calculations. The results of preliminary analysis is presented.

  7. Generating Rho-0 Cells Using Mesenchymal Stem Cell Lines

    PubMed Central

    Fernández-Moreno, Mercedes; Hermida-Gómez, Tamara; Gallardo, M. Esther; Dalmao-Fernández, Andrea; Rego-Pérez, Ignacio; Garesse, Rafael

    2016-01-01

    Introduction The generation of Rho-0 cells requires the use of an immortalization process, or tumor cell selection, followed by culture in the presence of ethidium bromide (EtBr), incurring the drawbacks its use entails. The purpose of this work was to generate Rho-0 cells using human mesenchymal stem cells (hMSCs) with reagents having the ability to remove mitochondrial DNA (mtDNA) more safely than by using EtBr. Methodology Two immortalized hMSC lines (3a6 and KP) were used; 143B.TK-Rho-0 cells were used as reference control. For generation of Rho-0 hMSCs, cells were cultured in medium supplemented with each tested reagent. Total DNA was isolated and mtDNA content was measured by real-time polymerase chain reaction (PCR). Phenotypic characterization and gene expression assays were performed to determine whether 3a6 Rho-0 hMSCs maintain the same stem properties as untreated 3a6 hMSCs. To evaluate whether 3a6 Rho-0 hMSCs had a phenotype similar to that of 143B.TK-Rho-0 cells, in terms of reactive oxygen species (ROS) production, apoptotic levels and mitochondrial membrane potential (Δψm) were measured by flow cytometry and mitochondrial respiration was evaluated using a SeaHorse XFp Extracellular Flux Analyzer. The differentiation capacity of 3a6 and 3a6 Rho-0 hMSCs was evaluated using real-time PCR, comparing the relative expression of genes involved in osteogenesis, adipogenesis and chondrogenesis. Results The results showed the capacity of the 3a6 cell line to deplete its mtDNA and to survive in culture with uridine. Of all tested drugs, Stavudine (dt4) was the most effective in producing 3a6-Rho cells. The data indicate that hMSC Rho-0 cells continue to express the characteristic MSC cell surface receptor pattern. Phenotypic characterization showed that 3a6 Rho-0 cells resembled 143B.TK-Rho-0 cells, indicating that hMSC Rho-0 cells are Rho-0 cells. While the adipogenic capability was higher in 3a6 Rho-0 cells than in 3a6 cells, the osteogenic and chondrogenic

  8. 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

  9. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

    Background Dorsal root ganglion neuron-derived immortal cell lines including ND7/23 and F-11 cells have been used extensively as in vitro model systems of native peripheral sensory neurons. However, while it is clear that some sensory neuron-specific receptors and ion channels are present in these cell lines, a systematic comparison of the molecular targets expressed by these cell lines with those expressed in intact peripheral neurons is lacking. Results In this study, we examined the expression of RNA transcripts in the human neuroblastoma-derived cell line, SH-SY5Y, and two dorsal root ganglion hybridoma cell lines, F-11 and ND7/23, using Illumina next-generation sequencing, and compared the results with native whole murine dorsal root ganglions. The gene expression profiles of these three cell lines did not resemble any specific defined dorsal root ganglion subclass. The cell lines lacked many markers for nociceptive sensory neurons, such as the Transient receptor potential V1 gene, but expressed markers for both myelinated and unmyelinated neurons. Global gene ontology analysis on whole dorsal root ganglions and cell lines showed similar enrichment of biological process terms across all samples. Conclusions This paper provides insights into the receptor repertoire expressed in common dorsal root ganglion neuron-derived cell lines compared with whole murine dorsal root ganglions, and illustrates the limits and potentials of these cell lines as tools for neuropharmacological exploration. PMID:27130590

  10. Development and characterization of a new human hepatic cell line.

    PubMed

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

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  11. Development and characterization of a new human hepatic cell line

    PubMed Central

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

    2015-01-01

    The increasing demand and hampered use of primary human hepatocytes for research purposes have urged scientists to search for alternative cell sources, such as immortalized hepatic cell lines. The aim of this study was to develop a human hepatic cell line using the combined overexpression of TERT and the cell cycle regulators cyclin D1 and mutant isoform CDK4R24C. Following transduction of adult human primary hepatocytes with the selected immortalization genes, cell growth was triggered and a cell line was established. When cultured under appropriate conditions, the cell line expressed several hepatocytic markers and liver-enriched transcription factors at the transcriptional and/or translational level, secreted liver-specific proteins and showed glycogen deposition. These results suggest that the immortalization strategy applied to primary human hepatocytes could generate a novel hepatic cell line that seems to retain some key hepatic characteristics. PMID:26869867

  12. Distinct differentiation characteristics of individual human embryonic stem cell lines

    PubMed Central

    Mikkola, Milla; Olsson, Cia; Palgi, Jaan; Ustinov, Jarkko; Palomaki, Tiina; Horelli-Kuitunen, Nina; Knuutila, Sakari; Lundin, Karolina; Otonkoski, Timo; Tuuri, Timo

    2006-01-01

    Background Individual differences between human embryonic stem cell (hESC) lines are poorly understood. Here, we describe the derivation of five hESC lines (called FES 21, 22, 29, 30 and 61) from frozen-thawed human embryos and compare their individual differentiation characteristic. Results The cell lines were cultured either on human or mouse feeder cells. The cells grew significantly faster and could be passaged enzymatically only on mouse feeders. However, this was found to lead to chromosomal instability after prolonged culture. All hESC lines expressed the established markers of pluripotent cells as well as several primordial germ cell (PGC) marker genes in a uniform manner. However, the cell lines showed distinct features in their spontaneous differentiation patterns. The embryoid body (EB) formation frequency of FES 30 cell line was significantly lower than that of other lines and cells within the EBs differentiated less readily. Likewise, teratomas derived from FES 30 cells were constantly cystic and showed only minor solid tissue formation with a monotonous differentiation pattern as compared with the other lines. Conclusion hESC lines may differ substantially in their differentiation properties although they appear similar in the undifferentiated state. PMID:16895598

  13. 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.

  14. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    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.

  15. VOLIN and KJON-Two novel hyperdiploid myeloma cell lines.

    PubMed

    Våtsveen, Thea Kristin; Børset, Magne; Dikic, Aida; Tian, Erming; Micci, Francesca; Lid, Ana H B; Meza-Zepeda, Leonardo A; Coward, Eivind; Waage, Anders; Sundan, Anders; Kuehl, W Michael; Holien, Toril

    2016-11-01

    Multiple myeloma can be divided into two distinct genetic subgroups: hyperdiploid (HRD) or nonhyperdiploid (NHRD) myeloma. Myeloma cell lines are important tools to study myeloma cell biology and are commonly used for preclinical screening and testing of new drugs. With few exceptions human myeloma cell lines are derived from NHRD patients, even though about half of the patients have HRD myeloma. Thus, there is a need for cell lines of HRD origin to enable more representative preclinical studies. Here, we present two novel myeloma cell lines, VOLIN and KJON. Both of them were derived from patients with HRD disease and shared the same genotype as their corresponding primary tumors. The cell lines' chromosomal content, genetic aberrations, gene expression, immunophenotype as well as some of their growth characteristics are described. Neither of the cell lines was found to harbor immunoglobulin heavy chain translocations. The VOLIN cell line was established from a bone marrow aspirate and KJON from peripheral blood. We propose that these unique cell lines may be used as tools to increase our understanding of myeloma cell biology. © 2016 Wiley Periodicals, Inc. PMID:27311012

  16. Cell lines used for the selection of recombinant baculovirus.

    PubMed

    Maruniak, J E; Garcia-Canedo, A; Rodrigues, J J

    1994-04-01

    Four insect cell lines were used to isolate two recombinant baculoviruses which had the beta-galactosidase (beta-gal) gene for colorimetric assay purposes. Plaque assays were performed using two Trichoplusia ni cell lines: BTI-TN-5B1-4 and TN-368, and two Spodptera frugiperda cell lines: IPLB-SF-21AE and SF9. The number of plaques (occlusion positive and blue beta-gal+ recombinants) formed in the Trichoplusia cells was higher than in the Spodoptera cells. The appearance of Autographa californica NPV polyhedra was also faster in the T. ni cell lines. The effect of cell passage on the plaque formation proved to be critical when two different passages of the SF9 cells were tested. The higher passage produced a lower viral titration. The size and time of appearance of the plaques was also different.

  17. Authentication of the R06E Fruit Bat Cell Line

    PubMed Central

    Jordan, Ingo; Munster, Vincent J.; Sandig, Volker

    2012-01-01

    Fruit bats and insectivorous bats are believed to provide a natural reservoir for a wide variety of infectious diseases. Several lines of evidence, including the successful isolation of infectious viruses, indicate that Marburg virus and Ravn virus have found a major reservoir in colonies of the Egyptian rousette (Rousettus aegyptiacus). To facilitate molecular studies on virus-reservoir host interactions and isolation of viruses from environmental samples, we established cell lines from primary cells of this animal. The cell lines were given to several laboratories until we realized that a contamination with Vero cells in one of the cultures had occurred. Here we describe a general diagnostic procedure for identification of cross-species contamination with the focus on Vero and Rousettus cell lines, and summarize newly discovered properties of the cell lines that may pertain to pathogen discovery. PMID:22754654

  18. Establishment and characterization of the Bombyx mandarina cell line.

    PubMed

    Iwanaga, Masashi; Arai, Rika; Shibano, Yuka; Kawasaki, Hideki; Imanishi, Shigeo

    2009-06-01

    A new cell line, designated as NIAS-Boma-529b, was established from the larval fat bodies of Bombyx mandarina (B. mandarina), which is believed to be an ancestor of Bombyx mori (B. mori). This cell line has been cultured for approximately 150 passages during 2years in an IPL-41 medium supplemented with 10% fetal bovine serum at a constant temperature of 26 degrees C. The morphology of this line includes adhesive round and spindle-shaped cells. Random-amplified polymorphic DNA analysis (RAPD) using 7 primers and a statistical analysis based on Nei's genetic distance revealed that this cell line was closely related to B. mori-derived cell lines. An infection study also revealed that this cell line was susceptible to B. mori nucleopolyhedrovirus (BmNPV); however, it had no apparent susceptibility to Autographa californica NPV (AcNPV), which is closely related to BmNPV. Nevertheless, cells infected with AcNPV showed an extensive cytopathic effect (CPE), including a rough cell surface, rounding, nuclear expansion, and cell blebbing. These results suggest that this cell line can be useful to clarify the mechanism of host range determination of BmNPV and AcNPV.

  19. 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.

  20. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    1994-12-31

    The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

  1. Hemin toxicity in a human epithelioid sarcoma cell line.

    PubMed

    Braverman, S; Helson, C; Helson, L

    1995-01-01

    The major cytotoxic component of hemin was identified as metal free protoporphyrin IX in an epithelioid sarcoma cell line (VA-ES-BJ) and a glioblastoma cell line (U-373 MG) by exposing the cell lines to the iron chelator deferoxamine, tin-protoporphyrin IX, and protoporphyrin IX. The contribution of lipid peroxidation and free radical generation to toxicity was examined using DL-buthionine-[S,R]-sulfoximine (BSO), and 21-aminosteroid (lazaroid, U74500A). Hemin caused significantly greater toxicity in VA-ES-BJ than in U-373 MG. While exogenous PpIX was more toxic than hemin in both cell lines, this toxicity was not due to iron depletion following intracellular heme formation since ferric citrate did not reverse PpIX toxicity. Pre-treatment with BSO enhanced hemin toxicity in the VA-ES-BJ cell line but not in U-373 MG, suggesting different modes of toxicity in the two cell lines. Exposure to lazaroid protected only VA-ES-BJ from protoporphyrin-induced toxicity implicating a specific sensitivity to lipid peroxidation and/or free radical generation by this cell line. These characteristics of the VA-ES-BJ cell line distinguish it from the glioblastoma and emphasize its utility for exploring cytotoxic effects of hemin and its precursors.

  2. Derivation of the human embryonic stem cell line RCM1.

    PubMed

    De Sousa, P A; Tye, B J; Sneddon, S; Bruce, K; Dand, P; Russell, G; Collins, D M; Greenshields, A; McDonald, K; Bradburn, H; Gardner, J; Downie, J M; Courtney, A; Brison, D R

    2016-03-01

    The human embryonic stem cell line RCM-1 was derived from a failed to fertilise egg undergoing parthenogenetic stimulation. The cell line shows normal pluripotency marker expression and differentiation to three germ layers in vitro and in vivo. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available. PMID:27346018

  3. Regulated expression of erythropoietin by two human hepatoma cell lines

    SciTech Connect

    Goldberg, M.A.; Glass, G.A.; Cunningham, J.M.; Bunn, H.F.

    1987-11-01

    The development of a cell culture system that produces erythropoietin (Epo) in a regulated manner has been the focus of much effort. The authors have screened multiple renal and hepatic cell lines for either constitutive or regulated expression of Epo. Only the human hepatoma cell lines, Hep3B and HepG2, made significant amounts of Epo as measured both by radioimmunoassay and in vitro bioassay (as much as 330 milliunits per 10/sup 6/ cells in 24 hr). The constitutive production of Epo increased dramatically as a function of cell density in both cell lines. At cell densities < 3.3 x 10/sup 5/ cells per cm/sup 2/, there was little constitutive release of Epo in the medium. With Hep3B cells grown at low cell densities, a mean 18-fold increase in Epo expression was seen in response to hypoxia and a 6-fold increase was observed in response to incubation in medium containing 50 ..mu..M cobalt(II) chloride. At similar low cell densities, Epo production in HepG2 cells could be enhanced an average of about 3-fold by stimulation with either hypoxia or cobalt(II) chloride. Upon such stimulation, both cell lines demonstrated markedly elevated levels of Epo mRNA. Hence, both Hep3B and HepG2 cell lines provide an excellent in vitro system in which to study the physiological regulation of Epo expression.

  4. 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.

  5. CACO-2 CELL LINES IN DRUG DISCOVERY- AN UPDATED PERSPECTIVE

    PubMed Central

    Kumar, Kalyan K.V; Karnati, Swathi; Reddy, Mamatha B; Chandramouli, R

    2010-01-01

    Cell lines are the invitro models used for the drug permeability studies in the preclinical and clinical phases of the drug discovery. Cell line models are simple and quick to use and avoids the usage of animal models for pharmacological and toxicological studies and hence cost effective, produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates. Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on CACO-2. By virtue of its merits, CACO-2 cell line development, transport experiments, automated assays, optimization of experimental conditions and mechanistic uses of CACO-2 cell lines dealt comprehensively in the following context. PMID:24825967

  6. Derivation of Genea057 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea057 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea057 was demonstrated with 97% of cells expressing Nanog, 81% Oct4, 75% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.59 and Novelty score of 1.32. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345782

  7. Derivation of Genea042 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea042 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea042 was demonstrated with 81% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 30.06, Novelty score of 1.24 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345994

  8. Derivation of Genea002 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea002 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype by CGH and male Allele pattern through STR analysis. Pluripotency of Genea002 was demonstrated with 75% of cells expressing Nanog, 93% Oct4, 83% Tra1-60 and 98% SSEA4, a Pluritest pluripotency score of 24.55, Novelty score of 1.39, teratomas with tissues from all embryonic germ layers and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345802

  9. Derivation of Genea052 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea052 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea052 was demonstrated with 85% of cells expressing Nanog, 87% Oct4, 60% Tra1-60 and 97% SSEA4, a PluriTest Pluripotency score of 27.21, Novelty score of 1.2 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345996

  10. Derivation of human embryonic stem cell line Genea023.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Goel, Divya; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea023 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea023 was demonstrated with 85% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 98% SSEA4, gave a Pluritest Pluripotency score of 42.76, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346015

  11. Derivation of Genea015 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea015 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea015 was demonstrated with 80% of cells expressing Nanog, 97% Oct4, 75% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 29.52, Novelty score of 1.3 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346028

  12. Derivation of human embryonic stem cell line Genea022.

    PubMed

    Dumevska, Biljana; Bosman, Alexis; McKernan, Robert; Schmidt, Uli; Peura, Teija

    2016-03-01

    The Genea022 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through CGH and STR analysis. Pluripotency of Genea022 was demonstrated with 84% of cells expressed Nanog, 98% Oct4, 55% Tra1-60 and 97% SSEA4, gave a Pluritest Pluripotency score of 42.95, Novelty of 1.23, demonstrated Alkaline Phosphatase activity and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and visible contamination. PMID:27346017

  13. Derivation of Genea047 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea047 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea047 was demonstrated with 88% of cells expressing Nanog, 95% Oct4, 59% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 30.86, Novelty score of 1.23 and tri-lineage teratoma formation. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345995

  14. Derivation of Genea043 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-01-01

    The Genea043 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XY karyotype and male allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea043 was demonstrated with 92% of cells expressing Nanog, 95% Oct4, 61% Tra1-60 and 99% SSEA4, a PluriTest Pluripotency score of 31.74, Novelty score of 1.2 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345801

  15. Derivation of Genea016 human embryonic stem cell line.

    PubMed

    Dumevska, Biljana; Chami, Omar; McKernan, Robert; Goel, Divya; Peura, Teija; Schmidt, Uli

    2016-01-01

    The Genea016 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated human feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype and female Allele pattern through traditional karyotyping, CGH and STR analysis. Pluripotency of Genea016 was demonstrated with 77% of cells expressing Nanog, 95% Oct4, 53% Tra1-60 and 98% SSEA4, a PluriTest Pluripotency score of 28.4, Novelty score of 1.37 and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27345780

  16. Differential effects of bisphosphonates on breast cancer cell lines.

    PubMed

    Verdijk, R; Franke, H R; Wolbers, F; Vermes, I

    2007-02-01

    Bisphosphonates may induce direct anti-tumor effects in breast cancer cells in vitro. In this study, six bisphosphonates were administered to three breast cancer cell lines. Cell proliferation was measured by quantification of the expression of Cyclin D1 mRNA. Apoptosis was determined by flow cytometry of a DNA fragmentation assay. We demonstrated that bisphosphonates have direct effects on cell proliferation and apoptosis in different breast cancer cell lines. However, not all bisphosphonates act equally on breast cancer cells in vitro. Zoledronate seems to be the most potent of the six bisphosphonates. This in vitro study showed that bisphosphonates possess promising anti-tumor potential.

  17. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced.

  18. Recombinant protein production from stable mammalian cell lines and pools.

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

    We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. PMID:27322762

  19. Deriving cell lines from zebrafish embryos and tumors.

    PubMed

    Choorapoikayil, Suma; Overvoorde, John; den Hertog, Jeroen

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months.

  20. Eternity and functionality - rational access to physiologically relevant cell lines.

    PubMed

    Lipps, Christoph; May, Tobias; Hauser, Hansjörg; Wirth, Dagmar

    2013-12-01

    In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines.

  1. Application of DNA fingerprints for cell-line individualization.

    PubMed Central

    Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

    1990-01-01

    DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells. Images p[504]-a PMID:1975479

  2. [DNA fingerprinting analysis of silkworm embryo cell lines].

    PubMed

    Pan, Min Hui; Feng, Zhen Yue; Tian, Zhi Qiang; Liu, Min; Lu, Cheng

    2006-12-01

    DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines.

  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. Development of a cell line from Echinococcus granulosus germinal layer.

    PubMed

    Albani, Clara María; Cumino, Andrea Carina; Elissondo, María Celina; Denegri, Guillermo María

    2013-10-01

    In vitro culture of parasitic helminths provides an important tool to study cell regeneration and physiology, as well as for molecular biology and genetic engineering studies. In the present study, we established in vitro propagation of cells from Echinococcus granulosus germinal cyst layer. E. granulosus germinal cells grew beyond 100 passages and showed no signs of reduced proliferation capacity. Microscopic analysis revealed that cells grew both attached to the substrate and in suspension, forming three-dimensional structures like mammalian stem cell aggregates. Examination of the chromosome number of attached germinal cells showed a high degree of heteroploidy, suggesting the occurrence of transformation during culture. Monolayer cells survived cryopreservation and were able to proliferate after thawing. Based on the characteristics displayed by E. granulosus germinal cells, we establish a cell line from the E. granulosus germinal layer. Furthermore, we propose that this cell line could be useful for drug screening and for obtaining parasite material.

  5. Vaccine production: upstream processing with adherent or suspension cell lines.

    PubMed

    Genzel, Yvonne; Rödig, Jana; Rapp, Erdmann; Reichl, Udo

    2014-01-01

    The production of viral vaccines in cell culture can be accomplished with primary, diploid, or continuous (transformed) cell lines. Each cell line, each virus type, and each vaccine preparation require the specific design of upstream and downstream processing. Media have to be selected as well as production vessels, cultivation conditions, and modes of operation. Many viruses only replicate to high titers in adherently growing cells, but similar to processes established for recombinant protein production, an increasing number of suspension cell lines is being evaluated for future use. Here, we describe key issues to be considered for the establishment of large-scale virus production in bioreactors. As an example upstream processing of cell culture-derived influenza virus production is described in more detail for adherently growing and for suspension cells. In particular, use of serum-containing, serum-free, and chemically defined media as well as choice of cultivation vessel are considered. PMID:24297427

  6. [Decontamination of continual cell lines spontaneously infected with mycoplasmas].

    PubMed

    Machatková, M; Jurmanová, K; Snejdar, V

    1986-07-01

    The continual cell lines of bovine kidneys MDBK and AUBEK, and porcine kidneys RPD and IBRS, spontaneously infected with Mycoplasma arginini and Acholeplasma laidlawii, were decontaminated by the method of selective elimination. Two elimination procedures were modified to be used for the decontamination: one based on the reduction of infection by the light treatment of the cultures, the other based on the selection of mycoplasma-free cell population through cell clonation. On the basis of a long-continued control of the cell clones a methodical procedure of the preparation of mycoplasma-free cell lines was worked out. PMID:3090766

  7. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014. PMID:25229492

  8. Development and characterization of a largemouth bass cell line.

    PubMed

    Getchell, Rodman G; Groocock, Geoffrey H; Cornwell, Emily R; Schumacher, Vanessa L; Glasner, Lindsay I; Baker, Barry J; Frattini, Stephen A; Wooster, Gregory A; Bowser, Paul R

    2014-09-01

    Abstract The development and characterization of a new cell line, derived from the ovary of Largemouth Bass Micropterus salmoides, is described. Gonad tissue was collected from Largemouth Bass that were electrofished from Oneida Lake, New York. The tissue was processed and grown in culture flasks at approximately 22°C for more than 118 passages during an 8-year period from 2004 to 2011. The identity of these cells as Largemouth Bass origin was confirmed by sequencing a portion of the cytochrome b gene. Growth rate at three different temperatures was documented. The cell line was susceptible to Largemouth Bass virus (LMBV) and its replication was compared with that of Bluegill Lepomis macrochirus fry (BF-2), one of the cell lines recommended for LMBV isolation by the American Fisheries Society Fish Health Section Blue Book. Quantitative PCR results from the replication trial showed the BF-2 cell line produced approximately 10-fold more LMBV copies per cell than the new Largemouth Bass cell line after 6 d, while the titration assay showed similar quantities in each cell line after 1 week. Received February 18, 2014; accepted April 16, 2014.

  9. Apoptotic effect of noscapine in breast cancer cell lines.

    PubMed

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

    Cancer is a public health problem in the world and breast cancer is the most frequently cancer in women. Approximately 15% of the breast cancers are triple-negative. Apoptosis regulates normal growth, homeostasis, development, embryogenesis and appropriate strategy to treat cancer. Bax is a protein pro-apoptotic enhancer of apoptosis in contrast to Bcl-2 with antiapoptotic properties. Initiator caspase-9 and caspase-8 are features of intrinsic and extrinsic apoptosis pathway, respectively. NF-κB is a transcription factor known to be involved in the initiation and progression of breast cancer. Noscapine, an alkaloid derived from opium is used as antitussive and showed antitumor properties that induced apoptosis in cancer cell lines. The aim of the present study was to determine the apoptotic effect of noscapine in breast cancer cell lines compared to breast normal cell line. Three cell lines were used: i) a control breast cell line MCF-10F; ii) a luminal-like adenocarcinoma triple-positive breast cell line MCF-7; iii) breast cancer triple-negative cell line MDA-MB-231. Our results showed that noscapine had lower toxicity in normal cells and was an effective anticancer agent that induced apoptosis in breast cancer cells because it increases Bax gene and protein expression in three cell lines, while decreases Bcl-xL gene expression, and Bcl-2 protein expression decreased in breast cancer cell lines. Therefore, Bax/Bcl-2 ratio increased in the three cell lines. This drug increased caspase-9 gene expression in breast cancer cell lines and caspase-8 gene expression increased in MCF-10F and MDA-MB-231. Furthermore, it increased cleavage of caspase-8, suggesting that noscapine-induced apoptosis is probably due to the involvement of extrinsic and intrinsic apoptosis pathways. Antiapoptotic gene and protein expression diminished and proapoptotic gene and protein expression increased noscapine-induced expression, probably due to decrease in NF-κB gene and protein expression

  10. Characterization of an epithelial cell line from bovine mammary gland.

    PubMed

    German, Tania; Barash, Itamar

    2002-05-01

    Elucidation of the bovine mammary gland's unique characteristics depends on obtaining an authentic cell line that will reproduce its function in vitro. Representative clones from bovine mammary cell populations, differing in their attachment capabilities, were cultured. L-1 cells showed strong attachment to the plate, whereas H-7 cells detached easily. Cultures established from these clones were nontumorigenic upon transplantation to an immunodeficient host; they exhibited the epithelial cell characteristics of positive cytokeratin but not smooth muscle actin staining. Both cell lines depended on fetal calf serum for proliferation. They exhibited distinct levels of differentiation on Matrigel in serum-free, insulin-supplemented medium on the basis of their organization and beta-lactoglobulin (BLG) secretion. H-7 cells organized into mammospheres, whereas L-1 cells arrested in a duct-like morphology. In both cell lines, prolactin activated phosphorylation of the signal transducer and activator of transcription, Stat5-a regulator of milk protein gene transcription, and of PHAS-I-an inhibitor of translation initiation in its nonphosphorylated form. De novo synthesis and secretion of BLG were detected in differentiated cultures: in L-1 cells, BLG was dependent on lactogenic hormones for maximal induction but was less stringently controlled than was beta-casein in the mouse CID-9 cell line. L-1 cells also encompassed a near-diploid chromosomal karyotype and may serve as a tool for studying functional characteristics of the bovine mammary gland.

  11. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease.

    PubMed

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-07-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the 'gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies.

  12. Molecular profiling reveals primary mesothelioma cell lines recapitulate human disease

    PubMed Central

    Chernova, T; Sun, X M; Powley, I R; Galavotti, S; Grosso, S; Murphy, F A; Miles, G J; Cresswell, L; Antonov, A V; Bennett, J; Nakas, A; Dinsdale, D; Cain, K; Bushell, M; Willis, A E; MacFarlane, M

    2016-01-01

    Malignant mesothelioma (MM) is an aggressive, fatal tumor strongly associated with asbestos exposure. There is an urgent need to improve MM patient outcomes and this requires functionally validated pre-clinical models. Mesothelioma-derived cell lines provide an essential and relatively robust tool and remain among the most widely used systems for candidate drug evaluation. Although a number of cell lines are commercially available, a detailed comparison of these commercial lines with freshly derived primary tumor cells to validate their suitability as pre-clinical models is lacking. To address this, patient-derived primary mesothelioma cell lines were established and characterized using complementary multidisciplinary approaches and bioinformatic analysis. Clinical markers of mesothelioma, transcriptional and metabolic profiles, as well as the status of p53 and the tumor suppressor genes CDKN2A and NF2, were examined in primary cell lines and in two widely used commercial lines. Expression of MM-associated markers, as well as the status of CDKN2A, NF2, the ‘gatekeeper' in MM development, and their products demonstrated that primary cell lines are more representative of the tumor close to its native state and show a degree of molecular diversity, thus capturing the disease heterogeneity in a patient cohort. Molecular profiling revealed a significantly different transcriptome and marked metabolic shift towards a greater glycolytic phenotype in commercial compared with primary cell lines. Our results highlight that multiple, appropriately characterised, patient-derived tumor cell lines are required to enable concurrent evaluation of molecular profiles versus drug response. Furthermore, application of this approach to other difficult-to-treat tumors would generate improved cellular models for pre-clinical evaluation of novel targeted therapies. PMID:26891694

  13. Mercury specifically induces LINE-1 activity in a human neuroblastoma cell line.

    PubMed

    Habibi, Laleh; Shokrgozar, Mohammad Ali; Tabrizi, Mina; Modarressi, Mohammad Hossein; Akrami, Seyed Mohammad

    2014-01-01

    L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time.

  14. Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

    PubMed Central

    Ertel, Adam; Verghese, Arun; Byers, Stephen W; Ochs, Michael; Tozeren, Aydin

    2006-01-01

    Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM). Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs), and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative phosphorylation. Signaling

  15. Global Conservation of Protein Status between Cell Lines and Xenografts.

    PubMed

    Biau, Julian; Chautard, Emmanuel; Court, Frank; Pereira, Bruno; Verrelle, Pierre; Devun, Flavien; De Koning, Leanne; Dutreix, Marie

    2016-08-01

    Common preclinical models for testing anticancer treatment include cultured human tumor cell lines in monolayer, and xenografts derived from these cell lines in immunodeficient mice. Our goal was to determine how similar the xenografts are compared with their original cell line and to determine whether it is possible to predict the stability of a xenograft model beforehand. We studied a selection of 89 protein markers of interest in 14 human cell cultures and respective subcutaneous xenografts using the reverse-phase protein array technology. We specifically focused on proteins and posttranslational modifications involved in DNA repair, PI3K pathway, apoptosis, tyrosine kinase signaling, stress, cell cycle, MAPK/ERK signaling, SAPK/JNK signaling, NFκB signaling, and adhesion/cytoskeleton. Using hierarchical clustering, most cell culture-xenograft pairs cluster together, suggesting a global conservation of protein signature. Particularly, Akt, NFkB, EGFR, and Vimentin showed very stable protein expression and phosphorylation levels highlighting that 4 of 10 pathways were highly correlated whatever the model. Other proteins were heterogeneously conserved depending on the cell line. Finally, cell line models with low Akt pathway activation and low levels of Vimentin gave rise to more reliable xenograft models. These results may be useful for the extrapolation of cell culture experiments to in vivo models in novel targeted drug discovery. PMID:27567954

  16. Guanylate-Binding Protein-1 protects ovarian cancer cell lines but not breast cancer cell lines from killing by paclitaxel.

    PubMed

    Tipton, Aaron R; Nyabuto, Geoffrey O; Trendel, Jill A; Mazur, Travis M; Wilson, John P; Wadi, Suzan; Justinger, Jacob S; Moore, Garret L; Nguyen, Peter T; Vestal, Deborah J

    2016-09-30

    Forced expression of the cytokine-induced large GTPase, human Guanylate-Binding Protein-1 (hGBP-1), in ovarian cancer cell lines increases resistance to paclitaxel. Elevated hGBP-1 RNA in ovarian tumors correlates with shorter recurrence-free survival. In contract, hGBP-1 is part of a gene signature predicting improved prognosis in all subtypes of breast cancers. hGBP-1 does not confer paclitaxel resistance on MCF-7 and TMX2-28 breast cancer cells. Expression of the isotype of the hGBP-1-interacting protein, PIM1, which may contribute to paclitaxel resistance when associated with hGBP-1, is different in breast and ovarian cancer cell lines. Breast cancer cell lines express the 44 kDa isoform of PIM-1, and ovarian cancer cell lines express the 33 kDa isoform. PMID:27590579

  17. 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.

  18. 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.

  19. Derivation of human embryonic stem cell lines, towards clinical quality.

    PubMed

    Hovatta, Outi

    2006-01-01

    Human embryonic stem (hES) cells offer an excellent source of cells for transplantation in the treatment of severe diseases. To be clinically safe, the lines have to be derived using strict quality criteria and good manufacturing practice. Animal proteins are immunogenic and may contain microbes, and they should not be used in establishing or propagating hES cells. Derivation systems have been improved towards clinical quality by establishing all 25 hES cell lines using human skin fibroblasts as feeder cells instead of mouse fibroblasts. A further 21 cell lines have been established using synthetic serum instead of fetal calf serum in the medium. In the five latest derivations, the inner cell mass was isolated mechanically instead of by immunosurgery (animal antibodies). Feeder-free derivation would be optimal, but it is not yet considered safe. Clinical-quality lines can be derived by establishing the skin fibroblast feeders in the good manufacturing practice laboratory with human serum in the medium, and by establishing the hES cells on such feeders. In this process, a serum replacement that contains only human protein can be used, the inner cell mass has to be isolated mechanically, and the colonies have to be split mechanically for passaging. Somatic cell nuclear transfer would help to overcome rejection of transplanted cells. PMID:17147930

  20. DNA fingerprinting of the NCI-60 cell line panel.

    PubMed

    Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

    2009-04-01

    The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

  1. Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines

    PubMed Central

    Gabrani, Reema; Jain, Ramya; Sharma, Anjali; Sarethy, Indira P.; Dang, Shweta; Gupta, S.

    2012-01-01

    Solanum nigrum is used in various traditional medical systems for antiproliferative, antiinflammatory, antiseizure and hepatoprotective activities. We have evaluated organic solvent and aqueous extracts obtained from berries of Solanum nigrum for antiproliferative activity on leukemic cell lines, Jurkat and HL-60 (Human promyelocytic leukemia cells). The cell viability after the treatment with Solanum nigrum extract was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay. Results indicated increased cytotoxicity with increasing extract concentrations. Comparative analysis indicated that 50% inhibitory concentration value of methanol extract is the lowest on both cell lines. PMID:23716874

  2. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

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

    PubMed Central

    Sadowska, Anna; Krętowski, Rafał; 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 μg/mL after 24 hours; 0.1, 10, 50, and 250 μg/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

  4. 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

  5. Regeneration of lateral line and inner ear vestibular cells.

    PubMed

    Jørgensen, J M

    1991-01-01

    Labelling experiments with [3H]thymidine demonstrate a continuous production of cells in the mechanoreceptive lateral line organs of the eel (Anguilla anguilla) and butterfly fish (Pantodon buchholzi) as well as in the electroreceptive ampullary organ of the transparent catfish (Kryptopterus bicirrhus). Shortly after [3H]thymidine injection many cells are labelled in the middle and basal parts of the sensory organ and after a few days' survival sensory cells are also labelled. The vestibular sensory organs of selected species of fishes, amphibians, reptiles and birds also show a continuous production of cells. In the budgerigar (Melopsittacus undulatus) labelled cells are found in the basal and middle layer of the sensory epithelium a few hours after injection with [3H]thymidine. A few days after the injection labelled cells are found in non-calyceal hair cells. After one month the calyceal cells are also labelled. Similar experiments with the bat Pipistrellus nathusii and with normal and gentamicin-treated mice (Mus musculus) show no labelled cells in the inner ear sensory epithelia. The lateral line organs and vestibular epithelia of non-mammalian vertebrates all contain a small number of dark cells with the characteristics of apoptotic cells. Macrophages and inclusions in some cells, thought to be remnants of apoptotic cells, are occasionally seen. Fixation at different osmolarities has little effect on the number of dark cells. It is suggested that the continually produced cells replace apoptotic dying cells.

  6. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

    Guerrero, Carlos A.; Guerrero, Rafael A.; Silva, Elver; Acosta, Orlando; Barreto, Emiliano

    2016-01-01

    A number of viruses show a naturally extended tropism for tumor cells whereas other viruses have been genetically modified or adapted to infect tumor cells. Oncolytic viruses have become a promising tool for treating some cancers by inducing cell lysis or immune response to tumor cells. In the present work, rotavirus strains TRF-41 (G5) (porcine), RRV (G3) (simian), UK (G6-P5) (bovine), Ym (G11-P9) (porcine), ECwt (murine), Wa (G1-P8), Wi61 (G9) and M69 (G8) (human), and five wild-type human rotavirus isolates were passaged multiple times in different human tumor cell lines and then combined in five different ways before additional multiple passages in tumor cell lines. Cell death caused by the tumor cell-adapted isolates was characterized using Hoechst, propidium iodide, 7-AAD, Annexin V, TUNEL, and anti-poly-(ADP ribose) polymerase (PARP) and -phospho-histone H2A.X antibodies. Multiple passages of the combined rotaviruses in tumor cell lines led to a successful infection of these cells, suggesting a gain-of-function by the acquisition of greater infectious capacity as compared with that of the parental rotaviruses. The electropherotype profiles suggest that unique tumor cell-adapted isolates were derived from reassortment of parental rotaviruses. Infection produced by such rotavirus isolates induced chromatin modifications compatible with apoptotic cell death. PMID:26828934

  7. 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

  8. Generation of stable Drosophila cell lines using multicistronic vectors.

    PubMed

    González, Monika; Martín-Ruíz, Itziar; Jiménez, Silvia; Pirone, Lucia; Barrio, Rosa; Sutherland, James D

    2011-01-01

    Insect cell culture is becoming increasingly important for applications including recombinant protein production and cell-based screening with chemical or RNAi libraries. While stable mammalian cell lines expressing a protein of interest can be efficiently prepared using IRES-based vectors or viral-based approaches, options for stable insect cell lines are more limited. Here, we describe pAc5-STABLEs, new vectors for use in Drosophila cell culture to facilitate stable transformation. We show that viral-derived 2A-like (or "CHYSEL") peptides function in Drosophila cells and can mediate the multicistronic expression of two or three proteins of interest under control of the Actin5C constitutive promoter. The current vectors allow mCherry and/or GFP fusions to be generated for positive selection by G418 resistance in cells and should serve as a flexible platform for future applications. PMID:22355594

  9. 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.

  10. 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

  11. Molecular dissection of AKT activation in lung cancer cell lines

    PubMed Central

    Guo, Yanan; Du, Jinyan; Kwiatkowski, David J

    2013-01-01

    AKT is a critical signaling node downstream of PI3K, which is often activated in cancer. We analyzed the state of activation of AKT in 80 human non-small cell lung cancer cell lines under serum starvation conditions. We identified 13 lines which showed persistent AKT activation in the absence of serum. In 12 of the 13 lines, AKT activation could be attributed to loss of PTEN, activating mutation in EGFR or PIK3CA, or amplification of ERBB2. HCC2429 was the only cell line that had no alterations in those genes, but had high phospho-AKT(Ser473) levels under serum starvation conditions. However, the activation of AKT in HCC2429 was PI3K- and mTORC2-dependent based upon use of specific inhibitors. Kinome tyrosine phosphorylation profiling showed that both Notch and SRC were highly activated in this cell line. Despite the activation of Notch, AKT activation and cell survival were not affected by Notch inhibitors DAPT or Compound E. In contrast, SRC inhibitors PP2 and dasatinib both significantly decreased pAKT(Ser473) levels and reduced cell survival by inducing apoptosis. Further, a combination of SRC and mTOR inhibition synergistically blocked activation of AKT and induced apoptosis. Over-expression of SRC has been identified previously in human lung cancers, and these results suggest that a combination of SRC and mTOR inhibitors may have unique therapeutic benefit for a subset of lung cancers with these molecular features. PMID:23319332

  12. Heterozygous Embryonic Stem Cell Lines Derived from Nonhuman Primate Parthenotes

    PubMed Central

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M. Cecilia T.; Wolf, Don; Mitalipov, Shoukhrat

    2009-01-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients. PMID:18192229

  13. Heterozygous embryonic stem cell lines derived from nonhuman primate parthenotes.

    PubMed

    Dighe, Vikas; Clepper, Lisa; Pedersen, Darlene; Byrne, James; Ferguson, Betsy; Gokhale, Sumita; Penedo, M Cecilia T; Wolf, Don; Mitalipov, Shoukhrat

    2008-03-01

    Monoparental parthenotes represent a potential source of histocompatible stem cells that should be isogenic with the oocyte donor and therefore suitable for use in cell or tissue replacement therapy. We generated five rhesus monkey parthenogenetic embryonic stem cell (PESC) lines with stable, diploid female karyotypes that were morphologically indistinguishable from biparental controls, expressed key pluripotent markers, and generated cell derivatives representative of all three germ layers following in vivo and in vitro differentiation. Interestingly, high levels of heterozygosity were observed at the majority of loci that were polymorphic in the oocyte donors. Some PESC lines were also heterozygous in the major histocompatibility complex region, carrying haplotypes identical to those of the egg donor females. Expression analysis revealed transcripts from some imprinted genes that are normally expressed from only the paternal allele. These results indicate that limitations accompanying the potential use of PESC-derived phenotypes in regenerative medicine, including aberrant genomic imprinting and high levels of homozygosity, are cell line-dependent and not always present. PESC lines were derived in high enough yields to be practicable, and their derivatives are suitable for autologous transplantation into oocyte donors or could be used to establish a bank of histocompatible cell lines for a broad spectrum of patients.

  14. Novel cell lines derived from transgenic mice expressing recombinant human proteins. Transgenic hepatoma-derived cell lines.

    PubMed

    Perraud, F; Dalemans, W; Ali-Hadji, D; Pavirani, A

    1992-01-01

    We have used transgenic mouse technology to establish immortalized hepatoma cell lines stably secreting heterologous proteins, such as human alpha 1-antitrypsin and human factor IX. Hepatocyte-specific regulatory DNA sequences were used to target both the expression of an onc gene and the gene coding for the human protein to the liver of transgenic mice which eventually developed hepatocellular carcinomas. Tumour cells were subsequently established as permanent cell lines, which maintained a differentiated phenotype under specific culture conditions, being capable of producing biologically active and correctly processed human alpha 1-antitrypsin and factor IX. Moreover, a preliminary analysis has shown that certain cell lines express elevated total cytochrome P450 activity. These cells could therefore represent a useful alternative to the use of animals or primary cultures in drug safety testing. PMID:1369183

  15. Germ line development: lessons learned from pluripotent stem cells.

    PubMed

    Martínez-Arroyo, Ana M; Medrano, Jose V; Remohí, José; Simón, Carlos

    2014-10-01

    Current knowledge about mammalian germ line development is mainly based on the mouse model and little is known about how this fundamental process occurs in humans. This review summarizes our current knowledge of genetic and epigenetic germ line development in mammals, mainly focusing on primordial germ cell (PGC) specification events, comparing the differences between mouse and human models. We also emphasize the knowledge derived from the most successful strategies used to generate germ cell-like cells in vitro in both models and major obstacles to obtaining bona fide in vitro-derived gametes are considered. PMID:25461452

  16. 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

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

    PubMed

    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

    2014-09-01

    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

  18. Derivation of human embryonic stem cell line Genea019.

    PubMed

    Dumevska, Biljana; Peura, Teija; McKernan, Robert; Goel, Divya; Schmidt, Uli

    2016-03-01

    The Genea019 human embryonic stem cell line was derived from a donated, fully commercially consented ART blastocyst, through ICM outgrowth on inactivated feeders. The line showed pluripotent cell morphology and genomic analysis verified a 46, XX karyotype, female Allele pattern and unaffected Htt CAG repeat length, compared to HD affected sibling Genea020. Pluripotency of Genea019 was demonstrated with 75% of cells expressing Nanog, 89% Oct4, 48% Tra1-60 and 85% SSEA4, a Pluritest Pluripotency score of 22.97, Novelty score of 1.42, tri-lineage teratoma formation and Alkaline Phosphatase activity. The cell line was negative for Mycoplasma and any visible contamination. PMID:27346002

  19. 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 φC31 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

  20. 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.

  1. 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

  2. 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

  3. Monoclonal antibodies against the human leukemia cell line K 562.

    PubMed

    Böttger, V; Hering, S; Jantscheff, P; Micheel, B

    1985-01-01

    Three monoclonal antibodies raised against K 562, a cell line originally established from a patient with chronic myeloid leukemia (CML) in terminal blast crisis, were selected according to their distinct reaction pattern. Whereas two antibodies (ZIK-C1-A/C5 and ZIK-C1-A/H5 also designated C and H) recognized antigens, present on K 562 cells and other immature and mature hematopoietic cells (cell lines and normal blood and bone marrow cells), antibody ZIK-C1-A/D9 also designated Y showed an exclusive binding to K 562 cells. The results obtained (here and in the following paper) indicate, that antibody ZIK-C1-A/D9 defines an early differentiation antigen of hematopoiesis or a leukemia-associated antigen.

  4. JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development

    PubMed Central

    Cai, Chengfu; Lin, Jinchao; Sun, Shaoyang; He, Yingzi

    2016-01-01

    JNK signaling is known to play a role in regulating cell behaviors such as cell cycle progression, cell proliferation, and apoptosis, and recent studies have suggested important roles for JNK signaling in embryonic development. However, the precise function of JNK signaling in hair cell development remains poorly studied. In this study, we used the small molecule JNK inhibitor SP600125 to examine the effect of JNK signaling abrogation on the development of hair cells in the zebrafish lateral line neuromast. Our results showed that SP600125 reduced the numbers of both hair cells and supporting cells in neuromasts during larval development in a dose-dependent manner. Additionally, JNK inhibition strongly inhibited the proliferation of neuromast cells, which likely explains the decrease in the number of differentiated hair cells in inhibitor-treated larvae. Furthermore, western blot and in situ analysis showed that JNK inhibition induced cell cycle arrest through induction of p21 expression. We also showed that SP600125 induced cell death in developing neuromasts as measured by cleaved caspase-3 immunohistochemistry, and this was accompanied with an induction of p53 gene expression. Together these results indicate that JNK might be an important regulator in the development of hair cells in the lateral line in zebrafish by controlling both cell cycle progression and apoptosis. PMID:26903805

  5. 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

  6. 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

  7. Comparative Metabolic Flux Profiling of Melanoma Cell Lines

    PubMed Central

    Scott, David A.; Richardson, Adam D.; Filipp, Fabian V.; Knutzen, Christine A.; Chiang, Gary G.; Ronai, Ze'ev A.; Osterman, Andrei L.; Smith, Jeffrey W.

    2011-01-01

    Metabolic rewiring is an established hallmark of cancer, but the details of this rewiring at a systems level are not well characterized. Here we acquire this insight in a melanoma cell line panel by tracking metabolic flux using isotopically labeled nutrients. Metabolic profiling and flux balance analysis were used to compare normal melanocytes to melanoma cell lines in both normoxic and hypoxic conditions. All melanoma cells exhibited the Warburg phenomenon; they used more glucose and produced more lactate than melanocytes. Other changes were observed in melanoma cells that are not described by the Warburg phenomenon. Hypoxic conditions increased fermentation of glucose to lactate in both melanocytes and melanoma cells (the Pasteur effect). However, metabolism was not strictly glycolytic, as the tricarboxylic acid (TCA) cycle was functional in all melanoma lines, even under hypoxia. Furthermore, glutamine was also a key nutrient providing a substantial anaplerotic contribution to the TCA cycle. In the WM35 melanoma line glutamine was metabolized in the “reverse” (reductive) direction in the TCA cycle, particularly under hypoxia. This reverse flux allowed the melanoma cells to synthesize fatty acids from glutamine while glucose was primarily converted to lactate. Altogether, this study, which is the first comprehensive comparative analysis of metabolism in melanoma cells, provides a foundation for targeting metabolism for therapeutic benefit in melanoma. PMID:21998308

  8. Morphology and growth of murine cell lines on model biomaterials.

    PubMed

    Godek, Marisha L; Duchsherer, Nichole L; McElwee, Quinn; Grainger, David W

    2004-01-01

    All biomaterial implants are assaulted by the host "foreign body" immune response. Understanding the complex, dynamic relationship between cells, biomaterials and milieu is an important first step towards controlling this reaction. Material surface chemistry dictates protein adsorption, and thus subsequent cell interactions. The cell-implant is a microenvironment involving 1) proteins that coat the surface and 2) cells that interact with these proteins. Macrophages and fibroblasts are two cell types that interact with proteins on biomaterials surfaces and play different related, but equally important, roles in biomaterials rejection and implant failure. Growth characteristics of four murine cell lines on model biomaterials surfaces were examined. Murine monocyte-macrophages (RAW 264.7 and J774A.1), murine macrophage (IC-21) and murine fibroblast (NIH 3T3) cell lines were tested to determine whether differences exist in adhesion, proliferation, differentiation, spreading, and fusion (macrophage lineages only) on these surfaces. Differences were observed in the ability of cells to adhere to and subsequently proliferate on polymer surfaces. (Monocyte-) macrophages grew well on all surfaces tested and growth rates were measured on three representative polymer biomaterials surfaces: tissue culture polystyrene (TCPS), polystyrene, and Teflon-AF. J774A.1 cultures grown on TCPS and treated with exogenous cytokines IL-4 and GM-CSF were observed to contain multinucleate cells with unusual morphologies. Thus, (monocyte-) macrophage cell lines were found to effectively attach to and interrogate each surface presented, with evidence of extensive spreading on Teflon-AF surfaces, particularly in the IC-21 cultures. The J774A.1 line was able to proliferate and/or differentiate to more specialized cell types (multinucleate/dendritic-like cells) in the presence of soluble chemokine cues. PMID:15133927

  9. Establishment of a rat nasal epithelial tumor cell line.

    PubMed

    Hood, A T; Currie, D; Garte, S J

    1987-04-01

    A new cell line designated NAS 2BL has been established from a rat nasal tumor induced by inhalation of the direct-acting carcinogen methylmethane sulfonate. The cells are epithelial in morphology, have a generation time of 34 h, require 10% fetal bovine serum for optimal growth, and exhibit keratinization at confluence. The karyotype is aneuploid, with several marker chromosomes, and the cells are transformed by the criterion of nude mouse tumorigenicity.

  10. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

    Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

    2014-05-01

    Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

  11. 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

    2016-01-01

    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

  12. 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.

  13. Characterization of a Novel Radiation-Induced Sarcoma Cell Line

    PubMed Central

    Lang, J.E.; Zhu, W.; Nokes, B.T.; Sheth, G.R.; Novak, P.; Fuchs, L.; Watts, G.S.; Futscher, B.W.; Mineyev, N.; Ring, A.; LeBeau, L.; Nagle, R.; Cranmer, L.D.

    2014-01-01

    Background Radiation-induced sarcoma (RIS) is a potential complication of cancer treatment. No widely available cell line models exist to facilitate studies of RIS. Methods We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a RIS. Results Short tandem repeat (STR) profiling of UACC-SARC1 was virtually identical to its parental tumor. Immunohistochemistry (IHC) analysis of the tumor and immunocytochemistry (ICC) analysis of UACC-SARC1 revealed shared expression of vimentin, osteonectin, CD68, Ki67 and PTEN but tumor-restricted expression of the histiocyte markers α1-antitrypsin and α1-antichymotrypsin. Karyotyping of the tumor demonstrated aneuploidy. Comparative genomic hybridization (CGH) provided direct genetic comparison between the tumor and UACC-SARC1. Sequencing of 740 mutation hotspots revealed no mutations in UACC-SARC1 nor in the tumor. NOD/SCID gamma mouse xenografts demonstrated tumor formation and metastasis. Clonogenicity assays demonstrated that 90% of single cells produced viable colonies. NOD/SCID gamma mice produced useful patient-derived xenografts for orthotopic or metastatic models. Conclusion Our novel RIS strain constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. UACC-SARC1 is an aneuploid cell line with complex genomics lacking common oncogenes or tumor suppressor genes as drivers of its biology. The UACC-SARC1 cell line will enable further studies of the drivers of RIS. Synopsis We derived a spontaneously immortalized primary human cell line, UACC-SARC1, from a radiation-induced sarcoma (RIS). Our novel RIS cell line constitutes a useful tool for pre-clinical studies of this rare, aggressive disease. PMID:25644184

  14. Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

    SciTech Connect

    Medina, D.; Oborn, C.J. ); Li, M.L.; Bissell, M.J. )

    1987-09-01

    The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of {beta}-casein mRNA in the presence or absence of prolactin. The inducibility of {beta}-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types.

  15. Human cell lines: A promising alternative for recombinant FIX production.

    PubMed

    de Sousa Bomfim, Aline; Cristina Corrêa de Freitas, Marcela; Picanço-Castro, Virgínia; de Abreu Soares Neto, Mário; Swiech, Kamilla; Tadeu Covas, Dimas; Maria de Sousa Russo, Elisa

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins.

  16. 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.

  17. Type II secretory phospholipase A2 binds to ischemic flip-flopped cardiomyocytes and subsequently induces cell death.

    PubMed

    Nijmeijer, R; Willemsen, M; Meijer, C J L M; Visser, C A; Verheijen, R H; Gottlieb, R A; Hack, C E; Niessen, H W M

    2003-11-01

    Type II secretory phospholipase A2 (sPLA2) is a cardiovascular risk factor. We recently found depositions of sPLA2 in the necrotic center of infarcted human myocardium and normally appearing cardiomyocytes adjacent to the border zone. The consequences of binding of sPLA2 to ischemic cardiomyocytes are not known. To explore a potential effect of sPLA2 on ischemic cardiomyocytes at a cellular level we used an in vitro model. The cardiomyocyte cell line H9c2 or adult cardiomyocytes were isolated from rabbits that were incubated with sPLA2 in the presence of metabolic inhibitors to mimic ischemia-reperfusion conditions. Cell viability was established with the use of annexin V and propidium iodide or 7-aminoactinomycin D. Metabolic inhibition induced an increase of the number of flip-flopped cells, including a population that did not stain with propidium iodide and that was caspase-3 negative. sPLA2 bound to the flip-flopped cells, including those negative for caspase-3. sPLA2 binding induced cell death in these latter cells. In addition, sPLA2 potentiated the binding of C-reactive protein (CRP) to these cells. We conclude that by binding to flip-flopped cardiomyocytes, including those that are caspase-3 negative and presumably reversibly injured, sPLA2 may induce cell death and tag these cells with CRP.

  18. Analysis of LINE-1 expression in human pluripotent cells.

    PubMed

    Muñoz-Lopez, Martin; Garcia-Cañadas, Marta; Macia, Angela; Morell, Santiago; Garcia-Perez, Jose L

    2012-01-01

    Half of the human genome is composed of repeated DNA, and some types are mobile within our genome (transposons and retrotransposons). Despite their abundance, only a small fraction of them are currently active in our genome (Long Interspersed Element-1 (LINE-1), Alu, and SVA elements). LINE-1 or L1 elements are a family of active non-LTR retrotransposons, the ongoing mobilization of which still impacts our genome. As selfish DNA elements, L1 activity is more prominent in early human development, where new insertions would be transmitted to the progeny. Here, we describe the conventional methods aimed to determine the expression level of LINE-1 elements in pluripotent human cells.

  19. Registration of Human Embryonic Stem Cell Lines: Korea, 2010

    PubMed Central

    Lee, Ji-Yoon; Lee, Dae-Yeon; Choi, Young-Sil; Lee, Kyoung-Jae; Kim, Yong-Ou

    2011-01-01

    In an effort to increase the credibility of human embryonic stem cell (hESC) lines established in Korea, obligatory registration was introduced by the Bioethics and Safety Act 2008, effective as of January 1, 2010. The DNA fingerprint, chromosome stability, expression of pluripotency markers, and contamination of mycoplasma of the submitted lines were analyzed by Korea Centers for Disease Control and Prevention (KCDC). The characterization data and ethical aspects, such as informed consent for donation of surplus embryos, were reviewed by a 10-member advisory review committee for stem cell registry. A total of 55 domestic hESC lines were submitted for registration in 2010; among them 51 were registered. Among these submitted lines, 26 were additionally characterized by KCDC, while 25 lines previously characterized by the Ministry of Education, Science and Technology were not additionally analyzed by KCDC. Registration completed an oversight system for embryo research by registering the products of licensed embryo research projects, making embryo research more transparent in Korea. Information about hESC lines is available at the website of the Korea Stem Cell Registry (kscr.nih.go.kr). PMID:24159464

  20. Isolation of dengue virus with a human promonocyte cell line.

    PubMed

    Liu, W T; Chen, C L; Lee, S S; Chan, C C; Lo, F L; Ko, Y C

    1991-05-01

    In October-November, 1988 there was an outbreak of dengue fever in the Kaoshiung area of southern Taiwan. We collected 100 serum samples from 96 patients at the onset of their fever for virus cultures and identification. A human promonocyte cell line (HL-CZ) established in our laboratory was used and proved to be susceptible for dengue virus propagation. Type 1 dengue virus in the HL-CZ cell culture was identified by immunofluorescence tests using monoclonal antibodies, and also by hemagglutination tests with goose red blood cells. The density of the virus particles, as measured by sucrose gradient ultracentrifugation, ranged from 1.186 to 1.224 g/ml. The virus yield from this cell culture is comparable with that from the C6/36 mosquito cell line. There was a significant correlation between the antibody responses tested with Western dot blots and hemagglutination inhibition techniques.

  1. Effect of Predatory Bacteria on Human Cell Lines

    PubMed Central

    Gupta, Shilpi; Tang, Chi; Tran, Michael; Kadouri, Daniel E.

    2016-01-01

    Predatory bacteria are Gram-negative bacteria that prey on other Gram-negative bacteria and have been considered as potential therapeutic agents against multi-drug resistant pathogens. In vivo animal models have demonstrated that predatory bacteria are non-toxic and non-immunogenic in rodents. In order to consider the use of predatory bacteria as live antibiotics, it is important to investigate their effect on human cells. The aim of this study was to determine the effect of Bdellovibrio bacteriovorus strains 109J and HD100, and Micavibrio aeruginosavorus strain ARL-13 on cell viability and inflammatory responses of five human cell lines, representative of clinically relevant tissues. We found that the predators were not cytotoxic to any of the human cell lines tested. Microscopic imaging showed no signs of cell detachment, as compared to predator-free cells. In comparison to an E. coli control, exposure to higher concentrations of the predators did not trigger a significant elevation of pro-inflammatory cytokines in four of the five human cell lines tested. Our work underlines the non-pathogenic attributes of predatory bacteria on human cells and highlights their potential use as live antibiotics against human pathogens. PMID:27579919

  2. Modeling adenovirus latency in human lymphocyte cell lines.

    PubMed

    Zhang, Yange; Huang, Wen; Ornelles, David A; Gooding, Linda R

    2010-09-01

    Species C adenovirus establishes a latent infection in lymphocytes of the tonsils and adenoids. To understand how this lytic virus is maintained in these cells, four human lymphocytic cell lines that support the entire virus life cycle were examined. The T-cell line Jurkat ceased proliferation and died shortly after virus infection. BJAB, Ramos (B cells), and KE37 (T cells) continued to divide at nearly normal rates while replicating the virus genome. Viral genome numbers peaked and then declined in BJAB cells below one genome per cell at 130 to 150 days postinfection. Ramos and KE37 cells maintained the virus genome at over 100 copies per cell over a comparable period of time. BJAB cells maintained the viral DNA as a monomeric episome. All three persistently infected cells lost expression of the cell surface coxsackie and adenovirus receptor (CAR) within 24 h postinfection, and CAR expression remained low for at least 340 days postinfection. CAR loss proceeded via a two-stage process. First, an initial loss of cell surface staining for CAR required virus late gene expression and a CAR-binding fiber protein even while CAR protein and mRNA levels remained high. Second, CAR mRNA disappeared at around 30 days postinfection and remained low even after virus DNA was lost from the cells. At late times postinfection (day 180), BJAB cells could not be reinfected with adenovirus, even when CAR was reintroduced to the cells via retroviral transduction, suggesting that the expression of multiple genes had been stably altered in these cells following infection. PMID:20573817

  3. Derivation of a Homozygous Human Androgenetic Embryonic Stem Cell Line.

    PubMed

    Ding, Chenhui; Huang, Sunxing; Qi, Quan; Fu, Rui; Zhu, Wanwan; Cai, Bing; Hong, Pingping; Liu, Zhengxin; Gu, Tiantian; Zeng, Yanhong; Wang, Jing; Xu, Yanwen; Zhao, Xiaoyang; Zhou, Qi; Zhou, Canquan

    2015-10-01

    Human embryonic stem cells (hESCs) have long been considered as a promising source for cell replacement therapy. However, one major obstacle for the use of these cells is immune compatibility. Histocompatible human parthenogenetic ESCs have been reported as a new method for generating human leukocyte antigen (HLA)-matched hESCs. To further investigate the possibility of obtaining histocompatible stem cells from uniparental embryos, we tried to produce androgenetic haploid human embryos by injecting a single spermatozoon into enucleated human oocyte, and establish human androgenetic embryonic stem (hAGES) cell lines from androgenetic embryos. In the present study, a diploid hAGES cell line has been established, which exhibits typical features of human ESCs, including the expression of pluripotency markers, having differentiation potential in vitro and in vivo, and stable propagation in an undifferentiated state (>P40). Bisulfite sequencing of the H19, Snrpn, Meg3, and Kv imprinting control regions suggested that hAGES cells maintained to a certain extent a sperm methylation pattern. Genome-wide single nucleotide polymorphism, short tandem repeat, and HLA analyses revealed that the hAGES cell genome was highly homozygous. These results suggest that hAGES cells from spermatozoon could serve as a useful tool for studying the mechanisms underlying genomic imprinting in humans. It might also be used as a potential resource for cell replacement therapy as parthenogenetic stem cells.

  4. Oral bioavailability of glyphosate: studies using two intestinal cell lines.

    PubMed

    Vasiluk, Luba; Pinto, Linda J; Moore, Margo M

    2005-01-01

    Glyphosate is a commonly used nonselective herbicide that inhibits plant growth through interference with the production of essential aromatic amino acids. In vivo studies in mammals with radiolabeled glyphosate have shown that 34% of radioactivity was associated with intestinal tissue 2 h after oral administration. The aim of our research was to investigate the transport, binding, and toxicity of glyphosate to the cultured human intestinal epithelial cell line, Caco-2, and the rat small intestinal crypt-derived cell line, ileum epithelial cells-18 (IEC-18). An in vitro analysis of the transport kinetics of [14C]-glyphosate showed that 4 h after exposure, approximately 8% of radiolabeled glyphosate moved through the Caco-2 monolayer in a dose-dependent manner. Binding of glyphosate to cells was saturable and approximately 4 x 10(11) binding sites/cell were estimated from bound [14C]. Exposure of Caco-2 cells to > or =10 mg/ml glyphosate reduced transmembrane electrical resistance (TEER) by 82 to 96% and increased permeability to [3H]-mannitol, indicating that paracellular permeability increased in glyphosate-treated cells. At 10-mg/ml glyphosate, both IEC-18 and Caco-2 cells showed disruption in the actin cytoskeleton. In Caco-2 cells, significant lactate dehydrogenase leakage was observed when cells were exposed to 15 mg/ml of glyphosate. These data indicate that at doses >10 mg/ml, glyphosate significantly disrupts the barrier properties of cultured intestinal cells.

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

    SciTech Connect

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

    1995-12-01

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

  6. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse.

  7. Innervation regulates synaptic ribbons in lateral line mechanosensory hair cells.

    PubMed

    Suli, Arminda; Pujol, Remy; Cunningham, Dale E; Hailey, Dale W; Prendergast, Andrew; Rubel, Edwin W; Raible, David W

    2016-06-01

    Failure to form proper synapses in mechanosensory hair cells, the sensory cells responsible for hearing and balance, leads to deafness and balance disorders. Ribbons are electron-dense structures that tether synaptic vesicles to the presynaptic zone of mechanosensory hair cells where they are juxtaposed with the post-synaptic endings of afferent fibers. They are initially formed throughout the cytoplasm, and, as cells mature, ribbons translocate to the basolateral membrane of hair cells to form functional synapses. We have examined the effect of post-synaptic elements on ribbon formation and maintenance in the zebrafish lateral line system by observing mutants that lack hair cell innervation, wild-type larvae whose nerves have been transected and ribbons in regenerating hair cells. Our results demonstrate that innervation is not required for initial ribbon formation but suggest that it is crucial for regulating the number, size and localization of ribbons in maturing hair cells, and for ribbon maintenance at the mature synapse. PMID:27103160

  8. LINEing germ and embryonic stem cells' silencing of retrotransposons.

    PubMed

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 1410-1428) and Castro-Diaz and colleagues (pp. 1397-1409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals.

  9. 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

  10. Mouse DRG Cell Line with Properties of Nociceptors.

    PubMed

    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

  11. 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

  12. Dengue-2 virus infection of human mononuclear cell lines and establishment of persistent infections.

    PubMed

    Kurane, I; Kontny, U; Janus, J; Ennis, F A

    1990-01-01

    Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K562, Jiyoye and Jurkat, respectively, showed the highest percentage of infected cells of these myelomonocytic, B and T cell lines. Antibody to dengue-2 virus at subneutralizing concentrations augmented dengue-2 virus infection of myelomonocytic cell lines, but not of B cell lines or of T cell lines. Persistent dengue-2 virus infection was established using a myelomonocytic cell line (K562), a B cell line (Raji), and a T cell line (HSB-2). These cell lines maintained a high percentage (more than 70%) of dengue-2 virus antigen-positive cells for at least 25 weeks. Very low titers of infectious dengue-2 virus were detected in the culture supernatant fluids of the persistently infected cells. Dengue-2 virus antigen-positive Raji cell clones were established from persistently-infected Raji cells using limiting dilutions and all of the cells in these clones were dengue-2 virus antigen-positive. These findings demonstrate that a variety of human mononuclear cell lines can be infected with dengue-2 virus and may be useful as models for the analysis of dengue virus-human cell interactions in dengue virus infections.

  13. Cytolytic activity against tumor cells by macrophage cell lines and augmentation by macrophage stimulants.

    PubMed

    Taniyama, T; Holden, H T

    1980-07-15

    Previous studies have shown that macrophage cell lines retained the ability to phagocytize, to secrete lysosomal enzymes, and to function as effector cells in antibody-dependent cellular cytoxicity. In this paper, the cytolytic activity of murine macrophage cell lines against tumor target cells was assessed using an 18-h 51Cr release assay. Of the macrophage cell lines tested, RAW 264, PU5-1.8 and IC-21 had intermediate to high levels of spontaneous cytolytic activity, P388D, and J774 had low to intermediate levels, while /WEHI-3 showed little or no cytolytic activity against RBL-5, MBL-2 and TU-5 target cells. Tumor-cell killing by macrophage cell lines could be augmented by the addition of macrophage stimulants, such as bacterial lipopolysaccharide and poly I:C, indicating that the activation of macrophages by these stimulants does not require the participation of other cell types. Treatment with interferon also augmented the tumor-cell killing by macrophage cell lines. Although the mechanism by which these cell lines exert their spontaneous or boosted cytotoxic activity is not clear, it does not appear to be due to depletion of nutrients since cell lines with high metabolic and proliferative activities, such as WEHI-3 and RBL-5, showed little or no cytotoxicity and supernatants from the macrophage cell lines did not exert any cytotoxic effects in their essay. Thus, it appears that the different macrophage cell lines represent different levels of activation and/or differentiation and may be useful for studying the development of these processes as well as providing a useful tool for analyzing the mechanisms of macrophage-mediated cytolysis. PMID:6165690

  14. Characterization of hepatocellular carcinoma cell lines based on cell adhesion molecules.

    PubMed

    Jung, Cheol Woong; Song, Tae-Jin; Lee, Kun-Ok; Choi, Sae Byeol; Kim, Wan Bae; Suh, Sung Ock; Kim, Young Chul; Choi, Sang Yong

    2012-06-01

    Many studies which focus on the molecules and mechanisms related to the characteristics of the cancer have been performed. In particular, cell adhesion molecules (CAMs) are known to play a central role in the adhesion of cancer cells to vascular endothelial cells. In this study, the expression of CAMs in hepatocellular carcinoma (HCC) cell lines was analyzed and correlated with the characteristics of various HCC cell lines. Eight human HCC cell lines were used in this study. We analyzed the expression of ICAM-1, E-selectin and the integrin subunits of HCC cell lines by western blot analysis and ELISA kit. We estimated the expression of integrin-α5 using western blot analysis and RT-PCR to compare the expression at the gene level with the protein level. In addition, we determined the expression of TGF-β1, as one of the markers for the cellular activity compared to the levels of expression with the expression of integrin-α3 and -α5. ICAM-1 was highly expressed in all of the cell lines except SNU398 and Hep3B, which exhibit a more aggressive nature among the studied HCC cell lines. E-selectin and integrin subunits varied in all HCC cell lines. In particular, integrin-β2 was highly expressed on all HCC cell lines. In conclusion, the levels of expression of the CAMs may not affect cellular activity, morphology or tumorigenicity. However, most HCC cell lines show various expressions of CAMs, suggesting that HCC cell lines expressing the major CAMs remain candidates for molecular targeted therapy, which may need to be patient-tailored for therapy according to the molecular profile.

  15. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

    Ljungkvist, Anna S.E. . E-mail: a.ljungkvist@rther.umcn.nl; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

    2005-07-15

    Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

  16. Cell membrane fatty acid composition differs between normal and malignant cell lines.

    PubMed

    Meng, Xialong; Riordan, Neil H; Riordan, Hugh D; Mikirova, Nina; Jackson, James; González, Michael J; Miranda-Massari, Jorge R; Mora, Edna; Trinidad Castillo, Waleska

    2004-06-01

    Twenty-eight fatty acids (C8:0 to C24:l n-9) were measured by gas chromatography in four normal cell lines (C3H / 10T1 / 2, CCD-18Co, CCD-25SK and CCD-37Lu) and seven cancer cell lines (C-41, Caov-3, LS-180, PC-3, SK-MEL-28, SK-MES-1 and U-87 MG). Results show differences in the content and proportions of fatty acids when comparing cancer cell lines with their normal counterparts. Cancer cell lines showed lower C20: 4 n-6, C24:1 n-9, polyunsaturated fatty acids (PUFA's) and ratios of C20:4 n-6 to C20:5 n-3 and C16:0 to C18:1 n-9 and stearic to oleic (SA/OA) than their normal counterparts. All cancer cell lines had SA/OA ratios lower than 7.0 while normal cell lines had ratios greater than 0.7 (p<0.05). In addition, the ratios of total saturated fatty acids (SFA) to PUFA'S and the concentration of C18:1 n-9, C18:2 n-6, C20:5 n-3 were higher in cancer cell lines as compared to normal cell lines. A positive correlation was detected between C16:0 and longer SFA'S (r = +0.511, p<0.05) in normal cell lines whereas a negative correlation (r=0.608, p<0.05) was obtained for malignant cell lines. Moreover, cancerous cell lines exhibited a particular desaturation defect and an abnormal incorporation of C18:2 n-6 and C20-4 n-6 fatty acids. PMID:15377057

  17. UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Cancer Institute’s Urologic Oncology Branch seeks parties to co-develop the UOK 262 immortalized cell line as research tool to study aggressive hereditary leiomyomatosis and renal cell carcinoma (HLRCC)-associated recurring kidney cancer.

  18. 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

  19. 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...

  20. Cryopreservation of specialized chicken lines using cultured primordial germ cells.

    PubMed

    Nandi, S; Whyte, J; Taylor, L; Sherman, A; Nair, V; Kaiser, P; McGrew, M J

    2016-08-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells ( PGCS: ). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- ( MHC-: ) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  1. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines.

    PubMed

    Contino, Gianmarco; Eldridge, Matthew D; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G; Edwards, Paul A W; Fitzgerald, Rebecca C

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines-ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4-all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  2. Cryopreservation of specialized chicken lines using cultured primordial germ cells

    PubMed Central

    Nandi, S.; Whyte, J.; Taylor, L.; Sherman, A.; Nair, V.; Kaiser, P.; McGrew, M. J.

    2016-01-01

    Biosecurity and sustainability in poultry production requires reliable germplasm conservation. Germplasm conservation in poultry is more challenging in comparison to other livestock species. Embryo cryopreservation is not feasible for egg-laying animals, and chicken semen conservation has variable success for different chicken breeds. A potential solution is the cryopreservation of the committed diploid stem cell precursors to the gametes, the primordial germ cells (PGCs). Primordial germ cells are the lineage-restricted cells found at early embryonic stages in birds and form the sperm and eggs. We demonstrate here, using flocks of partially inbred, lower-fertility, major histocompatibility complex- (MHC-) restricted lines of chicken, that we can easily derive and cryopreserve a sufficient number of independent lines of male and female PGCs that would be sufficient to reconstitute a poultry breed. We demonstrate that germ-line transmission can be attained from these PGCs using a commercial layer line of chickens as a surrogate host. This research is a major step in developing and demonstrating that cryopreserved PGCs could be used for the biobanking of specialized flocks of birds used in research settings. The prospective application of this technology to poultry production will further increase sustainability to meet current and future production needs. PMID:27099306

  3. 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...

  4. 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...

  5. Analysis of tumour cell composition in tumours composed of paired mixtures of mammary tumour cell lines.

    PubMed Central

    Miller, B. E.; Miller, F. R.; Wilburn, D. J.; Heppner, G. H.

    1987-01-01

    In order to quantitate the effects of tumour subpopulation interactions, we have devised a method to determine the subpopulation composition of tumours by using paired tumour cell lines able to grow in different selective media. Line 4T07 forms colonies in thioguanine but not in HAT and line 168 forms colonies in HAT but not in thioguanine. An independent technique of determining tumour cell content was used to validate this method: line 168 and 4T07 cells are distinguishable by flow cytometry after staining with propidium iodide for DNA content. Mixtures of cell suspensions prepared from each unmixed tumour, as well as from tumours arising from mixtures of these lines, were analysed by both the colony formation assay and by the DNA content assay. The colony formation assay yielded values in good agreement with the DNA content assay, but was considerably more sensitive in that it was able to quantitate minority subpopulations that constituted less than 10% of the tumour. Both methods revealed that in tumours arising from mixtures, the tumour cells were almost entirely line 4T07, even when the inoculum had contained a high proportion of 168 cells. Since line 168 cells are very tumorigenic per se, these results suggest that line 4T07 cells are capable of interfering with 168 proliferation in mixed tumours, either directly or through a host-mediated mechanism. PMID:3426919

  6. 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.

  7. Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines

    PubMed Central

    2014-01-01

    Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 μg/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p < 0.01) in cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the

  8. Mitochondrial DNA determines androgen dependence in prostate cancer cell lines

    PubMed Central

    Higuchi, M; Kudo, T; Suzuki, S; Evans, TT; Sasaki, R; Wada, Y; Shirakawa, T; Sawyer, JR; Gotoh, A

    2008-01-01

    Prostate cancer progresses from an androgen-dependent to androgen-independent stage after androgen ablation therapy. Mitochondrial DNA plays a role in cell death and metastatic competence. Further, heteroplasmic large-deletion mitochondrial DNA is verycommon in prostate cancer. To investigate the role of mitochondrial DNA in androgen dependence of prostate cancers, we tested the changes of normal and deleted mitochondrial DNA in accordance with the progression of prostate cancer. We demonstrated that the androgen-independent cell line C4-2, established byinoculation of the androgen-dependent LNCaP cell line into castrated mice, has a greatlyreduced amount of normal mitochondrial DNA and an accumulation of large-deletion DNA. Strikingly, the depletion of mitochondrial DNA from androgen-dependent LNCaP resulted in a loss of androgen dependence. Reconstitution of normal mitochondrial DNA to the mitochondrial DNA-depleted clone restored androgen dependence. These results indicate that mitochondrial DNA determines androgen dependence of prostate cancer cell lines. Further, mitochondrial DNA-deficient cells formed tumors in castrated athymic mice, whereas LNCaP did not. The accumulation of large deletion and depletion of mitochondrial DNA maythus playa role in the development of androgen independence, leading to progression of prostate cancers. PMID:16278679

  9. Cytotoxic effect of Plantago spp. on cancer cell lines.

    PubMed

    Gálvez, Marina; Martín-Cordero, Carmen; López-Lázaro, Miguel; Cortés, Felipe; Ayuso, María Jesús

    2003-10-01

    Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential.

  10. 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.

  11. 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

  12. 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.

  13. Cytotoxic effect of Plantago spp. on cancer cell lines.

    PubMed

    Gálvez, Marina; Martín-Cordero, Carmen; López-Lázaro, Miguel; Cortés, Felipe; Ayuso, María Jesús

    2003-10-01

    Methanolic extracts from seven Plantago species used in traditional medicine for the treatment of cancer, were evaluated for cytotoxic activity against three human cancer cell lines recommended by the National Cancer Institute (NCI, USA). The results showed that Plantago species exhibited cytotoxic activity, showing a certain degree of selectivity against the tested cells in culture. Since the flavonoids are able to strongly inhibit the proliferation of human cancer cell lines, we have identified luteolin-7-O-beta-glucoside as major flavonoid present in most of the Plantago species. Also, we have evaluated this compound and its aglycon, luteolin, for their cytotoxic and DNA topoisomerase I poisons activities. These results could justify the traditional use of the Plantago species and topoisomerase-mediated DNA damage might be a possible mechanism by which flavonoids of Plantago exert their cytotoxicity potential. PMID:12963131

  14. Over-expression of secreted proteins from mammalian cell lines

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

    Secreted mammalian proteins require the development of robust protein over-expression systems for crystallographic and biophysical studies of protein function. Due to complex disulfide bonds and distinct glycosylation patterns preventing folding and expression in prokaryotic expression hosts, many secreted proteins necessitate production in more complex eukaryotic expression systems. Here, we elaborate on the methods used to obtain high yields of purified secreted proteins from transiently or stably transfected mammalian cell lines. Among the issues discussed are the selection of appropriate expression vectors, choice of signal sequences for protein secretion, availability of fusion tags for enhancing protein stability and purification, choice of cell line, and the large-scale growth of cells in a variety of formats. PMID:24510886

  15. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    PubMed

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-01-01

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. PMID:26840224

  16. Ellipticine cytotoxicity to cancer cell lines — a comparative study

    PubMed Central

    Stiborová, Marie; Poljaková, Jitka; Martínková, Eva; Bořek-Dohalská, Lucie; Eckschlager, Tomáš; Kizek, Rene; Frei, Eva

    2011-01-01

    Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of action. This anticancer agent should be considered a pro-drug, whose pharmacological efficiency and/or genotoxic side effects are dependent on its cytochrome P450 (CYP)- and/or peroxidase-mediated activation to species forming covalent DNA adducts. Ellipticine can also act as an inhibitor or inducer of biotransformation enzymes, thereby modulating its own metabolism leading to its genotoxic and pharmacological effects. Here, a comparison of the toxicity of ellipticine to human breast adenocarcinoma MCF-7 cells, leukemia HL-60 and CCRF-CEM cells, neuroblastoma IMR-32, UKF-NB-3 and UKF-NB-4 cells and U87MG glioblastoma cells and mechanisms of its action to these cells were evaluated. Treatment of all cells tested with ellipticine resulted in inhibition of cell growth and proliferation. This effect was associated with formation of two covalent ellipticine-derived DNA adducts, identical to those formed by 13-hydroxy- and 12-hydroxyellipticine, the ellipticine metabolites generated by CYP and peroxidase enzymes, in MCF-7, HL-60, CCRF-CEM, UKF-NB-3, UKF-NB-4 and U87MG cells, but not in neuroblastoma UKF-NB-3 cells. Therefore, DNA adduct formation in most cancer cell lines tested in this comparative study might be the predominant cause of their sensitivity to ellipticine treatment, whereas other mechanisms of ellipticine action also contribute to its cytotoxicity to neuroblastoma UKF-NB-3 cells. PMID:21753906

  17. Highly efficient site-specific transgenesis in cancer cell lines

    PubMed Central

    2012-01-01

    Background Transgenes introduced into cancer cell lines serve as powerful tools for identification of genes involved in cancer. However, the random nature of genomic integration site of a transgene highly influences the fidelity, reliability and level of its expression. In order to alleviate this bottleneck, we characterized the potential utility of a novel PhiC31 integrase-mediated site-specific insertion system (PhiC31-IMSI) for introduction of transgenes into a pre-inserted docking site in the genome of cancer cells. Methods According to this system, a “docking-site” was first randomly inserted into human cancer cell lines and clones with a single copy were selected. Subsequently, an “incoming” vector containing the gene of interest was specifically inserted in the docking-site using PhiC31. Results Using the Pc-3 and SKOV-3 cancer cell lines, we showed that transgene insertion is reproducible and reliable. Furthermore, the selection system ensured that all surviving stable transgenic lines harbored the correct integration site. We demonstrated that the expression levels of reporter genes, such as green fluorescent protein and luciferase, from the same locus were comparable among sister, isogenic clones. Using in vivo xenograft studies, we showed that the genetically altered cancer cell lines retain the properties of the parental line. To achieve temporal control of transgene expression, we coupled our insertion strategy with the doxycycline inducible system and demonstrated tight regulation of the expression of the antiangiogenic molecule sFlt-1-Fc in Pc-3 cells. Furthermore, we introduced the luciferase gene into the insertion cassette allowing for possible live imaging of cancer cells in transplantation assays. We also generated a series of Gateway cloning-compatible intermediate cassettes ready for high-throughput cloning of transgenes and demonstrated that PhiC31-IMSI can be achieved in a high throughput 96-well plate format. Conclusions The novel

  18. Carbon nanoparticles for gene transfection in eukaryotic cell lines.

    PubMed

    Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

    2014-06-01

    For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests.

  19. Survival of different cell lines in alginate-agarose microcapsules.

    PubMed

    Orive, G; Hernández, R M; Gascón, A R; Igartua, M; Pedraz, J L

    2003-01-01

    Cell microencapsulation has emerged as a promising therapeutic strategy to treat a wide range of diseases. The optimisation of this technology depends on several critical issues such as the careful selection of the cell line, the controlled manufacture of microcapsules and the suitable adaptation of the construct design to the selected cell line. In this work, we studied the behavior of hybridoma cells once enclosed in solid and liquefied core alginate-agarose beads. Results show that hybridoma cells presented a better growing pattern and improved their viability and antibody production within liquefied beads. However, when these beads were evaluated with a compression resistance study, they were found to be mechanically more fragile than solid ones. To address this problem, we entrapped non-autologous cells (BHK fibroblast and C2C12 myoblast) in solid alginate-agarose beads and observed that they showed an improved growing profile and prolonged their viability up to 70 days in comparison to the 15 days seen for the hybridoma cells.

  20. Characterization of a transformed rat retinal ganglion cell line.

    PubMed

    Krishnamoorthy, R R; Agarwal, P; Prasanna, G; Vopat, K; Lambert, W; Sheedlo, H J; Pang, I H; Shade, D; Wordinger, R J; Yorio, T; Clark, A F; Agarwal, N

    2001-01-31

    The purpose of the present study was to establish a rat retinal ganglion cell line by transformation of rat retinal cells. For this investigation, retinal cells were isolated from postnatal day 1 (PN1) rats and transformed with the psi2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells. Expression of Thy-1 (a marker for RGC), glial fibrillary acidic protein (GFAP, a positive marker for Muller cells), HPC-1/syntaxin (a marker for amacrine cells), 8A1 (a marker for horizontal and ganglion cells) and neurotrophins was studied using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunocytochemistry. One of the retinal cell clones, designated RGC-5, was positive for Thy-1, Brn-3C, Neuritin, NMDA receptor, GABA-B receptor, and synaptophysin expression and negative for GFAP, HPC-1, and 8A1, suggesting that it represented a putative RGC clone. The results of RT-PCR analysis were confirmed by immunocytochemistry for Thy-1 and GFAP. Upon further characterization by immunoblotting, the RGC-5 clone was positive for Thy-1, negative for GFAP, 8A1 and syntaxin. RGC 5 cells were also positive for the expression of neurotrophins and their cognate receptors. To establish the physiological relevance of RGC-5, the effects of serum/trophic factor deprivation and glutamate toxicity were analyzed to determine if these cells would undergo apoptosis. The protective effects of neurotrophins on RGC-5 after serum deprivation was also investigated. Apoptosis was studied by terminal deoxynucleotidyl transferase-mediated fluoresceinated dUTP nick end labeling (TUNEL). Serum deprivation resulted in apoptosis and supplementation with both BDNF and NT-4 in the growth media, protected the RGC-5 cells from undergoing apoptosis. On differentiation with succinyl concanavalin A (sConA), RGC-5 cells became sensitive to glutamate toxicity, which could be reversed by inclusion of ciplizone (MK801

  1. Bioenergetic analysis of ovarian cancer cell lines: profiling of histological subtypes and identification of a mitochondria-defective cell line.

    PubMed

    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-1α 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

  2. Apoptosis inhibitor of macrophage (AIM) reduces cell number in canine histiocytic sarcoma cell lines

    PubMed Central

    UCHIDA, Mona; SAEKI, Kohei; MAEDA, Shingo; TAMAHARA, Satoshi; YONEZAWA, Tomohiro; MATSUKI, Naoaki

    2016-01-01

    Apoptosis inhibitor of macrophage (AIM) is initially reported to protect macrophages from apoptosis. In this study, we determined the effect of AIM on the macrophage-derived tumor, histiocytic sarcoma cell lines (HS) of dogs. Five HS and five other tumor cell lines were used. When recombinant canine AIM was applied to non-serum culture media, cell numbers of all the HS and two of other tumor cell lines decreased dose-dependently. The DNA fragmentation, TUNEL staining and flow cytometry tests revealed that AIM induced both of apoptosis and cell cycle arrest in the HS. Although AIM is known as an apoptosis inhibitor, these results suggest that a high dose of AIM could have an opposite function in HS and some tumor cell lines. PMID:27246397

  3. Beta-cell gene expression and functional characterisation of the human insulinoma cell line CM.

    PubMed

    Baroni, M G; Cavallo, M G; Mark, M; Monetini, L; Stoehrer, B; Pozzilli, P

    1999-04-01

    Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on beta-cells. In contrast, human insulinoma cell lines are rarely used because of difficulties in obtaining and culturing them for long periods. The aim of our study was to investigate, under different experimental conditions, the capacity of the human insulinoma cell line CM to retain beta-cell function, particularly the expression of constitutive beta-cell genes (insulin, the glucose transporters GLUT1 and GLUT2, glucokinase), intracellular and secreted insulin, beta-cell granules, and cAMP content. Results showed that CM cells from an early-passage express specific beta-cell genes in response to glucose stimulation, in particular the insulin and GLUT genes. Such capacity is lost at later passages when cells are cultured at standard glucose concentrations. However, if cultured at lower glucose concentration (0.8 mM) for a longer time, CM cells re-acquire the capacity to respond to glucose stimulation, as shown by the increased expression of beta-cell genes (insulin, GLUT2, glucokinase). Nonetheless, insulin secretion could not be restored under such experimental conditions despite the presence of intracellular insulin, although cAMP response to a potent activator of adenylate cyclase, forskolin, was present indicating a viable system. In conclusion, these data show that the human insulinoma cell line CM, at both early-passage and late-passage, posseses a functional glucose-signalling pathway and insulin mRNA expression similar to normal beta-cells, representing, therefore, a good model for studies concerning the signalling and expression of beta-cells. Furthermore, we have previously shown that it is also a good model for immunological studies. In this respect it is important to note that the CM cell line is one of the very few existing human beta-cell lines in long-term culture.

  4. Recycling of 5'-nucleotidase in a rat hepatoma cell line.

    PubMed Central

    van den Bosch, R A; du Maine, A P; Geuze, H J; van der Ende, A; Strous, G J

    1988-01-01

    Intracellular movement of cell surface 5'-nucleotidase was studied in H4S cells, a rat hepatoma cell line. Surface labelled cells were incubated for various periods at 37 degrees C and treated with neuraminidase at 0 degrees C. Removal of sialic acid residues from glycoproteins results in a change of their isoelectric points. Analysis with isoelectric focusing was then used to distinguish between cell surface and intracellular 5'-nucleotidase. Incubation of 125I-surface-labelled cells at 37 degrees C resulted in a gradual decrease of labelled 5'-nucleotidase at the plasma membrane until, at 60 to 90 min, a steady state was reached with 52% of the label on the cell surface and 48% intracellular. Pretreatment of the cells with the weak base primaquine had no influence on this distribution while at the same time uptake of iron via the transferrin receptor was inhibited. Using immunoelectron microscopy 5'-nucleotidase was found on the cell surface, in multivesicular endosomes and the Golgi complex. Preincubation of the cells in the presence of cycloheximide caused a reduction of labelling in the Golgi complex, whereas the label in the other compartments was retained. These results lead to the conclusion that 5'-nucleotidase does not recycle through the Golgi complex and that in contrast to the transferrin receptor the recycling of 5'-nucleotidase is not inhibited by primaquine. Images PMID:2850162

  5. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC. PMID:27594985

  6. Whole-genome sequencing of nine esophageal adenocarcinoma cell lines

    PubMed Central

    Contino, Gianmarco; Eldridge, Matthew D.; Secrier, Maria; Bower, Lawrence; Fels Elliott, Rachael; Weaver, Jamie; Lynch, Andy G.; Edwards, Paul A.W.; Fitzgerald, Rebecca C.

    2016-01-01

    Esophageal adenocarcinoma (EAC) is highly mutated and molecularly heterogeneous. The number of cell lines available for study is limited and their genome has been only partially characterized. The availability of an accurate annotation of their mutational landscape is crucial for accurate experimental design and correct interpretation of genotype-phenotype findings. We performed high coverage, paired end whole genome sequencing on eight EAC cell lines—ESO26, ESO51, FLO-1, JH-EsoAd1, OACM5.1 C, OACP4 C, OE33, SK-GT-4—all verified against original patient material, and one esophageal high grade dysplasia cell line, CP-D. We have made available the aligned sequence data and report single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number alterations, identified by comparison with the human reference genome and known single nucleotide polymorphisms (SNPs). We compare these putative mutations to mutations found in primary tissue EAC samples, to inform the use of these cell lines as a model of EAC.

  7. Fibronectin synthesized by a human hepatoma cell line

    SciTech Connect

    Glasgow, J.E.; Colman, R.W.

    1984-07-01

    Fibronectin is a family of immunologically similar glycoproteins which mediate a variety of cell-cell and cell-substratum interactions. It is a constituent of the extracellular matrix of connective tissue and circulates in plasma. When suspension and adherent cultures of a human hepatoma cell line (SK-HEP-1) were incubated in serum-free medium, the resulting conditioned medium contained material which was specifically immunoprecipitated by antisera to human plasma fibronectin. By double immunodiffusion, a component in the conditioned culture medium was shown to form a line of identity with fibronectin in human plasma and to migrate as an alpha 2- to beta-globulin during immunoelectrophoresis. Human fibronectin was quantified in conditioned medium by electroimmunodiffusion, and was found to increase for at least three days at about 0.1 micrograms/10(6) cells/day. Adherent cultures of SK-HEP-1 cells were incubated with L-(/sup 35/S)methionine to label newly synthesized proteins. Labeled fibronectin in conditioned medium or in cell extracts comigrated with fibronectin in human plasma as shown by autoradiography following crossed-immunoelectrophoresis. Fibronectin was demonstrated in the extra-cellular matrix of adherent SK-HEP-1 cultures by immunofluorescence. It was shown previously that SK-HEP-1 cells synthesize alpha 1-protease inhibitor, one of the products of normal hepatocytes. The finding that these hepatoma cells also synthesize fibronectin supports the concept that the hepatocyte may be one source of circulating fibronectin, a possibility consistent with the established role of this cell type in blood plasma protein synthesis.

  8. Cysteine modified polyaniline films improve biocompatibility for two cell lines.

    PubMed

    Yslas, Edith I; Cavallo, Pablo; Acevedo, Diego F; Barbero, César A; Rivarola, Viviana A

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering.

  9. Design of a stable cell line producing a recombinant monoclonal anti-TNFα antibody based on a CHO cell line.

    PubMed

    Voronina, E V; Seregin, Y A; Litvinova, N A; Shvets, V I; Shukurov, R R

    2016-01-01

    Recombinant monoclonal antibodies (mAbs) against tumor necrosis factor alpha are widely used in the biopharmaceutical therapy of autoimmune diseases. Currently, a large number of drugs based on these antibodies are available. Accordingly, the development of these products for the Russian market is an important goal. The aim of the current study is to describe the development of one such technology. CHO-DG44-derived cell lines producing mAb were developed using two strategies, one based on individual clones and the other based on cell pools. To obtain recombinant cell lines with highly amplified genes of interest, the clones underwent dihydrofolate reductase-mediated gene amplification. Using the best strategy for the selection and amplification of mAb-producing clones, we achieved the production of more than 1 g/L in small scale, non-optimized conditions. PMID:27652157

  10. Bovine viral diarrhea virus (BVDV) in cell lines used for somatic cell cloning.

    PubMed

    Stringfellow, David A; Riddell, Kay P; Givens, M Daniel; Galik, Patricia K; Sullivan, Eddie; Dykstra, Christine C; Robl, James; Kasinathan, Poothapillai

    2005-03-01

    Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus.

  11. Interaction of a mouse macrophage cell line with homologous erythrocytes.

    PubMed

    Singer, J A; Walker, W S; Morrison, M

    1982-06-01

    The interaction of the IC-21 murine macrophage cell line and homologous red blood cells (RBC) was assessed in the absence of exogenous opsonins. These results were used to evaluate this system as a potential model for macrophage-mediated clearance of old or damaged RBC. The binding and ingestion of density-separated and unseparated RBC by IC-21 cells were quantitated in assays that involved both 51Cr-labeled RBC and direct microscopy. The number of unseparated RBC that bound to IC-21 macrophages depended on the number of RBC added. Macrophages phagocytized an appreciable proportion of RBC within 3 hours with the ratio of RBC:macrophage of 10, a point at which the RBC-binding was not rate limiting. The mouse RBC were separated into dense- and less-dense fractions which are presumably enriched for old and young cells, respectively. When these RBC fractions were incubated with the IC-21 macrophage, significantly more of these dense cells were phagocytized. These results show that IC-21 macrophage cell line is a useful model for defining the processes whereby aged or damaged RBC are recognized and removed from circulation by macrophages. PMID:7120230

  12. 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

  13. Bisphosphonates induce apoptosis in human breast cancer cell lines

    PubMed Central

    Senaratne, S G; Pirianov, G; Mansi, J L; Arnett, T R; Colston, K W

    2000-01-01

    Breast cancer has a prodigious capacity to metastasize to bone. In women with advanced breast cancer and bone metastases, bisphosphonates reduce the incidence of hypercalcaemia and skeletal morbidity. Recent clinical findings suggest that some bisphosphonates reduce the tumour burden in bone with a consequent increase in survival, raising the possibility that bisphosphonates may have a direct effect on breast cancer cells. We have investigated the in vitro effects of bisphosphonates zoledronate, pamidronate, clodronate and EB 1053 on growth, viability and induction of apoptosis in three human breast cancer cell lines (MDA-MB-231, Hs 578T and MCF-7). Cell growth was monitored by crystal violet dye assay, and cell viability was quantitated by MTS dye reduction. Induction of apoptosis was determined by identification of morphological features of apoptosis using time-lapse videomicroscopy, identifying morphological changes in nucleis using Hoechst staining, quantitation of DNA fragmentation, level of expression of bcl-2 and bax proteins and identification of the proteolytic cleavage of Poly (ADP)-ribose polymerase (PARP). All four bisphosphonates significantly reduced cell viability in all three cell lines. Zoledronate was the most potent bisphosphonate with IC50values of 15, 20 and 3 μM respectively in MDA-MB-231, MCF-7 and Hs 578T cells. Corresponding values for pamidronate were 40, 35 and 25 μM, whereas clodronate and EB 1053 were more than two orders of magnitude less potent. An increase in the proportion of cells having morphological features characteristic of apoptosis, characteristic apoptotic changes in the nucleus, time-dependent increase in the percentage of fragmented chromosomal DNA, down-regulation in bcl-2 protein and proteolytic cleavage of PARP, all indicate that bisphosphonates have direct anti-tumour effects on human breast cancer cells. © 2000 Cancer Research Campaign PMID:10780527

  14. Identification and Characterization of CD133pos Subpopulation Cells From a Human Laryngeal Cancer Cell Line

    PubMed Central

    Qiu, Hai-ou; Wang, Huifang; Che, Na; Li, Dong; Mao, Yong; Zeng, Qiao; Ge, Rongming

    2016-01-01

    Background Recent research indicates that CD133 are expressed in several kinds of stem cells, among which, its high expression in laryngeal carcinoma has caused wide concern. To further explore efficaciously targeting drugs to laryngeal carcinoma stem cells (CSCs), we transplanted a solid tumor from CSCs into abdominal subcutaneous tissue of nude mice, and then compared the biological characteristics of laryngeal solid tumors with or without cisplatin intervention. Material/Methods In this study, the expression of CD133 was detected in the Hep-2 cell line by flow cytometry. By applying magnetic cell sorting (MACS) technology, we reported the results of purifying CD133-positive cells from a Hep-2 cell line. Cell proliferation, colony formation, and tumor-forming ability were examined in vitro and in vivo to identify the marker of CSCs in Hep-2 cell line. Results Upon flow cytometry analysis, CD133 was expressed constantly on 40.12±1.32% in Hep-2 cell line. Cell proliferation and colony formation ability were higher in CD133-positive cells compared to CD133-negative cells, and the in vivo tumorigenesis experiment showed the same results as in vitro assay. The 2 subpopulations cells were both sensitive to DDP, among which, the effect of DPP on proliferation ability and tumor-forming ability of CD133-positive cells was obviously greater than that of CD133-negative cells. Conclusions Above all, our study revealed that CD133-positive cells have properties of higher proliferation, colony formation, and tumorigenesis in Hep-2 cell line, indicating that CD133 could be a marker to characterize laryngeal cancer stem cells. PMID:27049928

  15. 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.

  16. Regulation of alkaline phosphatase expression in human choriocarcinoma cell lines.

    PubMed Central

    Hamilton, T A; Tin, A W; Sussman, H H

    1979-01-01

    The coincident expression of two structurally distinct isoenzymes of human alkaline phosphatase was demonstrated in two independently derived gestational choriocarcinoma cell lines. These proteins were shown to have enzymatic, antigenic, and physical-chemical properties resembling those of isoenzymes from term placenta and adult liver. The regulation of these isoenzymes has been studied during the exposure of both cell lines to 5-bromodeoxyuridine and dibutyryl cyclic AMP. The responses of the alkaline phosphatase isoenzymes to these agents have also been compared with the response of another protein phenotypic to placenta, the alpha subunit of chorionic gonadotropin. The results show that (i) the separate structural genes coding for placental and liver alkaline phosphatases are regulated in a noncoordinate fashion; (ii) both alkaline phosphatase genes respond independently of the alpha subunit; and (iii) the induction of the placental type isoenzyme occurs via at least two independent pathways. Images PMID:218197

  17. 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 β-cell transcription factors and pancreatic hormones. Exposure of Melligen and Huh7ins cells to proinflammatory cytokines (TNF-α, IL-1β, and IFN-γ) 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 β-cell cytotoxins was associated with the expression of NF-κB and antiapoptotic genes (such as BIRC3). This study describes the successful generation of an artificial β-cell line, which, if encapsulated to avoid allograft rejection, may offer a clinically applicable cure for T1D. PMID:26029722

  18. Restoration of WNT4 inhibits cell growth in leukemia-derived cell lines

    PubMed Central

    2013-01-01

    Background WNT signaling pathways are significantly altered during cancer development. Vertebrates possess two classes of WNT signaling pathways: the “canonical” WNT/β-catenin signaling pathway, and the “non-canonical” pathways including WNT/Ca2+ and WNT/Planar cell polarity [PCP] signaling. WNT4 influences hematopoietic progenitor cell expansion and survival; however, WNT4 function in cancer development and the resulting implications for oncogenesis are poorly understood. The aim of this study was twofold: first, to determine the expression of WNT4 in mature peripheral blood cells and diverse leukemia-derived cells including cell lines from hematopoietic neoplasms and cells from patients with leukemia; second, to identify the effect of this ligand on the proliferation and apoptosis of the blast-derived cell lines BJAB, Jurkat, CEM, K562, and HL60. Methods We determined WNT4 expression by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) and T- and B-lymphocytes from healthy individuals, as well as from five leukemia-derived cell lines and blasts derived from patients with leukemia. To analyze the effect of WNT4 on cell proliferation, PBMCs and cell lines were exposed to a commercially available WNT4 recombinant human protein. Furthermore, WNT4 expression was restored in BJAB cells using an inducible lentiviral expression system. Cell viability and proliferation were measured by the addition of WST-1 to cell cultures and counting cells; in addition, the progression of the cell cycle and the amount of apoptosis were analyzed in the absence or presence of WNT4. Finally, the expression of WNT-pathway target genes was measured by qRT-PCR. Results WNT4 expression was severely reduced in leukemia-derived cell lines and blasts derived from patients with leukemia. The exposure of cell lines to WNT4 recombinant protein significantly inhibited cell proliferation; inducing WNT4 expression in BJAB

  19. Effect of human procathepsin D on proliferation of human cell lines.

    PubMed

    Vĕtvicka, V; Vĕktvicková, J; Fusek, M

    1994-05-16

    We have used human procathepsin D isolated from supernatant of human breast cancer cell line ZR-75-1 to test its mitogenic activity for a broad spectrum of human-derived cell lines. These cell lines included: breast cancer cell lines ZR-75-1, MDA-MB-436, MBA-MD-483 and MDA-MB-231, B lymphoblastoid cell line Raji, the monocytoid cell line U937, T lymphoblastoid cell line 8402, epitheloid carcinoma cell line HELA, hepatocellular carcinoma cell line Hep G2, breast milk epithelial cell line HBL-100 and angiosarcoma cell line HAEND-1. We have tested the level of proliferation of these cell lines depending on the presence of procathepsin D in the medium. In parallel we have also measured the effect of insulin-like growth factor II under the same experimental conditions. We have found a significant difference between the influence of IGF II and that of procathepsin D. While IGF II promoted in practically the same way the proliferation of all cell lines tested, procathepsin D had a very pronounced effect on breast cancer cell lines only. This finding might help to explain some contradictory results of prognostic significance of procathepsin D in human breast cancer.

  20. Perhexiline maleate toxicity on human liver cell lines.

    PubMed Central

    Le Gall, J Y; Guillouzo, A; Glaise, D; Deugnier, Y; Messner, M; Bourel, M

    1980-01-01

    When added to the culture medium of human liver cell lines, perhexiline maleate induced formation of numerous myeloid bodies containing unicentric or multicentric smooth membranes within a few days. The nine lysosomal enzyme activities studied, except for beta-galactosidase which decreased, remained unchanged. These results indicate that on cultured human liver cells perhexiline maleate has an effect similar to that described on hepatocytes of some patients treated with this drug and suggest that myeloid body formation is not due to impairment of lysosomal enzyme activities. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 PMID:7192674

  1. 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 μm. Their morphology was similar to naïve 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.

  2. 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.

  3. MicroRNA profiles in various hepatocellular carcinoma cell lines

    PubMed Central

    Morishita, Asahiro; Iwama, Hisakazu; Fujihara, Shintaro; Sakamoto, Teppei; Fujita, Koji; Tani, Joji; Miyoshi, Hisaaki; Yoneyama, Hirohito; Himoto, Takashi; Masaki, Tsutomu

    2016-01-01

    Hepatocellular carcinoma (HCC) is one of the most common causes of cancer-associated mortality worldwide. Although surgery is considered the most effective treatment for patients with HCC, its indication is restricted by limited criteria and a high relapse rate following surgery; therefore, systemic chemotherapy is required for patients with advanced-stage HCC to prolong their survival. MicroRNAs (miRNAs) are endogenous non-coding RNAs of 18–22 nucleotides in length. It has been reported that aberrant expression of miRNAs is a feature shared by various types of human cancer. Previous studies have indicated that the modulation of non-coding RNAs, particularly miRNAs, may be a valuable therapeutic target for HCC. The aim of the present study was to elucidate the miRNA profiles associated with differentiation and hepatitis B virus (HBV) infection observed in HCC cell lines. The human Alex, Hep3B, HepG2, HuH1, HuH7, JHH1, JHH2, JHH5, JHH6, HLE, HLF and Li-7 HCC cell lines were used for an miRNA array. Replicate data were analyzed following their classification into: i) Poorly- and well-differentiated human HCC cells and ii) HBV-positive and -negative human HCC cells. Out of the 1,719 miRNAs, 4 were found to be significantly upregulated and 52 significantly downregulated in the poorly-differentiated cells, as compared with the well-differentiated cells. Conversely, in the HBV-positive cells 125 miRNAs were found to be upregulated and 2 downregulated, as compared with the HBV-negative cells. Unsupervised hierarchical clustering analysis with Pearson's correlation revealed that the miRNA expression levels were clustered both together and separately in each group. In conclusion, miRNA profile characterization based on various parameters may be a novel approach to determine the etiology of HCC. PMID:27588118

  4. Responding to hypoxia: lessons from a model cell line.

    PubMed

    Seta, K A; Spicer, Z; Yuan, Y; Lu, G; Millhorn, D E

    2002-08-20

    Mammalian cells require a constant supply of oxygen to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. It is, therefore, not surprising that sophisticated mechanisms have evolved that allow cells to adapt to hypoxia. "Oxygen-sensing" is a special phenotype that functions to detect changes in oxygen tension and to transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in various organisms. Oxygen-sensing cells can be segregated into two distinct cell types: those that functionally depolarize (excitable) and those that do not functionally depolarize (nonexcitable) in response to reduced oxygen. Theoretically, excitable cells have all the same signaling capabilities as the nonexcitable cells, but the nonexcitable cells cannot have all the signaling capabilities as excitable cells. A number of signaling pathways have been identified that regulate gene expression during hypoxia. These include the Ca2+-calmodulin pathway, the 3'-5' adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway, the p42 and p44 mitogen-activated protein kinase [(MAPK); also known as the extracellular signal-related kinase (ERK) for ERK1 and ERK2] pathway, the stress-activated protein kinase (SAPK; also known as p38 kinase) pathway, and the phosphatidylinositol 3-kinase (PI3K)-Akt pathway. In this review, we describe hypoxia-induced signaling in the model O2-sensing rat pheochromocytoma (PC12) cell line, the current level of understanding of the major signaling events that are activated by reduced O2, and how these signaling events lead to altered gene expression in both excitable and nonexcitable oxygen-sensing cells. PMID:12189251

  5. Boldine: a potential new antiproliferative drug against glioma cell lines.

    PubMed

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cañedo, Andrés; Pelegrini, Alessandra Luiza; Henriques, Amélia T; Lenz, Guido; Salbego, Christianne

    2009-12-01

    Malignant gliomas are the most common and devastating primary tumors of the central nervous system. Currently no efficient treatment is available. This study evaluated the effect and underlying mechanisms of boldine, an aporphine alkaloid of Peumus boldus, on glioma proliferation and cell death. Boldine decreased the cell number of U138-MG, U87-MG and C6 glioma lines at concentrations of 80, 250 and 500 muM. We observed that cell death caused by boldine was cell-type specific and dose-dependent. Exposure to boldine for 24 h did not activate key mediators of apoptosis. However, it induced alterations in the cell cycle suggesting a G(2)/M arrest in U138-MG cells. Boldine had no toxic effect on non-tumor cells when used at the same concentrations as those used on tumor cells. Based on these results, we speculate that boldine may be a promising compound for evaluation as an anti-cancer agent. PMID:19050827

  6. Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

    PubMed

    Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

    2012-10-01

    Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches. PMID:22511037

  7. Establishment and characterization of feeder-cell-dependent bovine fetal liver cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The establishment and initial characterization of bovine fetal liver cell lines is described. Bovine fetal hepatocytes were cultured from the liver of a 34-day bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO feeder layers and wer...

  8. Rapid micropatterning of cell lines and human pluripotent stem cells on elastomeric membranes.

    PubMed

    Paik, Isha; Scurr, David J; Morris, Bryan; Hall, Graham; Denning, Chris; Alexander, Morgan R; Shakesheff, Kevin M; Dixon, James E

    2012-10-01

    Tissue function during development and in regenerative medicine completely relies on correct cell organization and patterning at micro and macro scales. We describe a rapid method for patterning mammalian cells including human embryonic stem cells (HESCs) and induced pluripotent stem cells (iPSCs) on elastomeric membranes such that micron-scale control of cell position can be achieved over centimeter-length scales. Our method employs surface engineering of hydrophobic polydimethylsiloxane (PDMS) membranes by plasma polymerization of allylamine. Deposition of plasma polymerized allylamine (ppAAm) using our methods may be spatially restricted using a micro-stencil leaving faithful hydrophilic ppAAm patterns. We employed airbrushing to create aerosols which deposit extracellular matrix (ECM) proteins (such as fibronectin and Matrigel™) onto the same patterned ppAAm rich regions. Cell patterns were created with a variety of well characterized cell lines (e.g., NIH-3T3, C2C12, HL1, BJ6, HESC line HUES7, and HiPSC line IPS2). Individual and multiple cell line patterning were also achieved. Patterning remains faithful for several days and cells are viable and proliferate. To demonstrate the utility of our technique we have patterned cells in a variety of configurations. The ability to rapidly pattern cells at high resolution over macro scales should aid future tissue engineering efforts for regenerative medicine applications and in creating in vitro stem cell niches.

  9. Preparation of cell lines for single-cell analysis of transcriptional activation dynamics.

    PubMed

    Rafalska-Metcalf, Ilona U; Janicki, Susan M

    2013-01-01

    Imaging molecularly defined regions of chromatin in single living cells during transcriptional activation has the potential to provide new insight into gene regulatory mechanisms. Here, we describe a method for isolating cell lines with multi-copy arrays of reporter transgenes, which can be used for real-time high-resolution imaging of transcriptional activation dynamics in single cells.

  10. Primed Pluripotent Cell Lines Derived from Various Embryonic Origins and Somatic Cells in Pig

    PubMed Central

    Park, Jin-Kyu; Kim, Hye-Sun; Uh, Kyung-Jun; Choi, Kwang-Hwan; Kim, Hyeong-Min; Lee, Taeheon; Yang, Byung-Chul; Kim, Hyun-Jong; Ka, Hak-Hyun; Kim, Heebal; Lee, Chang-Kyu

    2013-01-01

    Since pluripotent embryonic stem cell (ESC) lines were first derived from the mouse, tremendous efforts have been made to establish ESC lines in several domestic species including the pig; however, authentic porcine ESCs have not yet been established. It has proven difficult to maintain an ESC-like state in pluripotent porcine cell lines due to the frequent occurrence of spontaneous differentiation into an epiblast stem cell (EpiSC)-like state during culture. We have been able to derive EpiSC-like porcine ESC (pESC) lines from blastocyst stage porcine embryos of various origins, including in vitro fertilized (IVF), in vivo derived, IVF aggregated, and parthenogenetic embryos. In addition, we have generated induced pluripotent stem cells (piPSCs) via plasmid transfection of reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) into porcine fibroblast cells. In this study, we analyzed characteristics such as marker expression, pluripotency and the X chromosome inactivation status in female of our EpiSC-like pESC lines along with our piPSC line. Our results show that these cell lines demonstrate the expression of genes associated with the Activin/Nodal and FGF2 pathways along with the expression of pluripotent markers Oct4, Sox2, Nanog, SSEA4, TRA 1–60 and TRA 1–81. Furthermore all of these cell lines showed in vitro differentiation potential, the X chromosome inactivation in female and a normal karyotype. Here we suggest that the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines. PMID:23326334

  11. Human Fucci Pancreatic Beta Cell Lines: New Tools to Study Beta Cell Cycle and Terminal Differentiation

    PubMed Central

    Carlier, Géraldine; Maugein, Alicia; Cordier, Corinne; Pechberty, Séverine; Garfa-Traoré, Meriem; Martin, Patrick; Scharfmann, Raphaël; Albagli, Olivier

    2014-01-01

    Regulation of cell cycle in beta cells is poorly understood, especially in humans. We exploited here the recently described human pancreatic beta cell line EndoC-βH2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-βH2 cells that stably harbor the 2 genes encoding the Fucci fluorescent indicators of cell cycle, either from two vectors, or from a unique bicistronic vector. In proliferating non-synchronized cells, the 2 Fucci indicators revealed cells in the expected phases of cell cycle, with orange and green cells being in G1 and S/G2/M cells, respectively, and allowed the sorting of cells in different substeps of G1. The Fucci indicators also faithfully red out alterations in human beta cell proliferative activity since a mitogen-rich medium decreased the proportion of orange cells and inflated the green population, while reciprocal changes were observed when cells were induced to cease proliferation and increased expression of some beta cell genes. In the last situation, acquisition of a more differentiated beta cell phenotype correlates with an increased intensity in orange fluorescence. Hence Fucci beta cell lines provide new tools to address important questions regarding human beta cell cycle and differentiation. PMID:25259951

  12. Cytotoxic Activity of New Acetoxycoumarin Derivatives in Cancer Cell Lines

    PubMed Central

    Musa, Musiliyu A.; Badisa, Veera L. D.; Latinwo, Lekan M.; Cooperwood, John; Sinclair, Andre; Abdullah, Ahkinyala

    2012-01-01

    Background Coumarin and their derivatives are important and useful compounds with diverse pharmacological properties. In the present study, we evaluated the in vitro cytotoxic activity of new acetoxycoumarin derivatives: 4-(7-methoxy-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (1), 4-(1-methyl-3-oxo-3H-benzo[f]chromen-2-yl)phenyl acetate (2), 4-(6-propionamido-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (3), 4-(7-acetoxy-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (4), 4-(2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (5), 4-(6-bromo-2-oxo-4-phenyl-2H-chromen-3-yl)phenyl acetate (6), 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7), 4-(6,8-dibromo-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (8) against A549 human lung cancer, CRL 1548 rat liver cancer and CRL 1439 normal rat liver cells. Materials and Methods The cytotoxic activity was evaluated by crystal violet dye-binding assay. The effect of compounds 5 and 7 on different phases of the cell cycle was determined using flow cytometry. Results In the A549 lung cancer cell line, the 50% lethal dose (LD50) values for compounds 1–4, 6 and 8 were found to be >100 μM while those for 5 and 7 were 89.3 and 48.1 μM, respectively after 48 h treatment. In the CRL 1548 liver cancer cell line, only compound 7 showed toxicity, with an LD50 of 45.1 μM. Compounds 5 and 7 caused different cell phase arrest in lung and liver cancer cell lines. Conclusion The results indicate that 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (7) had the highest cytotoxic activity in all of the examined cell lines. PMID:21737617

  13. Establishment of a novel human medulloblastoma cell line characterized by highly aggressive stem-like cells.

    PubMed

    Silva, Patrícia Benites Gonçalves da; Rodini, Carolina Oliveira; Kaid, Carolini; Nakahata, Adriana Miti; Pereira, Márcia Cristina Leite; Matushita, Hamilton; Costa, Silvia Souza da; Okamoto, Oswaldo Keith

    2016-08-01

    Medulloblastoma is a highly aggressive brain tumor and one of the leading causes of morbidity and mortality related to childhood cancer. These tumors display differential ability to metastasize and respond to treatment, which reflects their high degree of heterogeneity at the genetic and molecular levels. Such heterogeneity of medulloblastoma brings an additional challenge to the understanding of its physiopathology and impacts the development of new therapeutic strategies. This translational effort has been the focus of most pre-clinical studies which invariably employ experimental models using human tumor cell lines. Nonetheless, compared to other cancers, relatively few cell lines of human medulloblastoma are available in central repositories, partly due to the rarity of these tumors and to the intrinsic difficulties in establishing continuous cell lines from pediatric brain tumors. Here, we report the establishment of a new human medulloblastoma cell line which, in comparison with the commonly used and well-established cell line Daoy, is characterized by enhanced proliferation and invasion capabilities, stem cell properties, increased chemoresistance, tumorigenicity in an orthotopic metastatic model, replication of original medulloblastoma behavior in vivo, strong chromosome structural instability and deregulation of genes involved in neural development. These features are advantageous for designing biologically relevant experimental models in clinically oriented studies, making this novel cell line, named USP-13-Med, instrumental for the study of medulloblastoma biology and treatment.

  14. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers

    PubMed Central

    Qiu, Zhixin; Zou, Keke; Zhuang, Liping; Qin, Jianjie; Li, Hong; Li, Chao; Zhang, Zhengtao; Chen, Xiaotao; Cen, Jin; Meng, Zhiqiang; Zhang, Haibin; Li, Yixue; Hui, Lijian

    2016-01-01

    Hepatocellular carcinoma (HCC) cell lines are useful in vitro models for the study of primary HCCs. Because cell lines acquire additional mutations in culture, it is important to understand to what extent HCC cell lines retain the genetic landscapes of primary HCCs. Most HCC cell lines were established during the last century, precluding comparison between cell lines and primary cancers. In this study, 9 Chinese HCC cell lines with matched patient-derived cells at low passages (PDCs) were established in the defined culture condition. Whole genome analyses of 4 HCC cell lines showed that genomic mutation landscapes, including mutations, copy number alterations (CNAs) and HBV integrations, were highly stable during cell line establishment. Importantly, genetic alterations in cancer drivers and druggable genes were reserved in cell lines. HCC cell lines also retained gene expression patterns of primary HCCs during in vitro culture. Finally, sequential analysis of HCC cell lines and PDCs at different passages revealed their comparable and stable genomic and transcriptomic levels if maintained within proper passages. These results show that HCC cell lines largely retain the genomic and transcriptomic landscapes of primary HCCs, thus laying the rationale for testing HCC cell lines as preclinical models in precision medicine. PMID:27273737

  15. Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers.

    PubMed

    Qiu, Zhixin; Zou, Keke; Zhuang, Liping; Qin, Jianjie; Li, Hong; Li, Chao; Zhang, Zhengtao; Chen, Xiaotao; Cen, Jin; Meng, Zhiqiang; Zhang, Haibin; Li, Yixue; Hui, Lijian

    2016-06-07

    Hepatocellular carcinoma (HCC) cell lines are useful in vitro models for the study of primary HCCs. Because cell lines acquire additional mutations in culture, it is important to understand to what extent HCC cell lines retain the genetic landscapes of primary HCCs. Most HCC cell lines were established during the last century, precluding comparison between cell lines and primary cancers. In this study, 9 Chinese HCC cell lines with matched patient-derived cells at low passages (PDCs) were established in the defined culture condition. Whole genome analyses of 4 HCC cell lines showed that genomic mutation landscapes, including mutations, copy number alterations (CNAs) and HBV integrations, were highly stable during cell line establishment. Importantly, genetic alterations in cancer drivers and druggable genes were reserved in cell lines. HCC cell lines also retained gene expression patterns of primary HCCs during in vitro culture. Finally, sequential analysis of HCC cell lines and PDCs at different passages revealed their comparable and stable genomic and transcriptomic levels if maintained within proper passages. These results show that HCC cell lines largely retain the genomic and transcriptomic landscapes of primary HCCs, thus laying the rationale for testing HCC cell lines as preclinical models in precision medicine.

  16. KLN205--a murine lung carcinoma cell line.

    PubMed

    Kaneko, T; LePage, G A; Shnitka, T K

    1980-10-01

    KLN205 cells, a cloned cell line established from the Nettesheim lung carcinoma, grow in various synthetic media such as MEM, Fisher's or Roswell Park Memorial Institute Medium (RPMI) with the addition of 5 to 20% fetal bovine serum (FBS), calf-serum (CS) or horse serum (HS). They grow optimally in minimum Eagle's medium plus nonessential amino acids (NEAA) plus 5 to 10% FBS or HS. The cells are transplantable to DBA/2, BDF1, AKD2F1, and BALB/c, but not to C3H/He or ICR mice. The growth curves, plating efficiency, ultrastructural characteristics, modal number of chromosomes and transplantability to mice of various strains are almost the same for early and late passage of cells passaged in vitro. These parameters for 16th and 36th passages were: doubling time, 31 and 33 hr; plating efficiency, 12.4 +/- 1.2 and 14.6 +/- 2.6%; modal number of chromosomes, 73 and 76; lung colony formation in DBA/2, 50 and 45.9/mouse; and subcutaneous tumor diameter 24.5 and 27.4 mm, respectively. Only the numbers of lung colonies formed in BDF1 mice were different: 24.4/mouse with 16th passage cells, and 10.2/mouse with 36th passage cells. The results suggest that KLN205 is a relatively stable cultured cell line through 36 passages. As was expected, immunosuppression by higher concentrations of triaminolone acetonide (TA) enhanced lung colony formation in BDF1 mice. On the other hand, a low concentration of TA inhibited lung colony formation in DBA/2 mice, which was unexpected. These results suggest that KLN205 offers a model for investigations on metastases to lungs as well as chemotherapy for lung carcinoma.

  17. Single-walled carbon nanohorn (SWNH) aggregates inhibited proliferation of human liver cell lines and promoted apoptosis, especially for hepatoma cell lines

    PubMed Central

    Zhang, Jinqian; Sun, Qiang; Bo, Jian; Huang, Rui; Zhang, Mengran; Xia, Zhenglin; Ju, Lili; Xiang, Guoan

    2014-01-01

    Single-walled carbon nanohorns (SWNHs) may be useful as carriers for anticancer drugs due to their particular structure. However, the interactions between the material itself and cancerous or normal cells have seldom been studied. To address this problem, the effects of raw SWNH material on the biological functions of human liver cell lines were studied. Our results showed that unmodified SWNHs inhibited mitotic entry, growth, and proliferation of human liver cell lines and promoted their apoptosis, especially in hepatoma cell lines. Individual spherical SWNH particles were found inside the nuclei of human hepatoma HepG2 cells and the lysosomes of normal human liver L02 cells, implying that SWNH particles could penetrate into human liver cells_and the different interacted mechanisms on human normal cell lines compared to hepatoma cell lines. Further research on the mechanisms and application in treatment of hepatocellular carcinoma with SWNHs is needed. PMID:24523586

  18. Astaxanthin Inhibits Proliferation of Human Gastric Cancer Cell Lines by Interrupting Cell Cycle Progression

    PubMed Central

    Kim, Jung Ha; Park, Jong-Jae; Lee, Beom Jae; Joo, Moon Kyung; Chun, Hoon Jai; Lee, Sang Woo; Bak, Young-Tae

    2016-01-01

    Background/Aims Astaxanthin is a carotenoid pigment that has antioxidant, antitumoral, and anti-inflammatory properties. In this in vitro study, we investigated the mechanism of anticancer effects of astaxanthin in gastric carcinoma cell lines. Methods The human gastric adenocarcinoma cell lines AGS, KATO-III, MKN-45, and SNU-1 were treated with various concentrations of astaxanthin. A cell viability test, cell cycle analysis, and immunoblotting were performed. Results The viability of each cancer cell line was suppressed by astaxanthin in a dose-dependent manner with significantly decreased proliferation in KATO-III and SNU-1 cells. Astaxanthin increased the number of cells in the G0/G1 phase but reduced the proportion of S phase KATO-III and SNU-1 cells. Phosphorylated extracellular signal-regulated kinase (ERK) was decreased in an inverse dose-dependent correlation with astaxanthin concentration, and the expression of p27kip-1 increased the KATO-III and SNU-1 cell lines in an astaxanthin dose-dependent manner. Conclusions Astaxanthin inhibits proliferation by interrupting cell cycle progression in KATO-III and SNU-1 gastric cancer cells. This may be caused by the inhibition of the phosphorylation of ERK and the enhanced expression of p27kip-1. PMID:26470770

  19. The comparison of glycosphingolipids isolated from an epithelial ovarian cancer cell line and a nontumorigenic epithelial ovarian cell line using MALDI-MS and MALDI-MS/MS.

    PubMed

    Rajanayake, Krishani K; Taylor, William R; Isailovic, Dragan

    2016-08-01

    Glycosphingolipids (GSLs) are important biomolecules, which are linked to many diseases such as GSL storage disorders and cancer. Consequently, the expression of GSLs may be altered in ovarian cancer cell lines in comparison to apparently healthy cell lines. Here, differential expressions of GSLs in an epithelial ovarian cancer cell line SKOV3 and a nontumorigenic epithelial ovarian cell line T29 were studied using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and MALDI-MS/MS. The isolation of GSLs from SKOV3 and T29 cell lines was carried out using Folch partition. GSLs were successfully detected by MALDI-MS, and structurally assigned by a comparison of their MALDI-MS/MS fragmentation patterns with MS/MS data found in SimLipid database. Additionally, LIPID MAPS was used to assign GSL ion masses in MALDI-MS spectra. Seventeen neutral GSLs were identified in Folch partition lower (chloroform/methanol) phases originating from both cell lines, while five globo series neutral GSLs were identified only in the Folch partition lower phase of SKOV3 cell line. Several different sialylated GSLs were detected in Folch partition upper (water/methanol) phases of SKOV3 and T29 cell lines. Overall, this study demonstrates the alteration and increased glycosylation of GSLs in an epithelial ovarian cancer cell line in comparison to a nontumorigenic epithelial ovarian cell line. PMID:27267063

  20. Interactions of Streptococcus iniae with phagocytic cell line.

    PubMed

    El Aamri, Fatima; Remuzgo-Martínez, S; Acosta, Félix; Real, Fernando; Ramos-Vivas, José; Icardo, José M; Padilla, Daniel

    2015-04-01

    Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1α, IL-1β, and TNF-α, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection.

  1. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; 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.

  2. Mechanisms of prodigiosin cytotoxicity in human neuroblastoma cell lines.

    PubMed

    Francisco, Roser; Pérez-Tomás, Ricardo; Gimènez-Bonafé, Pepita; Soto-Cerrato, Vanessa; Giménez-Xavier, Pol; Ambrosio, Santiago

    2007-10-31

    Prodigiosin is a bacterial red pigment with cytotoxic properties and potential antitumor activity that has been tested against different cancerous cells. In this study we report the effect and mechanisms of action of prodigiosin against different human neuroblastoma cell lines: SH-SY5Y, LAN-1, IMR-32 (N-type) and SK-N-AS (S-type). We compare the anticancerous effect of prodigiosin with that of cisplatin at different concentrations during 24 h of exposure. Prodigiosin is more potent, with IC50 values lower than 1.5 microM in N-type neuroblastoma cells and around 7 microM in the S-type neuroblastoma cell line. We describe prodigiosin as a proton sequestering agent that destroys the intracellular pH gradient, and propose that its main cytotoxic effect could be related to its action on mitochondria, where it exerts an uncoupling effect on the electronic chain transport of protons to mitochondrial ATP synthase. As a result of this action, ATP production is reduced but without decreasing in oxygen consumption. This mechanism of action differs from those induced by conventional chemotherapeutic drugs, suggesting a possible role for prodigiosin to enhance the effect of antitumor agents in the treatment of neuroblastoma.

  3. 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.

  4. Airway goblet cells: responsive and adaptable front-line defenders.

    PubMed

    Rogers, D F

    1994-09-01

    development of a hypersecretory epithelium include excessive discharge of mucus and increased expression of airway mucin messenger ribonucleic acid (mRNA). Cessation of chronic airway stress rapidly reverses the increased number of goblet cells. Irritant-induced increases in number of goblet cells can be inhibited by a variety of drugs with anti-inflammatory and mucoregulatory properties, and the reversal to normal numbers after cessation of the irritation is speeded by these drugs. The ability of goblet cells to be progenitors of ciliated cells, to rapidly produce vast quantities of mucus in response to acute airway insult, and to change in number according to variations in chronic insult indicates that these cells are vitally important responsive and adaptable front-line defenders of the airways. PMID:7995400

  5. Genetically-defined novel oral squamous cell carcinoma cell lines for the development of molecular therapies

    PubMed Central

    Fadlullah, Muhammad Zaki Hidayatullah; Chiang, Ivy Kim-Ni; Dionne, Kalen R.; Yee, Pei San; Gan, Chai Phei; Sam, Kin Kit; Tiong, Kai Hung; Ng, Adrian Kwok Wen; Martin, Daniel; Lim, Kue Peng; Kallarakkal, Thomas George; Mustafa, Wan Mahadzir Wan; Lau, Shin Hin; Abraham, Mannil Thomas; Zain, Rosnah Binti; Rahman, Zainal Ariff Abdul; Molinolo, Alfredo; Patel, Vyomesh; Gutkind, J. Silvio; Tan, Aik Choon; Cheong, Sok Ching

    2016-01-01

    Emerging biological and translational insights from large sequencing efforts underscore the need for genetically-relevant cell lines to study the relationships between genomic alterations of tumors, and therapeutic dependencies. Here, we report a detailed characterization of a novel panel of clinically annotated oral squamous cell carcinoma (OSCC) cell lines, derived from patients with diverse ethnicity and risk habits. Molecular analysis by RNAseq and copy number alterations (CNA) identified that the cell lines harbour CNA that have been previously reported in OSCC, for example focal amplications in 3q, 7p, 8q, 11q, 20q and deletions in 3p, 5q, 8p, 18q. Similarly, our analysis identified the same cohort of frequently mutated genes previously reported in OSCC including TP53, CDKN2A, EPHA2, FAT1, NOTCH1, CASP8 and PIK3CA. Notably, we identified mutations (MLL4, USP9X, ARID2) in cell lines derived from betel quid users that may be associated with this specific risk factor. Gene expression profiles of the ORL lines also aligned with those reported for OSCC. By focusing on those gene expression signatures that are predictive of chemotherapeutic response, we observed that the ORL lines broadly clustered into three groups (cell cycle, xenobiotic metabolism, others). The ORL lines noted to be enriched in cell cycle genes responded preferentially to the CDK1 inhibitor RO3306, by MTT cell viability assay. Overall, our in-depth characterization of clinically annotated ORL lines provides new insight into the molecular alterations synonymous with OSCC, which can facilitate in the identification of biomarkers that can be used to guide diagnosis, prognosis, and treatment of OSCC. PMID:27050151

  6. 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.

  7. 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.

  8. Integrative proteomic profiling of ovarian cancer cell lines reveals precursor cell associated proteins and functional status

    PubMed Central

    Coscia, F.; Watters, K. M.; Curtis, M.; Eckert, M. A.; Chiang, C. Y.; Tyanova, S.; Montag, A.; Lastra, R. R.; Lengyel, E.; Mann, M.

    2016-01-01

    A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumour of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here, we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumours, immortalized ovarian surface epithelial cells and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantification of >10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II) and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic data set, as well as a confirmatory publicly available CPTAC/TCGA tumour proteome data set, into a predominantly epithelial and mesenchymal HGSOC tumour cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumours indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium. PMID:27561551

  9. Creation and characterization of a cell-death reporter cell line for hepatitis C virus infection

    PubMed Central

    Chen, Zhilei; Simeon, Rudo; Chockalingam, Karuppiah; Rice, Charles M.

    2010-01-01

    The present study describes the creation and characterization of a hepatoma cell line, n4mBid, that supports all stages of the hepatitis C virus (HCV) life cycle and strongly reports HCV infection by a cell-death phenotype. The n4mBid cell line is derived from the highly HCV-permissive Huh-7.5 hepatoma cell line and contains a modified Bid protein (mBid) that is cleaved and activated by the HCV serine protease NS3-4A. N4mBid exhibited a 10–20 fold difference in cell viability between the HCV-infected and mock-infected states, while the parental Huh-7.5 cells showed <2 fold difference under the same conditions. The pronounced difference in n4mBid cell viability between the HCV- and mock-infected states in a 96-well plate format points to its usefulness in cell survival-based high-throughput screens for anti-HCV molecules. The degree of cell death was found to be proportional to the intracellular load of HCV. HCV-low n4mBid cells, expressing an anti-HCV short hairpin RNA, showed a significant growth advantage over naïve cells and could be rapidly enriched after HCV infection, suggesting the possibility of using n4mBid cells for the cell survival-based selection of genetic anti-HCV factors. PMID:20188762

  10. Characterization of hybrids between bovine (MDBK) and mouse (L-cell) cell lines.

    PubMed

    Chinchar, V G; Floyd, A D; Chinchar, G D; Taylor, M W

    1979-02-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient mutants of a bovine kidney cell line (MDBK) were selected following mutagenesis with ethylmethane sulfonate or ICR-170G. MDBK mutants were hybridized to thymidine kinase-deficient L cells and selected in HAT medium. Parental and hybrid cells were characterized for isozyme patterns of lactic dehydrogenase malate dehydrogenase, glucose-6-phosphate dehydrogenase, and glutamate oxalate transaminase. Chromosomes of MDBK can be distinguished from mouse L cells by configuration and by fluorescent staining with Hoechst 33-258 stain. Hybrid cells contained both MDBK and L-cell chromosomes and had elevated DNA content. MDBK cells are normally restrictive for mengovirus replication. Both permissive and restrictive hybrids were found. Our data indicate that there was preferential loss of MDBK chromosomes in the hybrid cell lines.

  11. Internalization of cystatin C in human cell lines.

    PubMed

    Ekström, Ulf; Wallin, Hanna; Lorenzo, Julia; Holmqvist, Bo; Abrahamson, Magnus; Avilés, Francesc X

    2008-09-01

    Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors.

  12. Melatonin decreases cell proliferation, impairs myogenic differentiation and triggers apoptotic cell death in rhabdomyosarcoma cell lines.

    PubMed

    Codenotti, Silvia; Battistelli, Michela; Burattini, Sabrina; Salucci, Sara; Falcieri, Elisabetta; Rezzani, Rita; Faggi, Fiorella; Colombi, Marina; Monti, Eugenio; Fanzani, Alessandro

    2015-07-01

    Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance.

  13. Proteomic patterns of cervical cancer cell lines, a network perspective

    PubMed Central

    2011-01-01

    Background Cervical cancer is a major mortality factor in the female population. This neoplastic is an excellent model for studying the mechanisms involved in cancer maintenance, because the Human Papilloma Virus (HPV) is the etiology factor in most cases. With the purpose of characterizing the effects of malignant transformation in cellular activity, proteomic studies constitute a reliable way to monitor the biological alterations induced by this disease. In this contextual scheme, a systemic description that enables the identification of the common events between cell lines of different origins, is required to distinguish the essence of carcinogenesis. Results With this study, we sought to achieve a systemic perspective of the common proteomic profile of six cervical cancer cell lines, both positive and negative for HPV, and which differ from the profile corresponding to the non-tumourgenic cell line, HaCaT. Our objectives were to identify common cellular events participating in cancer maintenance, as well as the establishment of a pipeline to work with proteomic-derived results. We analyzed by means of 2D SDS-PAGE and MALDI-TOF mass spectrometry the protein extracts of six cervical cancer cell lines, from which we identified a consensus of 66 proteins. We call this group of proteins, the "central core of cervical cancer". Starting from this core set of proteins, we acquired a PPI network that pointed, through topological analysis, to some proteins that may well be playing a central role in the neoplastic process, such as 14-3-3ζ. In silico overrepresentation analysis of transcription factors pointed to the overexpression of c-Myc, Max and E2F1 as key transcription factors involved in orchestrating the neoplastic phenotype. Conclusions Our findings show that there is a "central core of cervical cancer" protein expression pattern, and suggest that 14-3-3ζ is key to determine if the cell proliferates or dies. In addition, our bioinformatics analysis suggests that

  14. Heparanase augments inflammatory chemokine production from colorectal carcinoma cell lines.

    PubMed

    Tsunekawa, Naoki; Higashi, Nobuaki; Kogane, Yusuke; Waki, Michihiko; Shida, Hiroaki; Nishimura, Yoshio; Adachi, Hayamitsu; Nakajima, Motowo; Irimura, Tatsuro

    2016-01-22

    To explore possible roles of heparanase in cancer-host crosstalk, we examined whether heparanase influences expression of inflammatory chemokines in colorectal cancer cells. Murine colorectal carcinoma cells incubated with heparanase upregulated MCP-1, KC, and RANTES genes and released MCP-1 and KC proteins. Heparanase-dependent production of IL-8 was detected in two human colorectal carcinoma cell lines. Addition of a heparanase inhibitor Heparastatin (SF4) did not influence MCP-1 production, while both latent and mature forms of heparanase augmented MCP-1 release, suggesting that heparanase catalytic activity was dispensable for MCP-1 production. In contrast, addition of heparin to the medium suppressed MCP-1 release in a dose-dependent manner. Similarly, targeted suppression of Ext1 by RNAi significantly suppressed cell surface expression of heparan sulfate and MCP-1 production in colon 26 cells. Taken together, it is concluded that colon 26 cells transduce the heparanase-mediated signal through heparan sulfate binding. We propose a novel function for heparanase independent of its endoglycosidase activity, namely as a stimulant for chemokine production. PMID:26713365

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

    SciTech Connect

    Xi, Wenjun

    2008-01-01

    A capillary electrophoresis (CE) method is developed to determine both NAD+ and NADH levels in a single cell, based on an enzymatic cycling reaction. The detection limit can reach down to 0.2 amol NAD+ 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+ and NADH were separated, incubated with the cycling buffer, and quantified by the amount of fluorescent product generated. NADH and NAD+ levels of single cells of three cell lines and primary astrocyte culture were determined using this method. Comparing cellular NAD+ and NADH levels with and without exposure to oxidative stress induced by H2O2, it was found that H9c2 cells respond to the stress by reducing both cellular NAD+ and NADH levels, while astrocytes respond by increasing cellular NADH/NAD+ ratio.

  16. 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.

  17. Cycle reset in a melanoma cell line caused by cooling.

    PubMed

    Dewey, D L

    1987-11-01

    When cells in culture are released from G0 into cycle by diluting into fresh medium there is a delay of many hours before they re-enter the cycle and start DNA synthesis. A mouse melanoma cell line designated HP2 has been used to investigate the effects of non-standard temperatures between the time of plating and DNA synthesis. When the cells were incubated in a 5% CO2 box at 8 degrees C for periods during the G0-G1 transition there was an extra delay before the start of S, approximately equal to the time that the cells were held at 8 degrees C and independent of the time when the cold pulse was administered. When the cells were cooled to 25 degrees C the delay was longer than the time for which the cells had been kept at 25 degrees C, and this extra delay was also dependent on the point in G0-G1 when the cells were cooled, as though the cells could be reset to an earlier time by this treatment. It is suggested that a labile substance required for progression is destroyed faster than it is made at 25 degrees C but at 8 degrees C the rate of destruction is very low. Another phenomenon noted during these cooling experiments was that the peak height of the S phase profile, as measured by frequent pulse-thymidine incorporation experiments, was substantially higher for cells which had been cooled at a later stage in the G0-G1 transition, even though the overall times at 37 degrees C and at the colder temperature were identical. By varying the temperature of the cold pulse it was possible to separate the change in the peak height and the delay as separate entities. PMID:3502929

  18. Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells

    PubMed Central

    Kurita, Ryo; Suda, Noriko; Sudo, Kazuhiro; Miharada, Kenichi; Hiroyama, Takashi; Miyoshi, Hiroyuki; Tani, Kenzaburo; Nakamura, Yukio

    2013-01-01

    Transfusion of red blood cells (RBCs) is a standard and indispensable therapy in current clinical practice. In vitro production of RBCs offers a potential means to overcome a shortage of transfusable RBCs in some clinical situations and also to provide a source of cells free from possible infection or contamination by microorganisms. Thus, in vitro production of RBCs may become a standard procedure in the future. We previously reported the successful establishment of immortalized mouse erythroid progenitor cell lines that were able to produce mature RBCs very efficiently. Here, we have developed a reliable protocol for establishing immortalized human erythroid progenitor cell lines that are able to produce enucleated RBCs. These immortalized cell lines produce functional hemoglobin and express erythroid-specific markers, and these markers are upregulated following induction of differentiation in vitro. Most importantly, these immortalized cell lines all produce enucleated RBCs after induction of differentiation in vitro, although the efficiency of producing enucleated RBCs remains to be improved further. To the best of our knowledge, this is the first demonstration of the feasibility of using immortalized human erythroid progenitor cell lines as an ex vivo source for production of enucleated RBCs. PMID:23533656

  19. Sourcing human embryos for embryonic stem cell lines: Problems & perspectives

    PubMed Central

    Mehta, Rajvi H.

    2014-01-01

    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

  20. Development of buffalo (Bubalus bubalis) embryonic stem cell lines from somatic cell nuclear transferred blastocysts.

    PubMed

    Shah, Syed Mohmad; Saini, Neha; Ashraf, Syma; Singh, Manoj K; Manik, Radheysham; Singla, Suresh K; Palta, Prabhat; Chauhan, Manmohan Singh

    2015-11-01

    We developed buffalo embryonic stem cell lines from somatic cell nuclear transfer derived blastocysts, produced by hand-guided cloning technique. The inner cell mass of the blastocyst was cut mechanically using a Microblade and cultured onto feeder cells in buffalo embryonic stem (ES) cell culture medium at 38 °C in a 5% CO2 incubator. The stem cell colonies were characterized for alkaline phosphatase activity, karyotype, pluripotency and self-renewal markers like OCT4, NANOG, SOX2, c-Myc, FOXD3, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 and CD90. The cell lines also possessed the capability to differentiate across all the three germ layers under spontaneous differentiation conditions. PMID:26987926

  1. Development and Characterization of Six New Human Papillary Thyroid Carcinoma Cell Lines

    PubMed Central

    Henderson, Ying C.; Ahn, Soon-Hyun; Ryu, Junsun; Chen, Yunyun; Williams, Michelle D.; El-Naggar, Adel K.; Gagea, Mihai; Schweppe, Rebecca E.; Haugen, Bryan R.; Lai, Stephen Y.

    2015-01-01

    Context: Cell lines are a widely used tool in cancer research. However, despite the relatively high incidence of papillary thyroid carcinoma (PTC), there are only four PTC cell lines available for international research audience. Objective: The objective of this study was to establish and characterize new PTC cell lines that represent primary tumor biology. Surgical specimens were obtained to generate PTC cell lines. Short tandem repeat profiling was used to confirm the uniqueness of the cell lines against databases of known cell lines and mutations were assessed using Sequenom. The expression of thyroid-specific genes was examined using real-time PCR. Tumorigenicity was determined using an orthotopic thyroid xenograft tumor mouse model. Results: Six PTC cell lines (five conventional PTCs and one follicular variant of PTC) were generated and found to be unique when compared by short tandem repeat profiling against databases of all existing cell lines. The five conventional PTC cell lines carry the BRAF V600E mutation and the follicular variant of PTC cell line had an NRAS mutation. Five of the six cell lines had a mutation in the promoter of the human telomerase reverse transcriptase gene. None of the cell lines have RET/PTC rearrangements. Three cell lines were tumorigenic in the orthotopic thyroid xenograft tumor mouse model. Conclusions: These five characterized conventional PTC cell lines and the unique follicular variant of PTC cell line should be valuable reagents for thyroid cancer research. The three tumorigenic cell lines can be used for in vivo testing of targeted therapeutic and novel agents. PMID:25427145

  2. Erythropoietin modulates intracellular calcium in a human neuroblastoma cell line

    PubMed Central

    Assandri, Roberta; Egger, Marcel; Gassmann, Max; Niggli, Ernst; Bauer, Christian; Forster, Ian; Görlach, Agnes

    1999-01-01

    Recent investigations have shown that the glycoprotein erythropoietin (Epo) and its specific receptor (EpoR) are present in the mammalian brain including human, monkey and mouse. These findings suggest a local action of Epo in the nervous system. The aim of this study was to elucidate a possible functional interaction of Epo with neuronal cells. To examine the influence of externally applied Epo on Ca2+ homeostasis the human neuroblastoma cell line SK-N-MC was chosen as a suitable in vitro model for undifferentiated neuronal cells. Expression of the EpoR in SK-N-MC cells was detected by reverse transcription-PCR, Western blot and immunofluorescence analysis. Patch-clamp studies of SK-N-MC cells confirmed the expression of T-type Ca2+ channels, whose peak macroscopic current was increased by the addition of recombinant human Epo (rhEpo) to the bathing medium. Confocal laser scanning microscopy analysis of SK-N-MC cells confirmed a transient increase in intracellular free [Ca2+] in response to externally applied rhEpo. The transient response to Epo was dependent on external Ca2+ and remained even after depletion of internal Ca2+ stores by caffeine or thapsigargin. However, after depletion the response to Epo was absent when cells were superfused with the T-type Ca2+ channel blocker flunarizine. This study demonstrates that Epo can interact with neuronal cells by affecting Ca2+ homeostasis through an increase in Ca2+ influx via plasma membrane T-type voltage-dependent Ca2+ channels. PMID:10087335

  3. Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions.

    PubMed

    Pan, Cuiping; Kumar, Chanchal; Bohl, Sebastian; Klingmueller, Ursula; Mann, Matthias

    2009-03-01

    Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions.

  4. Novel antiproliferative flavonoids induce cell cycle arrest in human prostate cancer cell lines.

    PubMed

    Haddad, A Q; Venkateswaran, V; Viswanathan, L; Teahan, S J; Fleshner, N E; Klotz, L H

    2006-01-01

    Epidemiologic studies have demonstrated an inverse association between flavonoid intake and prostate cancer (PCa) risk. The East Asian diet is very high in flavonoids and, correspondingly, men in China and Japan have the lowest incidence of PCa worldwide. There are thousands of different naturally occurring and synthetic flavonoids. However, only a few have been studied in PCa. Our aim was to identify novel flavonoids with antiproliferative effect in PCa cell lines, as well as determine their effects on cell cycle. We have screened a representative subgroup of 26 flavonoids for antiproliferative effect on the human PCa (LNCaP and PC3), breast cancer (MCF-7), and normal prostate stromal cell lines (PrSC). Using a fluorescence-based cell proliferation assay (Cyquant), we have identified five flavonoids, including the novel compounds 2,2'-dihydroxychalcone and fisetin, with antiproliferative and cell cycle arresting properties in human PCa in vitro. Most of the flavonoids tested exerted antiproliferative effect at lower doses in the PCa cell lines compared to the non-PCa cells. Flow cytometry was used as a means to determine the effects on cell cycle. PC3 cells were arrested in G2/M phase by flavonoids. LNCaP cells demonstrated different cell cycle profiles. Further studies are warranted to determine the molecular mechanism of action of 2,2'-DHC and fisetin in PCa, and to establish their effectiveness in vivo.

  5. Sirtuin-3 (SIRT3) protein attenuates doxorubicin-induced oxidative stress and improves mitochondrial respiration in H9c2 cardiomyocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electro...

  6. Characterization of UV radiation sensitive frog cell lines

    SciTech Connect

    Smith-Stein, A.C.

    1983-01-01

    Twenty-one subclones of nine frog cell isolates were tested for sensitivity to a panel of DNA damaging agents. Two clones were identified which had a greater than wild type level of sensitivity to UV radiation but had a wild type level of sensitivity to the other agents. These clones were the haploid RRP602-7 and the diploid RRP802-1. RRP802-1 was found to be unstable with respect to UV sensitivity. The line was cloned in order to isolate stable sensitive and wild type derivatives. RRP802-1-16, a UV sensitive clone and RRP802-1-13, a clone with a wild type level of sensitivity to UV radiation, were isolated. The UV radiation sensitivity of RRP602-7, RRP802-1 and RRP802-1-16 did not correlate with cell size, cell shape, cell cycle distribution or ploidy. The cell cycle distribution after UV irradiation, the rate of DNA synthesis after UV-irradiation, the DNA polymerase ..cap alpha.. activity and the sister chromatid exchange frequency were all measured in RRP602-7, RRP802-1 and RRP802-1-16 in order to examine the DNA repair capacity. The presence of DNA repair pathways was examined directly in RRP602-7, RRP802-1 and RRP802-1-16. All were found to be proficient in photo-reactivation repair and postreplication repair of UV elicited DNA damage.

  7. Response of a mouse hybridoma cell line to heat shock, agitation, and sparging

    NASA Technical Reports Server (NTRS)

    Passini, Cheryl A.; Goochee, Charles F.

    1989-01-01

    A mouse hybridoma cell line is used as a model system for studying the effect of environmental stress on attachment-independent mammalian cells. The full time course of recovery for a mouse hybridoma cell line from both a mild and intermediate heat shock is examined. The pattern of intracellular synthesis is compared for actively growing, log phase cells and nondividing, stationary phase cells.

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. 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...

  13. 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

  14. 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.

  15. Development of a cell line from the American eel brain expressing endothelial cell properties.

    PubMed

    Bloch, Sophia R; Vo, Nguyen T K; Walsh, Sarah K; Chen, Cici; Lee, Lucy E J; Hodson, Peter V; Bols, Niels C

    2016-04-01

    A cell line (eelB) was developed from the outgrowth of adherent cells from brain explants of the American eel, Anguilla rostrata (Lesueur). EelB cells have been grown routinely in L-15 with 10% fetal bovine serum (FBS), undergone over 100 passages, and cryopreserved successfully. The cells from late-passage cultures (>45) were polygonal, formed capillary-like structures (CLS) on Matrigel, and stained immunocytochemically for von Willebrand factor (vWF) and for three tight junction proteins, zonula occludens-1 (ZO-1), claudin 3, and claudin 5. These results suggest that eelB is an endothelial cell line, one of the few from fish and the first from the brain. Despite this, eelB did not respond to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with the induction of CYP1A protein. The cells from early-passage cultures (<20) had more varied shapes and did not form CLS on Matrigel. Only cells from early-passage cultures formed in suspension three-dimensional aggregates that had some cells expressing alkaline phosphatase and nestin. These cells are thought to be neural stem cells and the aggregates neurospheres. The emergence of endothelial-like cells upon the continued subcultivation of cells from early-passage cultures that had neural stem cells has been described previously for mammals, but this is a first for teleosts. Remarkably, cells from all passage levels were stained strongly for senescence-associated β-galactosidase (SA β-Gal) activity.

  16. 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

  17. 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.

  18. 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.

  19. In vitro generation of hematopoietic stem cells from an embryonic stem cell line.

    PubMed Central

    Palacios, R; Golunski, E; Samaridis, J

    1995-01-01

    Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC. Images Fig. 3 PMID:7638225

  20. Induction of cellular glutathione-linked enzymes and catalase by the unique chemoprotective agent, 3H-1,2-dithiole-3-thione in rat cardiomyocytes affords protection against oxidative cell injury.

    PubMed

    Peng, Xingxiang; Li, Yunbo

    2002-06-01

    Considerable evidence suggests that reactive oxygen species (ROS) are crucially involved in the pathogenesis of cardiovascular diseases, such as myocardial ischemia-reperfusion injury. Consistent with this notion, administration of exogenous antioxidative compounds has been shown to provide protection against oxidative cardiac injury. However, whether induction of endogenous cellular antioxidants by chemicals (drugs) also offers protection against oxidative cardiac injury has not been extensively investigated. In the present study, with rat cardiomyocyte H9C2 cells as an in vitro model, we have investigated the induction of cellular antioxidants by the unique chemoprotective agent, 3 H -1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-induced cellular antioxidants against ROS-mediated injury in cardiac cells. Incubation of H9C2 cells with micromolar concentrations of D3T for 24 h resulted in a significant induction of a battery of cellular antioxidants, including reduced glutathione (GSH), GSH peroxidase, GSSG reductase, GSH S-transferase and catalase. To further examine the protective effects of the induced endogenous antioxidants against oxidative cell injury, H9C2 cells were pre-treated with D3T and then incubated with xanthine oxidase (XO) plus xanthine, a system that generates ROS. We observed that D3T pre-treatment of H9C2 cells led to significant protection against XO/xanthine-induced cytotoxicity as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction and morphological changes. Taken together, this study demonstrates for the first time that a number of endogenous antioxidants in cardiomyocytes can be induced by exposure to D3T, and that this chemical (drug) induction of cellular antioxidants is accompanied by markedly increased resistance to ROS-mediated cardiac cell injury.

  1. Characterisation and manipulation of docetaxel resistant prostate cancer cell lines

    PubMed Central

    2011-01-01

    Background There is no effective treatment strategy for advanced castration-resistant prostate cancer. Although Docetaxel (Taxotere®) represents the most active chemotherapeutic agent it only gives a modest survival advantage with most patients eventually progressing because of inherent or acquired drug resistance. The aims of this study were to further investigate the mechanisms of resistance to Docetaxel. Three Docetaxel resistant sub-lines were generated and confirmed to be resistant to the apoptotic and anti-proliferative effects of increasing concentrations of Docetaxel. Results The resistant DU-145 R and 22RV1 R had expression of P-glycoprotein and its inhibition with Elacridar partially and totally reversed the resistant phenotype in the two cell lines respectively, which was not seen in the PC-3 resistant sublines. Resistance was also not mediated in the PC-3 cells by cellular senescence or autophagy but multiple changes in pro- and anti-apoptotic genes and proteins were demonstrated. Even though there were lower basal levels of NF-κB activity in the PC-3 D12 cells compared to the Parental PC-3, docetaxel induced higher NF-κB activity and IκB phosphorylation at 3 and 6 hours with only minor changes in the DU-145 cells. Inhibition of NF-κB with the BAY 11-7082 inhibitor reversed the resistance to Docetaxel. Conclusion This study confirms that multiple mechanisms contribute to Docetaxel resistance and the central transcription factor NF-κB plays an immensely important role in determining docetaxel-resistance which may represent an appropriate therapeutic target. PMID:21982118

  2. Oxidative stress induces hypomethylation of LINE-1 and hypermethylation of the RUNX3 promoter in a bladder cancer cell line.

    PubMed

    Wongpaiboonwattana, Wikrom; Tosukhowong, Piyaratana; Dissayabutra, Thasinas; Mutirangura, Apiwat; Boonla, Chanchai

    2013-01-01

    Increased oxidative stress and changes in DNA methylation are frequently detected in bladder cancer patients. We previously demonstrated a relationship between increased oxidative stress and hypomethylation of the transposable long-interspersed nuclear element-1 (LINE-1). Promoter hypermethylation of a tumor suppressor gene, runt-related transcription factor 3 (RUNX3), may also be associated with bladder cancer genesis. In this study, we investigated changes of DNA methylation in LINE-1 and RUNX3 promoter in a bladder cancer cell (UM-UC-3) under oxidative stress conditions, stimulated by challenge with H2O2 for 72 h. Cells were pretreated with an antioxidant, tocopheryl acetate for 1 h to attenuate oxidative stress. Methylation levels of LINE-1 and RUNX3 promoter were measured by combined bisulfite restriction analysis PCR and methylation-specific PCR, respectively. Levels of LINE-1 methylation were significantly decreased in H2O2-treated cells, and reestablished after pretreated with tocopheryl acetate. Methylation of RUNX3 promoter was significantly increased in cells exposed to H2O2. In tocopheryl acetate pretreated cells, it was markedly decreased. In conclusion, hypomethylation of LINE-1 and hypermethylation of RUNX3 promoter in bladder cancer cell line was experimentally induced by reactive oxygen species (ROS). The present findings support the hypothesis that oxidative stress promotes urothelial cell carcinogenesis through modulation of DNA methylation. Our data also imply that mechanistic pathways of ROS-induced alteration of DNA methylation in a repetitive DNA element and a gene promoter might differ.

  3. The MDCK epithelial cell line expresses a cell surface antigen of the kidney distal tubule

    PubMed Central

    1982-01-01

    Monoclonal antibodies were prepared against the Madin-Darby canine kidney (MDCK) cell line to identify epithelial cell surface macromolecules involved in renal function. Lymphocyte hybrids were generated by fusing P3U-1 myeloma cells with spleen cells from a C3H mouse immunized with MDCK cells. Hybridomas secreting anti-MDCK antibodies were obtained and clonal lines isolated in soft agarose. We are reporting on one hybridoma line that secretes a monoclonal antibody that binds to MDCK cells at levels 20-fold greater than background binding. Indirect immunofluorescence microscopy was utilized to study the distribution of antibody binding on MDCK cells and on frozen sections of dog kidney and several nonrenal tissues. In the kidney the fluorescence staining pattern demonstrates that the antibody recognizes an antigenic determinant that is expressed only on the epithelial cells of the thick ascending limb of Henle's loops and the distal convoluted tubule and appears to be localized on the basolateral plasma membrane. This antigen also has a unique distribution in non-renal tissues and can only be detected on cells known to be active in transepithelial ion movements. These results indicate the probable distal tubule origin of MDCK and suggest that the monoclonal antibody recognizes a cell surface antigen involved in physiological functions unique to the kidney distal tubule and transporting epithelia of nonrenal tissues. PMID:6178742

  4. Epigenetic alterations differ in phenotypically distinct human neuroblastoma cell lines

    PubMed Central

    2010-01-01

    Background Epigenetic aberrations and a CpG island methylator phenotype have been shown to be associated with poor outcomes in children with neuroblastoma (NB). Seven cancer related genes (THBS-1, CASP8, HIN-1, TIG-1, BLU, SPARC, and HIC-1) that have been shown to have epigenetic changes in adult cancers and play important roles in the regulation of angiogenesis, tumor growth, and apoptosis were analyzed to investigate the role epigenetic alterations play in determining NB phenotype. Methods Two NB cell lines (tumorigenic LA1-55n and non-tumorigenic LA1-5s) that differ in their ability to form colonies in soft agar and tumors in nude mice were used. Quantitative RNA expression analyses were performed on seven genes in LA1-5s, LA1-55n and 5-Aza-dC treated LA1-55n NB cell lines. The methylation status around THBS-1, HIN-1, TIG-1 and CASP8 promoters was examined using methylation specific PCR. Chromatin immunoprecipitation assay was used to examine histone modifications along the THBS-1 promoter. Luciferase assay was used to determine THBS-1 promoter activity. Cell proliferation assay was used to examine the effect of 5-Aza-dC on NB cell growth. The soft agar assay was used to determine the tumorigenicity. Results Promoter methylation values for THBS-1, HIN-1, TIG-1, and CASP8 were higher in LA1-55n cells compared to LA1-5s cells. Consistent with the promoter methylation status, lower levels of gene expression were detected in the LA1-55n cells. Histone marks associated with repressive chromatin states (H3K9Me3, H3K27Me3, and H3K4Me3) were identified in the THBS-1 promoter region in the LA1-55n cells, but not the LA1-5s cells. In contrast, the three histone codes associated with an active chromatin state (acetyl H3, acetyl H4, and H3K4Me3) were present in the THBS-1 promoter region in LA1-5s cells, but not the LA1-55n cells, suggesting that an accessible chromatin structure is important for THBS-1 expression. We also show that 5-Aza-dC treatment of LA1-55n cells

  5. Origin of the U87MG glioma cell line: Good news and bad news.

    PubMed

    Allen, Marie; Bjerke, Mia; Edlund, Hanna; Nelander, Sven; Westermark, Bengt

    2016-08-31

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin.

  6. Origin of the U87MG glioma cell line: Good news and bad news.

    PubMed

    Allen, Marie; Bjerke, Mia; Edlund, Hanna; Nelander, Sven; Westermark, Bengt

    2016-08-31

    Human tumor-derived cell lines are indispensable tools for basic and translational oncology. They have an infinite life span and are easy to handle and scalable, and results can be obtained with high reproducibility. However, a tumor-derived cell line may not be authentic to the tumor of origin. Two major questions emerge: Have the identity of the donor and the actual tumor origin of the cell line been accurately determined? To what extent does the cell line reflect the phenotype of the tumor type of origin? The importance of these questions is greatest in translational research. We have examined these questions using genetic profiling and transcriptome analysis in human glioma cell lines. We find that the DNA profile of the widely used glioma cell line U87MG is different from that of the original cells and that it is likely to be a bona fide human glioblastoma cell line of unknown origin. PMID:27582061

  7. Human embryonic stem cell lines derived from single blastomeres of two 4-cell stage embryos

    PubMed Central

    Geens, Mieke; Mateizel, Ileana; Sermon, Karen; De Rycke, Martine; Spits, Claudia; Cauffman, Greet; Devroey, Paul; Tournaye, Herman; Liebaers, Inge; Van de Velde, Hilde

    2009-01-01

    BACKGROUND Recently, we demonstrated that single blastomeres of a 4-cell stage human embryo are able to develop into blastocysts with inner cell mass and trophectoderm. To further investigate potency at the 4-cell stage, we aimed to derive pluripotent human embryonic stem cells (hESC) from single blastomeres. METHODS Four 4-cell stage embryos were split on Day 2 of preimplantation development and the 16 blastomeres were individually cultured in sequential medium. On Day 3 or 4, the blastomere-derived embryos were plated on inactivated mouse embryonic fibroblasts (MEFs). RESULTS Ten out of sixteen blastomere-derived morulae attached to the MEFs, and two produced an outgrowth. They were mechanically passaged onto fresh MEFs as described for blastocyst ICM-derived hESC, and shown to express the typical stemness markers by immunocytochemistry and/or RT–PCR. In vivo pluripotency was confirmed by the presence of all three germ layers in the teratoma obtained after injection in immunodeficient mice. The first hESC line displays a mosaic normal/abnormal 46, XX, dup(7)(q33qter), del(18)(q23qter) karyotype. The second hESC line displays a normal 46, XY karyotype. CONCLUSION We report the successful derivation and characterization of two hESC lines from single blastomeres of four split 4-cell stage human embryos. These two hESC lines were derived from distinct embryos, proving that at least one of the 4-cell stage blastomeres is pluripotent. PMID:19633307

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

    PubMed

    Restelli, Valentina; Chilà, 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

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

    PubMed Central

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

    2015-01-01

    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

  10. "Fluorescent Cell Chip" for immunotoxicity testing: development of the c-fos expression reporter cell lines.

    PubMed

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ullerås, Erik; Dastych, Jarosław

    2005-09-01

    The Fluorescent Cell Chip for in vitro immunotoxicity testing employs cell lines derived from lymphocytes, mast cells, and monocytes-macrophages transfected with various EGFP cytokine reporter gene constructs. While cytokine expression is a valid endpoint for in vitro immunotoxicity screening, additional marker for the immediate-early response gene expression level could be of interest for further development and refinement of the Fluorescent Cell Chip. We have used BW.5147.3 murine thymoma transfected with c-fos reporter constructs to obtain reporter cell lines expressing ECFP under the control of murine c-fos promoter. These cells upon serum withdrawal and readdition and incubation with heavy metal compounds showed paralleled induction of c-Fos expression as evidenced by Real-Time PCR and ECFP fluorescence as evidenced by computer-supported fluorescence microscopy. In conclusion, we developed fluorescent reporter cell lines that could be employed in a simple and time-efficient screening assay for possible action of chemicals on c-Fos expression in lymphocytes. The evaluation of usefulness of these cells for the Fluorescent Cell Chip-based detection of immunotoxicity will require additional testing with a larger number of chemicals.

  11. PACAP protects against TNFα-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 TNFα. Live slices of neonatal mouse OE were treated with 40 ng/ml TNFα ± 40 nM PACAP for 6 hours and dying cells were live-labeled with 0.5% propidium iodide. TNFα significantly increased the percentage of dying cells while co-incubation with PACAP prevented cell death. PACAP also prevented TNFα-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 TNFα 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 TNFα 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

  12. Different toxic effects of YTX in tumor K-562 and lymphoblastoid cell lines

    PubMed Central

    Fernández-Araujo, Andrea; Sánchez, Jon A.; Alfonso, Amparo; Vieytes, Mercedes R.; Botana, Luis M.

    2015-01-01

    Yessotoxin (YTX) modulates cellular phosphodiesterases (PDEs). In this regard, opposite effects had been described in the tumor model K-562 cell line and fresh human lymphocytes in terms of cell viability, cyclic adenosine 3',5'-cyclic monophosphate (cAMP) production and protein expression after YTX treatment. Studies in depth of the pathways activated by YTX in K-562 cell line, have demonstrated the activation of two different cell death types, apoptosis, and autophagy after 24 and 48 h of treatment, respectively. Furthermore, the key role of type 4A PDE (PDE4A) in both pathways activated by YTX was demonstrated. Therefore, taking into account the differences between cellular lines and fresh cells, a study of cell death pathways activated by YTX in a non-tumor cell line with mitotic activity, was performed. The cellular model used was the lymphoblastoid cell line that represents a non-tumor model with normal apoptotic and mitotic machinery. In this context, cell viability and cell proliferation, expression of proteins involved in cell death activated by YTX and mitochondrial mass, were studied after the incubation with the toxin. Opposite to the tumor model, no cell death activation was observed in lymphoblastoid cell line in the presence of YTX. In this sense, variations in apoptosis hallmarks were not detected in the lymphoblastoid cell line after YTX incubation, whereas this type I of programmed cell death was observed in K-562 cells. On the other hand, autophagy cell death was triggered in this cellular line, while other autophagic process is suggested in lymphoblastoid cells. These YTX effects are related to PDE4A in both cellular lines. In addition, while cell death is triggered in K-562 cells after YTX treatment, in lymphoblastoid cells the toxin stops cellular proliferation. These results point to YTX as a specific toxic compound of tumor cells, since in the non-tumor lymphoblastoid cell line, no cell death hallmarks are observed. PMID:26136685

  13. 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

  14. 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

  15. Mechanisms of lymphocyte adhesion to endothelial cells: studies using a LFA-1-deficient cell line.

    PubMed Central

    Haskard, D O; Strobel, S; Thornhill, M; Pitzalis, C; Levinsky, R J

    1989-01-01

    In order to investigate the role of lymphocyte function-associated antigen 1 (LFA-1) in lymphocyte adhesion to endothelial cells (EC), we have studied the adhesion of a LFA-1-deficient lymphoblastoid cell line, ICH-KM, which has < 10% of the cell surface LFA-1 expressed on a normal lymphoblastoid cell line, ICH-BJ. The adhesion of ICH-KM cells to unstimulated EC was 49.9 +/- 8.6% (mean +/- SD) that of ICH-BJ cells. Moreover, phorbol ester-stimulated ICH-KM cells showed a considerably weaker increase in adhesion to unstimulated EC compared with ICH-BJ cells (mean +/- SD increase in percentage adhesion, 3.8 +/- 2.3 compared with 18.5 +/- 8.0; P<0.025). In contrast, there was no significant difference between the enhanced adhesion of ICH-KM cells and ICH-BJ cells to interleukin-1 (IL-1)-stimulated EC. Thus ICH-KM cells showed a 22.7 +/- 11.0 (mean +/- SD) increase in percentage adhesion to IL-1-stimulated EC compared with the 24.8 +/- 8.5 increase in percentage adhesion of ICH-BJ cells. Anti-LFA-1 monoclonal antibodies had no effect on the enhanced adhesion of ICH-KM and ICH-BJ cells to IL-1-stimulated EC but abolished the differences in adhesion between the two cell lines. The study therefore indicates that although a major part of unstimulated and phorbol ester-stimulated lymphocyte-EC adhesion is dependent upon LFA-1, the enhanced adhesion due to stimulation of EC with IL-1 is not dependent upon this molecule. The data therefore supports the existence of cytokine-inducible LFA-1-independent adhesion molecules for lymphocytes on EC. PMID:15493272

  16. Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

    PubMed Central

    Welker, Alessandra M.; Jaros, Brian D.; Puduvalli, Vinay K.; Imitola, Jaime; Kaur, Balveen; Beattie, Christine E.

    2016-01-01

    ABSTRACT Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for

  17. Establishment of a highly differentiated thyroid cancer cell line of Hürthle cell origin.

    PubMed

    Zielke, A; Tezelman, S; Jossart, G H; Wong, M; Siperstein, A E; Duh, Q Y; Clark, O H

    1998-06-01

    Hürthle cell carcinomas (HCC) of the thyroid are a variant of follicular thyroid tumors. In contrast to follicular thyroid carcinoma, HCC rarely take up radioiodine and frequently metastasize to the lymph nodes. Histologically they are indistinguishable from Hürthle cell adenomas except for evidence of invasion and metastasis. How these carcinomas develop and why they behave differently than other follicular tumors is not known. Although some differentiated thyroid cancer cell lines exist, none are from Hürthle cell tumors. We have established a well-differentiated thyroid cancer cell line from a metastasis of a HCC, designated XTC.UC1. In vitro, XTC cells display epitheloid morphology, grow with a population doubling time of 4.3 +/- 0.3 days, migrate, and invade through reconstituted basement membranes. The cells are immunoreactive for and release thyroglobulin, respond to thyrotropin (TSH) with increase of intracellular cyclic adenosine monophosphate (cAMP), proliferation, and invasion of reconstituted basement membrane, thus exhibiting characteristics of well-differentiated thyroid carcinoma. In vivo, xenografted XTC cells grow with a doubling time of 9.8 +/- 0.8 days. Tumors spontaneously metastasize to the lymph nodes and less frequently to the lungs and the liver. The cells retained their differentiated function in vivo as assessed by human thyroglobulin (hTG) secretion and immunohistochemistry. This is a first report of the establishment of a unique, highly differentiated thyroid carcinoma cell line derived from an HCC. Based on the ability to invade through reconstituted basement membrane in vitro and the potential to metastasize in vivo, this cell line may provide a unique model to study invasion and metastazation of well-differentiated thyroid cancer.

  18. Natural Killer Cells for Immunotherapy – Advantages of the NK-92 Cell Line over Blood NK Cells

    PubMed Central

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

    Natural killer (NK) cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells from a patient’s blood since they represent only 10% of the lymphocytes and are often dysfunctional. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T cells to prevent graft-versus-host reactions. Cytotoxic cell lines have been established from patients with clonal NK-cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. Except for the NK-92 cell line, though, none of the other six known NK cell lines has consistently and reproducibly shown high antitumor cytotoxicity. Only NK-92 cells can easily be genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through antibody-dependent cellular cytotoxicity. NK-92 is also the only cell line product that has been infused into patients with advanced cancer with clinical benefit and minimal side effects. PMID:27014270

  19. Side population cells isolated from KATO III human gastric cancer cell line have cancer stem cell-like characteristics

    PubMed Central

    She, Jun-Jun; Zhang, Peng-Ge; Wang, Xuan; Che, Xiang-Ming; Wang, Zi-Ming

    2012-01-01

    AIM: To investigate whether the side population (SP) cells possess cancer stem cell-like characteristics in vitro and the role of SP cells in tumorigenic process in gastric cancer. METHODS: We analyzed the presence of SP cells in different human gastric carcinoma cell lines, and then isolated and identified the SP cells from the KATO III human gastric cancer cell line by flow cytometry. The clonogenic ability and self-renewal were evaluated by clone and sphere formation assays. The related genes were determined by reverse transcription polymerase chain reaction. To compare tumorigenic ability, SP and non-side population (NSP) cells from the KATO III human gastric cancer cell line were subcutaneously injected into nude mice. RESULTS: SP cells from the total population accounted for 0.57% in KATO III, 1.04% in Hs-746T, and 0.02% in AGS (CRL-1739). SP cells could grow clonally and have self-renewal capability in conditioned media. The expression of ABCG2, MDRI, Bmi-1 and Oct-4 was different between SP and NSP cells. However, there was no apparent difference between SP and NSP cells when they were injected into nude mice. CONCLUSION: SP cells have some cancer stem cell-like characteristics in vitro and can be used for studying the tumorigenic process in gastric cancer. PMID:22969237

  20. Identification of Replication Competent Murine Gammaretroviruses in Commonly Used Prostate Cancer Cell Lines

    PubMed Central

    Sfanos, Karen Sandell; Aloia, Amanda L.; Hicks, Jessica L.; Esopi, David M.; Steranka, Jared P.; Shao, Wei; Sanchez-Martinez, Silvia; Yegnasubramanian, Srinivasan; Burns, Kathleen H.; Rein, Alan; De Marzo, Angelo M.

    2011-01-01

    A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral “contamination”, much like routine mycoplasma testing. PMID:21698104

  1. Cell cycle analysis and cytotoxic potential of Ruta graveolens against human tumor cell lines.

    PubMed

    Varamini, P; Soltani, M; Ghaderi, A

    2009-01-01

    There are reports on the presence of various compounds exerting different biological activities in Ruta graveolens, a plant of Rutaceae family. The aim of the present study was to evaluate in vitro cytotoxicity of the total extract of R. graveolens against tumor cell lines of different origin. Aerial parts of the plant was extracted with 70% ethanol by sonication method and cytotoxic activity was examined on RAJI, RAMOS, RPMI8866, U937, Jurkat, MDA-MB-453, MCF-7, LNCap-FGC-10, 5637, HeLa, SK-OV-3, A549, Mehr-80 and also peripheral blood mononuclear cells (PBMC) by the use of WST-1 assay. Results were expressed as IC(50) values. R. graveolens extract showed high cytotoxic activity against RAJI and RAMOS, two Burkitt's lymphoma cell lines, with an IC(50) equal to 24.3 microg/ml and 35.2 microg/ml respectively and LNCap-FGC-10, a prostate adenocarcinoma cell line with an IC(50) equal to 27.6 microg/ml as well as Mehr-80, a newly established Large Cell Lung Carcinoma (IC(50)=46.2 microg/ml). No significant anti-proliferative activity was observed on other cell lines including MCF-7, MDA-MB-453, SK-OV-3, HeLa, 5637, JURKAT and RPMI8866. Adverse cytotoxic effect of R. graveolens was investigated against PBMCs and a significantly lower effect of this extract (IC(50)=104 microg/ml) was seen on normal cells compared with RAJI and RAMOS, two haematopoietic cell lines.

  2. A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

    PubMed Central

    Ruben, Jurjen M.; Visser, Lindy L.; Heinhuis, Kimberley M.; O’Toole, Tom; Bontkes, Hetty J.; Westers, Theresia M.; Ossenkoppele, Gert J.; de Gruijl, Tanja D.; van de Loosdrecht, Arjan A.

    2015-01-01

    The CD34+ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8+ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8+ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality. PMID:26252775

  3. A cell-permeable fluorescent polymeric thermometer for intracellular temperature mapping in mammalian cell lines.

    PubMed

    Hayashi, Teruyuki; Fukuda, Nanaho; Uchiyama, Seiichi; Inada, Noriko

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature.

  4. Establishment and characterization of a clear cell carcinoma cell line, designated NOCC, derived from human ovary.

    PubMed

    Ohyama, Akihiro; Toyomura, Junko; Tachibana, Toshiaki; Isonishi, Seiji; Takahashi, Haruka; Ishikawa, Hiroshi

    2016-10-01

    A cell line, designated NOCC, was established from the ascites of a patient with clear cell adenocarcinoma of the ovary. The cell line has been grown without interruption and continuously propagated by serial passaging (more than 76 times) over 7 years. The cells are spherical to polygonal-shaped, display neoplastic, and pleomorphic features, and grow in a jigsaw puzzle-like pattern while forming monolayers without contact inhibition. The cells proliferate rapidly, but are easily floated as a cell sheet. The population doubling time is about 29 h. The number of chromosomes ranges from 60 to 83. The modal number of chromosomes is 70-74 at the 30th passage. NOCC cells secreted 750.5 ng/ml of VEGF over 3 days of culture. Hypoxia inducible factor-1α (HIF-1α) is a primary regulator of VEGF under hypoxic conditions. NOCC cells were not sensitive to the anticancer drugs BEV, DOX, GEM, ETP, CDDP, or TXT. The graft of NOCC cells to a scid mouse displayed similar histological aspects to the original tumor. Both the NOCC cells and the graft of the NOCC cells gave a positive PAS reaction. PMID:27541369

  5. 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.

  6. In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

    PubMed Central

    Damstrup, L.; Rude Voldborg, B.; Spang-Thomsen, M.; Brünner, N.; Skovgaard Poulsen, H.

    1998-01-01

    Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines. Images Figure 1 Figure 3 Figure 4 PMID:9744504

  7. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines

    DOE PAGESBeta

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; Brown, Ben; Cherbas, Peter

    2016-01-15

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest suchmore » atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. In conclusion, this atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly.« less

  8. Comparison of antibody molecules produced from two cell lines with contrasting productivities and aggregate contents.

    PubMed

    Ishii, Yoichi; Imamoto, Yasufumi; Yamamoto, Rie; Tsukahara, Masayoshi; Wakamatsu, Kaori

    2015-01-01

    Cell culture processes that produce therapeutic antibodies with high productivity (titer) and low aggregate content reduce the risk of adverse effects and expense to patients. To elucidate the mechanism of aggregate formation, we compared trastuzumab samples produced from two contrasting cell lines: cell line A, which exhibits high titer and low aggregate content, and cell line B, which exhibits low titer and high aggregate content. Cell line B produced significantly fewer (approximately 1/3) antibodies compared with cell line A and contained higher (approximately 3-fold) percentages of aggregates. The aggregates of antibodies found in the protein A-purified samples of cell line B were associated mostly with noncovalent interactions. Cell line B exhibited a low content of monomers/dimers of light chains in the medium and within cells. Because light chains are essential for the correct folding of heavy chains and secretion of mature antibodies, the characteristics of cell line B may be attributed to low levels of light chain production. In addition, protein A-purified antibodies from cell line B (but not those from cell line A) contained fragments that are expected to expose the hydrophobic CH3 domain, which may serve as nuclei for aggregation. PMID:25501618

  9. Lipid analysis of eight human breast cancer cell lines with ToF-SIMS

    PubMed Central

    Robinson, Michael A.; Graham, Daniel J.; Morrish, Fionnuala; Hockenbery, David; Gamble, Lara J.

    2015-01-01

    In this work, four triple negative (TN) cell lines, three ER+ and PR+ receptor positive (RP) cell lines, and one ER+, PR+, and HER2+ cell line were chemically distinguished from one another using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and principal component analysis (PCA). PCA scores separation was observed between the individual cell lines within a given classification (TN and RP) and there were distinctly different trends found in the fatty acid and lipid compositions of the two different classifications. These trends indicated that the RP cell lines separated out based on the carbon chain length of the lipids while the TN cell lines showed separation based on cholesterol-related peaks (in the positive ion data). Both cell types separated out by trends in fatty acid chain length and saturation in the negative ions. These chemical differences may be manifestations of unique metabolic processes within each of the different cell lines. Additionally, the HER2+ cell line was distinguished from three other RP cell types as having a unique distribution of fatty acids including anticorrelation to 18-carbon chain fatty acids. As these cell lines could not be grown in the same growth media, a combination of chemical fixation, rinsing, C60+ presputtering, and selection of cellular regions-of-interest is also presented as a successful method to acquire ToF-SIMS data from cell lines grown in different media. PMID:26319020

  10. 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...

  11. Derivation and characterization of matched cell lines from primary and recurrent serous ovarian cancer

    PubMed Central

    2012-01-01

    Background Cell line models have proven to be effective tools to investigate a variety of ovarian cancer features. Due to the limited number of cell lines, particularly of the serous subtype, the heterogeneity of the disease, and the lack of cell lines that model disease progression, there is a need to further develop cell line resources available for research. This study describes nine cell lines derived from three ovarian cancer cases that were established at initial diagnosis and at subsequent relapse after chemotherapy. Methods The cell lines from three women diagnosed with high-grade serous ovarian cancer (1369, 2295 and 3133) were derived from solid tumor (TOV) and ascites (OV), at specific time points at diagnosis and relapse (R). Primary treatment was a combination of paclitaxel/carboplatin (1369, 3133), or cisplatin/topotecan (2295). Second line treatment included doxorubicin, gemcitabine and topotecan. In addition to molecular characterization (p53, HER2), the cell lines were characterized based on cell growth characteristics including spheroid growth, migration potential, and anchorage independence. The in vivo tumorigenicity potential of the cell lines was measured. Response to paclitaxel and carboplatin was assessed using a clonogenic assay. Results All cell lines had either a nonsense or missense TP53 mutations. The ability to form compact spheroids or aggregates was observed in six of nine cell lines. Limited ability for migration and anchorage independence was observed. The OV3133(R) cell line, formed tumors at subcutaneous sites in SCID mice. Based on IC50 values and dose response curves, there was clear evidence of acquired resistance to carboplatin for TOV2295(R) and OV2295(R2) cell lines. Conclusion The study identified nine new high-grade serous ovarian cancer cell lines, derived before and after chemotherapy that provides a unique resource for investigating the evolution of this common histopathological subtype of ovarian cancer. PMID:22931248

  12. Detection of antigens specific for B-lymphoid cultured cell lines with human alloantisera.

    PubMed

    Mann, D L; Abelson, L; Harris, S; Amos, D B

    1975-07-01

    Human sera were tested for cytotoxicity to pairs of long-term tissue-cultured cell lines. Each pair had been derived from the same individual and one of the pairs possessed the characteristics of either "T" or "B" cells. The alloantisera used were HL-A-typing reagents or sera obtained from Amish multiparas. Selected cytotoxicity was found against the B-cell lines by direct testing. Cytotoxicity was abolished by absorption with B-cell line but not by absorption with the T-cell lines. The results suggest that a group of allotypic antigens may be expressed exculsively on human B cells. PMID:1080182

  13. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines.

    PubMed

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I (2) statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating "hub genes" - heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  14. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility.

  15. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

    Tian, Sunny; Bertelsmann, Karina; Yu, Linda; Sun, Shuying

    2016-01-01

    Heterogeneous DNA methylation patterns are linked to tumor growth. In order to study DNA methylation heterogeneity patterns for breast cancer cell lines, we comparatively study four metrics: variance, I2 statistic, entropy, and methylation state. Using the categorical metric methylation state, we select the two most heterogeneous states to identify genes that directly affect tumor suppressor genes and high- or moderate-risk breast cancer genes. Utilizing the Gene Set Enrichment Analysis software and the ConsensusPath Database visualization tool, we generate integrated gene networks to study biological relations of heterogeneous genes. This analysis has allowed us to contribute 19 potential breast cancer biomarker genes to cancer databases by locating “hub genes” – heterogeneous genes of significant biological interactions, selected from numerous cancer modules. We have discovered a considerable relationship between these hub genes and heterogeneously methylated oncogenes. Our results have many implications for further heterogeneity analyses of methylation patterns and early detection of breast cancer susceptibility. PMID:27688708

  16. Effects of feeder cells (human cancer cell lines) on the development of mouse embryos by co-culture.

    PubMed

    Ishiwata, I; Tokieda, Y; Ishiwata, C; Okane, N; Iguchi, M; Sato, K; Ishikawa, H

    1997-12-01

    In order to establish the best co-culture system on embryogenesis such as egg fertilization, egg cleavage, blastocyst formation, hatching and implantation etc., several kinds of cell lines as a feeder cell and mouse fertilized eggs (zygotes) were co-cultured in the organ culture dish, and embryotrophic effects of feeder cells were investigated. Best feeder cell on the embryogenesis was SKG-II cell line derived from squamous cell carcinoma of human uterine cervix which was chosen from 10 of the human tumor cell lines. Furthermore, in order to isolate and determinate embryotrophic factors produced by feeder cells, we established a SKG-II SF subline which was grown in serum free medium derived from SKG-II cell line. The SKG-II SF cell line secreted an epidermal growth factor (EGF) into the medium. Also, cleavaged egg produced and secreted interleukin (IL)-1 alpha into the medium. PMID:9573483

  17. 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.

  18. 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.

  19. Quality Check in Oral Cell Lines: The Need for Molecular Characterization.

    PubMed

    Patil, Shankargouda; Rao, Roopa S; Raj, A Thirumal

    2015-11-01

    Oral cell lines have provided valuable insights into the various molecular pathways in oral carcinogenesis. Several landmark studies in oral oncology have utilized commercially available normal, dysplastic and cancer cell lines to decode the genetic alterations leading to the development of oral cancer. Most of these studies have shown a significant degree of variation in their mutation landscapes. These variations were thought to represent the heterogeneity of oral cancer.(1) But in a recent study, Dickman et al have shown that normal and dysplastic cell lines carry specific genetic alterations within the parent cell line, thus questioning the authenticity of several published mutation profiles. These genetic alterations in the commercial cell lines have been attributed to several factors, the most common being immortalization. Normal and dysplastic cell lines unlike cancer cell lines attain senescence following limited number of replication. Immortalization of the normal and dysplastic cell lines would aid the researcher in maintaining a viable population of cells for further studies. Ideally, the immortalized cell line must possess potential for indefinite replication and must retain the genetic makeup of its parent cell line.(2). PMID:26718303

  20. Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts.

    PubMed

    Xin, Hong; Wang, Ke; Hu, Gang; Xie, Fubo; Ouyang, Kedong; Tang, Xuzhen; Wang, Minjun; Wen, Danyi; Zhu, Yizhun; Qin, Xiaoran

    2014-01-01

    Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening.

  1. Biological characteristics of a novel giant cell tumor cell line derived from spine.

    PubMed

    Zhou, Zhenhua; Li, Yan; Xu, Leqin; Wang, Xudong; Chen, Su; Yang, Cheng; Xiao, Jianru

    2016-07-01

    Giant cell tumor of bone(GCTB) is a special bone tumor for it consists of various cell types, and its biological characteristics is different from common benign or malignant neoplasm. In the present study, we report the biological features of a primary Asian GCTB cell line named GCTB28. We analyzed extensive properties of the GCTB28 cells including morphological observations, growth, cell cycle, karyotype, proliferation, proteins expression, surface biomarker verification, and tumorigenicity in nude mice. We found that the stromal cells of GCTB were endowed with self-renewal capacity and played dominant roles in GCTB development. Moreover, we confirmed that GCTB cells can be CD33(-)CD14(-) phenotype which was not in accord with previous study. This study provides an in vitro model system to investigate pathogenic mechanisms and molecular characteristics of GCTB and also provides a useful tool for researching the therapeutic targeting of GCTB.

  2. 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.

  3. A bioassay for metals utilizing a human cell line.

    PubMed

    Shea, J; Moran, T; Dehn, P F

    2008-06-01

    The purpose of this study was to assess the ability of the HepG2 cell line to function as a bioassay for metal contamination in sediments, using metallothionein (MT) as a biomarker of exposure. Sediments were collected from the eastern and western ends of Lake Erie, extracted using EPA method 200.7, and analyzed for cadmium (Cd), mercury (Hg) and lead (Pb) levels using ICP-AES. Sediment extracts were neutralized then used at a 2.5% final concentration in the exposure medium. MT levels were measured using the cadmium-hemoglobin affinity assay after a 48 h exposure. Fortified blanks from the ICP protocol served as positive controls. Also, HepG2 cells were exposed to Cd, Pb or combinations of Cd and Pb to determine whether or not induction of MT observed in cells exposed to sediment extracts was due to a single metal, combinations of metals, pH, or some other factor. Additionally, cells were exposed to a range of Cd concentrations approximating the levels found in the extracts (0.0005-0.1mg/L) to determine if a concentration-response occurred. Total metal levels ranged from 527 to 33.5mg/kg with lead the predominant metal, accounting for 100-88.9% of the total quantifiable metals in the sediments. The biomarker response (MT induction) was strongly correlated (r2=0.9919, r2=0.990) with total metal and lead levels in the sediments, respectively, which supports recent field studies indicating the biomarker can discern differences in the strength of the inducing agent. Statistically significant MT induction was associated with sediments which contained measurable Cd concentrations and no significant differences were observed when comparing Cd only and Cd+Pb exposed cells indicating no interactions between Cd and Pb were occurring and supporting our finding that Cd was the main inducing agent in sediment extracts. MT levels also increased significantly in a concentration-dependent manner when cells were exposed only to Cd. Results suggest this human bioassay and the MT

  4. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    SciTech Connect

    Nakajima, Hideaki . E-mail: hnakajim@ims.u-tokyo.ac.jp; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-02-03

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

  5. HIG-82: an established cell line from rabbit periarticular soft tissue, which retains the "activatable" phenotype.

    PubMed

    Georgescu, H I; Mendelow, D; Evans, C H

    1988-10-01

    We have isolated a continuous cell line from soft tissue lining the knee joints of rabbits. Designated HIG-82, this line was produced by spontaneous establishment of an aging, late-passage culture of primary cells. Like unpassaged, primary cells, HIG-82 cells can be activated by a number of stimuli, including phorbol myristate acetate (PMA), interleukin-1 (IL-1), and the endocytosis of latex beads. Activated cells secrete collagenase, gelatinase, caseinase (stromelysin), and prostaglandin E2 (PGE2) into their culture medium. Pseudodiploid, HIG-82 cells combine a high plating efficiency with a doubling time of approximately 24 h. As primary tissue of this origin is difficult to obtain in large quantities and shows cellular heterogeneity, the HIG-82 cell line should facilitate research into the biology and biochemistry of the fibroblastic cells that line the diarthrodial joints of mammals. Such cells are likely to be important in the pathophysiology of various arthritides.

  6. Structure of retroviral RNAs produced by cell lines derived from spontaneous lymphomas of AKR mice.

    PubMed Central

    Pedersen, F S; Crowther, R L; Hays, E F; Nowinski, R C; Haseltine, W A

    1982-01-01

    The retrovirus expression of eight independent lymphoid cell lines derived from spontaneous thymomas of AKR mice was investigated. The RNase T1 fingerprints of viral 70S RNA produced by these cell lines were compared with genome structures of the non-leukemogenic Akv virus and with two types of cloned leukemogenic viruses derived from one of the thymoma cell lines. Viral RNAs from three cell lines, SL3, 4, and 7, were indistinguishable from one another. The fingerprint patterns indicated that these cell lines produce equal amounts of two prototype, leukomogenic SL viruses that were previously isolated from the SL3 cell line. Viral RNA produced by the SL1 and SL2 cell lines contained similar components, but at a different ratio. Two other cell lines (SL5 and SL11) produced viral RNAs that resemble those of AKR mink cell focus-forming viruses. One additional line, SL9, produced viral RNA of a novel structure. The complex pattern of viral RNA expression observed for these lymphoid cell lines can be interpreted in terms of recombination among three types of endogenous viral sequences: the Akv virus, a xenotropic virus, and an SL (for spontaneous leukemia) virus. Images PMID:7086955

  7. Characterization of novel low passage primary and metastatic colorectal cancer cell lines

    PubMed Central

    Boot, Arnoud; van Eendenburg, Jaap; Crobach, Stijn; Ruano, Dina; Speetjens, Frank; Calame, Jan; Oosting, Jan; Morreau, Hans; van Wezel, Tom

    2016-01-01

    In vitro models are essential to understanding the molecular characteristics of colorectal cancer (CRC) and the testing of therapies for CRC. Many efforts to establish and characterize primary CRC cell lines have been published, most describing a small number of novel cell lines. However, there remains a lack of a large panel of uniformly established and characterized cell lines. To this end we established 20 novel CRC cell lines, of which six were derived from liver metastases. Genetic, genomic and transcriptomic profiling was performed in order to characterize these new cell lines. All data are made publically available upon publication. By combining mutation profiles with CNA and gene expression profiles, we generated an overall profile of the alterations in the major CRC-related signaling pathways. The combination of mutation profiles with genome, transcriptome and methylome data means that these low passage cell lines are among the best characterized of all CRC cell lines. This will allow researchers to select model cell lines appropriate to specific experiments, facilitating the optimal use of these cell lines as in vitro models for CRC. All cell lines are available for further research. PMID:26894854

  8. Selective migration of neuralized embryonic stem cells to stem cell factor and media conditioned by glioma cell lines

    PubMed Central

    Serfozo, Peter; Schlarman, Maggie S; Pierret, Chris; Maria, Bernard L; Kirk, Mark D

    2006-01-01

    Background Pluripotent mouse embryonic stem (ES) cells can be induced in vitro to become neural progenitors. Upon transplantation, neural progenitors migrate toward areas of damage and inflammation in the CNS. We tested whether undifferentiated and neuralized mouse ES cells migrate toward media conditioned by glioma cell lines (C6, U87 & N1321) or Stem Cell Factor (SCF). Results Cell migration assays revealed selective migration by neuralized ES cells to conditioned media as well as to synthetic SCF. Migration of undifferentiated ES cells was extensive, but not significantly different from that of controls (Unconditioned Medium). RT-PCR analysis revealed that all the three tumor cell lines tested synthesized SCF and that both undifferentiated and neuralized ES cells expressed c-kit, the receptor for SCF. Conclusion Our results demonstrate that undifferentiated ES cells are highly mobile and that neural progenitors derived from ES cells are selectively attracted toward factors produced by gliomas. Given that the glioma cell lines synthesize SCF, SCF may be one of several factors that contribute to the selective migration observed. PMID:16436212

  9. Integromic analysis of the NCI-60 cancer cell lines.

    PubMed

    Weinstein, John N

    2004-01-01

    Microarray-based transcript profiling has become exceedingly popular, particularly for breast cancer. However, other 'omic' profiling technologies at the DNA, RNA, protein, functional, and pharmacological levels are also becoming increasingly practical. We define 'integromics' as the melding of such diverse types of data from different experimental platforms. The whole can sometimes be more than the sum of its parts. We describe here a set of integromic studies in which we have profiled the 60 human cancer cell lines (the NCI-60) used by the National Cancer Institute to screen >100,000 chemical compounds over the last 13 years. Patterns of potency in the screen can be mapped into molecular structures of the compounds or into molecular characteristics of the cells. Here we discuss conceptual and experimental aspects of the profiling, as well as a number of bioinformatic computer programs (CIMminer, MedMiner, MatchMiner, and GoMiner) that we have developed for biological interpretation of the profiles. As briefly reviewed here, we have used the combination of NCI-60 data types to identify markers for distinguishing tumor types and to obtain pharmacogenomic clues for possible individualization of a cancer therapy. PMID:15687693

  10. 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.

  11. The endocannabinoid system in the human granulosa cell line KGN.

    PubMed

    Ernst, Jana; Grabiec, Urszula; Greither, Thomas; Fischer, Bernd; Dehghani, Faramarz

    2016-03-01

    Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions. Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis. PMID:26773729

  12. 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

  13. 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.

  14. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    PubMed

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide. PMID:27241584

  15. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    PubMed

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

  16. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.

    PubMed

    Martin, G R

    1981-12-01

    This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

  17. Absence of keratins 8 and 18 expression in rodent epithelial cell lines associates with keratin gene mutation and DNA methylation: cell line selective effects on cell invasion

    PubMed Central

    Omary, M. Bishr

    2016-01-01

    Epithelial-mesenchymal transition (EMT) in carcinoma is associated with dramatic up-regulation of vimentin and down-regulation of the simple-type keratins 8 and 18 (K8/K18), but the mechanisms of these changes are poorly understood. We demonstrate that two commonly-studied murine (CT26) and rat (IEC-6) intestinal cell lines have negligible K8/K18 but high vimentin protein expression. Proteasome inhibition led to a limited increase in K18 but not K8 stabilization, thereby indicating that K8/K18 absence is not due, in large part, to increased protein turnover. CT26 and IEC-6 cells had <10% of normal K8/K18 mRNA and exhibited decreased mRNA stability, with K8 being higher in IEC-6 versus CT26 and K18 being higher in CT26 versus IEC-6 cells. Keratin gene sequencing showed that KRT8 in CT26 cells had a 21-nucleotide deletion while K18 in IEC-6 cells had a 9-amino acid in-frame insertion. Furthermore, the KRT8 promoter in CT26 and the KRT18 promoter in IEC-6 are hypermethylated. Inhibition of DNA methylation using 5-azacytidine increased K8 or K18 in some but all the tested rodent epithelial cell lines. Restoring K8 and K18 by lentiviral transduction reduced CT26 but not IEC-6 cell matrigel invasion. K8/K18 re-introduction also decreased E-cadherin expression in IEC-6 but not CT26 cells, suggesting that the effect of keratin expression on epithelial to mesenchymal transition is cell-line dependent. Therefore, some commonly utilized rodent epithelial cell lines, unexpectedly, manifest barely detectable keratin expression but have high levels of vimentin. In the CT26 and IEC-6 intestinal cell lines, keratin expression correlates with keratin gene insertion or deletion and with promoter methylation, which likely suppress keratin transcription or mRNA stability. PMID:25882495

  18. Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

    PubMed Central

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

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

  19. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  20. 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.

  1. 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.

  2. Evaluation of effect of triterpenes and limonoids on cell growth, cell cycle and apoptosis in human tumor cell line.

    PubMed

    Cazal, Cristiane M; Choosang, Kantima; Severino, Vanessa Gisele P; Soares, Marcio S; Sarria, Andre Lucio F; Fernandes, Joao B; Silva, Maria Fatima G F; Vieira, Paulo Cezar; Pakkong, Pannee; Almeida, Gabriela M; Vasconcelos, M Helena; Nascimento, Maria S J; Pinto, Madalena M M

    2010-12-01

    Six triterpenes and eight limonoids were evaluated for their capacity to inhibit the growth of three human tumour cell lines, breast adenocarcinoma (MCF-7), non-small cell lung cancer (NCI-H460) and melanoma (A375-C5). The mechanisms involved in the observed cell growth arrest of the four most potent compounds were carried out by studying their effect in cell cycle profile and programmed cell death. The results showed that one triterpene (odoratol) and two limonoids (gedunin and cedrelone) caused cell cycle arrest while only the limonoids gedunin and cedrelone were found to be very potent inducers of apoptosis. PMID:21269253

  3. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  4. CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

    PubMed Central

    Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

    2016-01-01

    Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

  5. Expression of HOX genes in acute leukemia cell lines with and without MLL translocations.

    PubMed

    Quentmeier, Hilmar; Dirks, Wilhelm G; Macleod, Roderick A F; Reinhardt, Julia; Zaborski, Margarete; Drexler, Hans G

    2004-03-01

    In primary cells from acute leukemia patients, expression of the genes MEIS1, HOXA5, HOXA7 and HOXA9 has been reported to be correlated with the occurrence of MLL translocations. It was our aim to find out whether MLL mutant (MLLmu) and MLL wild-type (MLLwt) acute leukemia-derived cell lines might likewise be discriminated on the basis of HOX gene expression. Southern blot analysis, performed to verify the MLL status of the cells, showed that NOMO-1 was the only cell line not tested previously carrying a rearranged MLL gene. Fluorescence in situ hybridization analysis demonstrated that this cell line exhibited a reciprocal t(9;11)(q23;p22). Sequencing of RT-PCR products thereof identified unique MLL exon 10/AF-9 exon 5 fusion transcripts. We divided the acute leukemia-derived cell lines (n = 37) according to the results of Southern blot analysis into MLLmu (n = 19) and MLLwt (n = 18). Expression of HOX genes was then analyzed by applying reverse transcriptase-polymerase chain reaction, Northern and Western blot analyses. Acute myeloid leukemia (AML) cell lines expressed the HOX genes significantly more often than acute lymphoblastic (ALL) cell lines. In ALL, cells with MLL translocations expressed the genes 4 times more often than MLLwt cells. Most distinct was the correlation between MLL status and MEIS1 expression in ALL-derived cell lines: 8/8 MLLmu but 0/10 MLLwt cell lines expressed MEIS1. Northern and Western blot analysis confirmed that also HOXA9 and FLT3 were significantly more often and stronger expressed in MLLmu than in MLLwt ALL cell lines. These results suggest that MLL aberrations may regulate MEIS1 and HOXA9 gene expression in ALL-derived cell lines, while AML-derived cell lines express these genes independently of the MLL status. PMID:15160920

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

    PubMed

    Dai, Haiping; Ehrentraut, Stefan; 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

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

    PubMed

    Dai, Haiping; Ehrentraut, Stefan; 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

  8. 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

  9. Cell and membrane lipid analysis by proton magnetic resonance spectroscopy in five breast cancer cell lines.

    PubMed

    Le Moyec, L; Tatoud, R; Eugène, M; Gauvillé, C; Primot, I; Charlemagne, D; Calvo, F

    1992-10-01

    The lipid composition of five human breast cancer cell lines (MCF-7, T47D, ZR-75-1, SKBR3 and MDA-MB231) was assessed by proton magnetic resonance spectroscopy (MRS) in whole cells and membrane-enriched fractions. The proportions of the three main lipid resonances in 1D spectra were different for each cell line. These resonances included mobile methyl and methylene functions from fatty acids of triglycerides and phospholipids and N-trimethyl from choline of phospholipids. T47D and ZR-75-1 cells presented a high methylene/methyl ratio (6.02 +/- 0.35 and 6.28 +/- 0.90). This ratio was significantly lower for SKBR3, MCF-7 and MDA-MB231 cells (2.76 +/- 0.22, 2.27 +/- 0.57 and 1.39 +/- 0.39). The N-trimethyl/methyl ratio was high for MDA-MB231 and SKBR3 cells (1.38 +/- 0.54 and 0.86 +/- 0.32), but lower for MCF-7, T47D and ZR-75-1 cells (0.49 +/- 0.11, 0.16 +/- 0.07 and 0.07 +/- 0.03). 2D COSY spectra confirmed these different proportions in mobile lipids. From 1D spectra obtained on membrane preparations, T47D and ZR-75-1 were the only cell lines to retain a signal from mobile methylene functions. These differences might be related to the heterogeneity found for several parameters of these cells (tumorigenicity, growth rate, hormone receptors); an extended number of cases from fresh samples might enable clinical correlations. PMID:1329906

  10. Establishment of an indicator cell line for monitoring bovine immunodeficiency virus infection and inhibitor susceptibility.

    PubMed

    Yao, Xue; Su, Yang; Liu, Chang; Tan, Juan; Liu, Li; Geng, Yun-Qi; Qiao, Wen-Tao

    2010-01-01

    Indicator cell lines are useful biological tools for monitoring virus infection. In order to monitor infection with bovine immunodeficiency virus (BIV) in vitro, an indicator cell line derived from baby hamster kidney cells which contains integrated copies of an enhanced green fluorescent protein gene driven by the BIV long terminal repeat was constructed. The BIV indicator cell line, designated BIVE, can detect BIV infection more easily and effectively than the established method, which involves the observation of cell cytopathic effects. Furthermore, viral titration using an assay based on the indicator cells is 100 times more sensitive than the assay based on cytopathic effect. The finding that BIV can infect the hamster cell line expands the known host range of BIV in vitro. The BIV indicator cell line could also be used for the evaluation of the inhibitory effect of antiviral agents. The fusion inhibition effect of the heptad repeat 2 region of the BIV envelope protein could also be quantified.

  11. Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

    PubMed Central

    Ortega, Francisco G; Fernández-Baldo, Martín A; Fernández, Jorge G; Serrano, María J; Sanz, María I; Diaz-Mochón, Juan J; Lorente, José A; Raba, Julio

    2015-01-01

    In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors. PMID:25844035

  12. Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro.

    PubMed

    Matsumoto, Asako; Harada, Hidemitsu; Saito, Masahiro; Taniguchi, Akiyoshi

    2011-01-01

    Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal