Sample records for h9c2 cell line

  1. Leptin affects adenylate cyclase activity in H9c2 cardiac cell line: effects of short- and long-term exposure.

    PubMed

    Illiano, Gennaro; Naviglio, Silvio; Pagano, Mario; Spina, Annamaria; Chiosi, Emilio; Barbieri, Michelangela; Paolisso, Giuseppe

    2002-07-01

    Leptin has been hypothesized to be a pathophysiologic link between obesity and cardiovascular diseases. Because the adenylate cyclase (AC) system is a main effector of beta-adrenergic receptors and leptin has been shown to modulate AC activity in other cell lines, a leptin impact on cardiac AC activity was hypothesized. Therefore, acute and chronic effects of leptin on a rat cardiac cell line (H9c2) were investigated. Leptin affected both basal (+ 13% at 30 min and -16.4% after 18 h v untreated cells) and catecholamine-stimulated AC activity (isoproterenol + leptin at 30 min or 18 h was +21% v untreated cells; norepinephrine + leptin at 30 min was +38.8% v untreated cells; and norepinephrine + leptin at 18 h was +6% v untreated cells). Thus, long-term leptin treatment was associated with a reduced AC activity and a different responsiveness to catecholamines. The AC activity on leptin treatment was accompanied by changes in levels of proteins structurally or functionally related to AC complex (AC, Gas, Gai, p21-ras). These data indicate that the AC complex is profoundly affected at more than one level by leptin treatment in the H9c2 cardiac cell line. Differences in AC activity after short- and long-term exposure to leptin and the interaction between leptin and catecholamine might provide further insight to the understanding of the development of hypertension and congestive heart failure in obese patients. PMID:12118913

  2. 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; Coďsson, 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

  3. Herba leonurine attenuates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells

    Microsoft Academic Search

    Hong Xin; Xin Hua Liu; Yi Zhun Zhu

    2009-01-01

    Doxorubicin (DOX) is a highly effective antineoplastic drug. However, DOX-induced apoptosis in cardiomyocytes leads to irreversible degenerative cardiomyopathy and heart failure, which limits DOX clinical application. Leonurine is a special alkaloid for Herba leonuri, a traditional herb with cardioprotective effects. In current study, we investigated possible protective effects of Leonurine against DOX-induced cardiomyopathy in H9c2 cells. DOX-injured H9c2 cell model

  4. Synthesis and cardio protective biological applications of glucodendrimers by H9C2 cell studies.

    PubMed

    Rajakumar, Perumal; Anandhan, Ramasamy; Vadla, Gangadhara Prasadachari; Vellaichamy, Elangovan

    2014-01-01

    Novel glucodendrimers scaffolds containing ?-d-glucopyranoside at the surface and triazole as bridging unit have been synthesized. Cardiomyocytes were exposed to normal and high glucose level in the presence and absence of glucodendrimers. Cytotoxicity studies were also carried out and the expression of metalloproteinases mainly MMP-2 and 9 was confirmed with gelatine zymography and RT-PCR gene expression studies. Cardio protective efficiency of the synthesized glucodendrimers against high glucose induced toxicity on mettalloproteinase-2 and 9 and also on H9C2 cell lines revealed that higher generation glucodendrimers 6 and 8 are more effective than the lower generation glucodendrimers. PMID:24274524

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

    E-print Network

    Hammock, Bruce D.

    PPARd signaling mediates the cytotoxicity of DHA in H9c2 cells Victor Samokhvalov a,1 , Igor T S Investigated the mechanisms of DHA mediated cytotoxicity in H9c2 cardiac cells. DHA treatment decreased cell viability and triggered apoptosis in H9c2 cells. DHA induced cell death involved PPARd signaling. DHA

  6. Herba leonurine attenuates doxorubicin-induced apoptosis in H9c2 cardiac muscle cells.

    PubMed

    Xin, Hong; Liu, Xin Hua; Zhu, Yi Zhun

    2009-06-10

    Doxorubicin (DOX) is a highly effective antineoplastic drug. However, DOX-induced apoptosis in cardiomyocytes leads to irreversible degenerative cardiomyopathy and heart failure, which limits DOX clinical application. Leonurine is a special alkaloid for Herba leonuri, a traditional herb with cardioprotective effects. In current study, we investigated possible protective effects of Leonurine against DOX-induced cardiomyopathy in H9c2 cells. DOX-injured H9c2 cell model was made by application of 2 microM DOX. Leonurine was added to cells 2 h before DOX treatment. Pre-treated with Leonurine could attenuate DOX-induced apoptotic death of H9c2 cell, reduce MDA formation and intracellular Ca2+ overload. Leonurine also attenuated DOX-induced high expression of Bax, increased Bcl-2 expression in both protein and mRNA level. Myocardial mitochondrion is the target organelle of DOX-induced toxicity in cardiomyocytes. Leonurine moderated the dissipation of mitochondrial membrane potential (DeltaPsim) caused by DOX treatment. Our results indicated that Leonurine attenuated DOX-induced apoptosis in H9c2 cell by increasing anti-oxidant, anti-apoptotic ability and protecting mitochondrial function. PMID:19356731

  7. Vital imaging of H9c2 myoblasts exposed to tert-butylhydroperoxide – characterization of morphological features of cell death

    PubMed Central

    Sardăo, Vilma A; Oliveira, Paulo J; Holy, Jon; Oliveira, Catarina R; Wallace, Kendall B

    2007-01-01

    Background When exposed to oxidative conditions, cells suffer not only biochemical alterations, but also morphologic changes. Oxidative stress is a condition induced by some pro-oxidant compounds, such as by tert-butylhydroperoxide (tBHP) and can also be induced in vivo by ischemia/reperfusion conditions, which is very common in cardiac tissue. The cell line H9c2 has been used as an in vitro cellular model for both skeletal and cardiac muscle. Understanding how these cells respond to oxidative agents may furnish novel insights into how cardiac and skeletal tissues respond to oxidative stress conditions. The objective of this work was to characterize, through vital imaging, morphological alterations and the appearance of apoptotic hallmarks, with a special focus on mitochondrial changes, upon exposure of H9c2 cells to tBHP. Results When exposed to tBHP, an increase in intracellular oxidative stress was detected in H9c2 cells by epifluorescence microscopy, which was accompanied by an increase in cell death that was prevented by the antioxidants Trolox and N-acetylcysteine. Several morphological alterations characteristic of apoptosis were noted, including changes in nuclear morphology, translocation of phosphatidylserine to the outer leaflet of the cell membrane, and cell blebbing. An increase in the exposure period or in tBHP concentration resulted in a clear loss of membrane integrity, which is characteristic of necrosis. Changes in mitochondrial morphology, consisting of a transition from long filaments to small and round fragments, were also detected in H9c2 cells after treatment with tBHP. Bax aggregates near mitochondrial networks were formed after short periods of incubation. Conclusion Vital imaging of alterations in cell morphology is a useful method to characterize cellular responses to oxidative stress. In the present work, we report two distinct patterns of morphological alterations in H9c2 cells exposed to tBHP, a pro-oxidant agent frequently used as model to induce oxidative stress. In particular, dynamic changes in mitochondrial networks could be visualized, which appear to be centrally involved in how these cells respond to oxidative stress. The data also indicate that the cause of H9c2 cell death following tBHP exposure is increased intracellular oxidative stress. PMID:17362523

  8. N-Acetylsphingosine stimulates phosphatidylglycerolphosphate synthase activity in H9c2 cardiac cells.

    PubMed Central

    Xu, F Y; Kelly, S L; Hatch, G M

    1999-01-01

    Cardiolipin and phosphatidylglycerol biosynthesis were examined in H9c2 cells incubated with short-chain ceramides. Incubation of cells with N-acetylsphingosine or N-hexanoylsphingosine stimulated [1, 3-3H]glycerol incorporation into phosphatidylglycerol and cardiolipin, with N-acetylsphingosine having the greater effect. The mechanism for the ceramide-mediated stimulation of de novo phosphatidylglycerol and cardiolipin biosynthesis appeared to be an increase in the activity of phosphatidylglycerolphosphate synthase, the committed step of phosphatidylglycerol and cardiolipin biosynthesis. The presence of the potent protein phosphatase inhibitors calyculin A or okadaic acid attenuated the N-acetylsphingosine-mediated stimulation of phosphatidylglycerolphosphate synthase activity and of phosphatidylglycerol and cardiolipin biosynthesis, indicating the involvement of a ceramide-activated protein phosphatase(s). The presence of 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) stimulated enzyme activity and [1,3-3H]glycerol incorporation into phosphatidylglycerol and cardiolipin. The effects of CPT-cAMP and N-acetylsphingosine on phosphatidylglycerol and cardiolipin biosynthesis and on phosphatidylglycerolphosphate synthase activity were additive. Phosphatidylglycerol biosynthesis from sn-[14C]glycerol 3-phosphate in permeabilized H9c2 cells was stimulated by preincubation with N-acetylsphingosine, and this was attenuated by okadaic acid. N-Acetylsphingosine treatment of cells elevated mitochondrial phospholipase A2 activity. Since the pool sizes of phosphatidylglycerol and cardiolipin were unaltered in these cells, the observed increase in phosphatidylglycerolphosphate synthase activity may be a compensatory mechanism for the N-acetylsphingosine-mediated elevation of mitochondrial phospholipase A2 activity. Finally, addition of tumour necrosis factor alpha to H9c2 cells resulted in an elevation of both phosphatidylglycerolphosphate synthase and phospholipase A2 activities. The results suggest that phosphatidylglycerol and cardiolipin metabolism in H9c2 cells may be regulated by intracellular ceramide signalling. PMID:9895291

  9. Acetaminophen Induced Cytotoxicity and Altered Gene Expression in Cultured Cardiomyocytes of H9C2 Cells

    PubMed Central

    Jin, Seon Mi

    2012-01-01

    Objectives Hepatotoxicity of acetaminophen has been widely studied. However, the adverse effects on the heart have not been sufficiently evaluated. This study was performed to investigate cytotoxicity and alterations of gene expression in cultured cardiomyocytes (H9C2 cells) after exposure to acetaminophen. Methods H9C2 cells were incubated in a 10 mM concentration of acetaminophen for the designated times (6, 12, and 24 hours), and cytotoxicity was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Alteration of gene expression was observed by microarray analysis, and RT-PCR was performed for the three representative oxidative stress-related genes at 24 hours after treatment. Results It revealed that acetaminophen was toxic to cardiomyocytes, and numerous critical genes were affected. Induced genes included those associated with oxidative stress, DNA damage, and apoptosis. Repressed genes included those associated with cell proliferation, myocardial contraction, and cell shape control. Conclusions These findings provide the evidences of acetaminophen-induced cytotoxicity and changes in gene expression in cultured cardiomyocytes of H9C2 cells. PMID:22639738

  10. 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 (?M) 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. PMID:24968061

  11. Cardioprotective Effect of Propofol against Oxygen Glucose Deprivation and Reperfusion Injury in H9c2 Cells

    PubMed Central

    Zhao, Dandan; Li, Qing; Huang, Qiuping; Li, Xuguang; Yin, Min; Wang, Zejian; Hong, Jiang

    2015-01-01

    Background. The intravenous anesthetic propofol is reported to be a cardioprotective agent against ischemic-reperfusion injury in the heart. However, the regulatory mechanism still remains unclear. Methods. In this study, we used H9c2 cell line under condition of oxygen glucose deprivation (OGD) followed by reperfusion (OGD/R) to induce in vitro cardiomyocytes ischemia-reperfusion injury. Propofol (5, 10, and 20??M) was added to the cell cultures before and during the OGD/R phases to investigate the underlying mechanism. Results. Our data showed that OGD/R decreased cell viability, and increased lactate dehydrogenase leakage, and reactive oxygen species and malondialdehyde production in H9c2 cells, all of which were significantly reversed by propofol. Moreover, we found that propofol increased both the activities and protein expressions of superoxide dismutase and catalase. In addition, propofol increased FoxO1 expression in a dose-dependent manner and inhibited p-AMPK formation significantly. Conclusions. These results indicate that the propofol might exert its antioxidative effect through FoxO1 in H9c2 cells, and it has a potential therapeutic effect on cardiac disorders involved in oxidative stress. PMID:25821553

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

    PubMed

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

    2014-04-01

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

  13. Icariin Protects Rat Cardiac H9c2 Cells from Apoptosis by Inhibiting Endoplasmic Reticulum Stress

    PubMed Central

    Zhang, Qiufang; Li, Hongliang; Wang, Shanshan; Liu, Ming; Feng, Yibin; Wang, Xuanbin

    2013-01-01

    Endoplasmic reticulum stress (ERS) is one of the mechanisms of apoptotic cell death. Inhibiting the apoptosis induced by ERS may be a novel therapeutic target in cardiovascular diseases. Icariin, a flavonoid isolated from Epimedium brevicornum Maxim, has been demonstrated to have cardiovascular protective effects, but its effects on ERS are unknown. In the present study, we focused on icariin and investigated whether it might protect the cardiac cell from apoptosis via inhibition of ERS. In H9c2 rat cardiomyoblast cells, pretreatment of icariin significantly inhibited cell apoptosis by tunicamycin, an ERS inducer. Icariin also decreased generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential and activation of caspase-3. Moreover, icariin inhibited upregulation of endoplasmic reticulum markers, GRP78, GRP94 and CHOP, elicited by tunicamycin. These results indicated that icariin could protect H9c2 cardiomyoblast cells from ERS-mitochondrial apoptosis in vitro, the mechanisms may be associated with its inhibiting of GRP78, GRP94 and CHOP and decreasing ROS generation directly. It may be a potential agent for treating cardiovascular disease. PMID:23999590

  14. 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 [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 (China); Lu Wei [Department of Chemistry and Institute of Medicinal Chemistry, East China Normal University, Shanghai, 200062 (China); Yang Bo, E-mail: yang924@zju.edu.c [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 (China); He Qiaojun, E-mail: qiaojunhe@zju.edu.c [Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058 (China); Center for Drug Safety Evaluation and Research of Zhejiang University, Hangzhou, 310058 (China)

    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.

  15. Efficiency of DNA Transfection of Rat Heart Myoblast Cells H9c2(2-1) by Either Polyethyleneimine or Electroporation

    Microsoft Academic Search

    Yu Chia Liu; Win Yu Lin; Ya Ru Jhang; Sing Hui Huang; Chean Ping Wu; Hsi Tien Wu

    2011-01-01

    Expression of exogenous DNA in vitro is significantly affected by the particular transfection method utilized. In this study,\\u000a we evaluated the efficiency of two transfection methods, chemically mediated polyethyleneimine (PEI) treatment and physically\\u000a mediated electroporation, on a rat heart myoblast cell line, H9c2(2-1). After PEI transfection of pPgk-1\\/EGFP into H9c2(2-1) cells, EGFP expression could be easily detected by fluorospectrometer after

  16. Exogenous hydrogen sulfide alleviates high glucose-induced cardiotoxicity via inhibition of leptin signaling in H9c2 cells.

    PubMed

    Zhuang, Xiao-Dong; Hu, Xun; Long, Ming; Dong, Xiao-Bian; Liu, Dong-Hong; Liao, Xin-Xue

    2014-06-01

    Hydrogen sulfide (H?S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin-p38 MAPK pathway is involved in HG-induced injury and whether exogenous H2S prevents the HG-induced insult through inhibition of the leptin-p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (?? m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ?? m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 ?M NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin-p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H?S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin-p38 MAPK pathway. PMID:24687304

  17. Protein kinase C inhibitors attenuate protective effect of high glucose against hypoxic injury in H9c2 cardiac cells.

    PubMed

    Moon, C H; Jung, Y S; Kim, M H; Park, R M; Lee, S H; Baik, E J

    2000-12-01

    In this study, we demonstrated that PKC inhibitors significantly attenuated the cardioprotective effect produced by high-glucose (22 mM) treatment for 48 h against hypoxic injury in H9c2 cardiac cells. PKC activators mimicked the cardioprotective effect of high glucose. These results suggest a possible role of PKC activation in high-glucose--induced protection. PMID:11173560

  18. Sulforaphane prevents doxorubicin-induced oxidative stress and cell death in rat H9c2 cells

    PubMed Central

    LI, BO; KIM, DO SUNG; YADAV, RAJ KUMAR; KIM, HYUNG RYONG; CHAE, HAN JUNG

    2015-01-01

    Sulforaphane, a natural isothiocyanate compound found in cruciferous vegetables, has been shown to exert cardioprotective effects during ischemic heart injury. However, the effects of sulforaphane on cardiotoxicity induced by doxorubicin are unknown. Thus, in the present study, H9c2 rat myoblasts were pre-treated with sulforaphane and its effects on cardiotoxicity were then examined. The results revealed that the pre-treatment of H9c2 rat myoblasts with sulforaphane decreased the apoptotic cell number (as shown by trypan blue exclusion assay) and the expression of pro-apoptotic proteins (Bax, caspase-3 and cytochrome c; as shown by western blot analysis and immunostaining), as well as the doxorubicin-induced increase in mitochondrial membrane potential (measured by JC-1 assay). Furthermore, sulforaphane increased the mRNA and protein expression of heme oxygenase-1 (HO-1, measured by RT-qPCR), which consequently reduced the levels of reactive oxygen species (ROS, measured using MitoSOX Red reagent) in the mitochondria which were induced by doxorubicin. The cardioprotective effects of sulforaphane were found to be mediated by the activation of the Kelch-like ECH-associated protein 1 (Keap1)/NF-E2-related factor-2 (Nrf2)/antioxidant-responsive element (ARE) pathway, which in turn mediates the induction of HO-1. Taken together, the findings of this study demonstrate that sulforaphane prevents doxorubicin-induced oxidative stress and cell death in H9c2 cells through the induction of HO-1 expression. PMID:25936432

  19. Protective effects of extendin?4 on hypoxia/reoxygenation?induced injury in H9c2 cells.

    PubMed

    Lu, Kai; Chang, Guanglei; Ye, Lin; Zhang, Peng; Li, Yong; Zhang, Dongying

    2015-08-01

    Glucagon-like peptide-1 (GLP-1) analogues are likely to exert cardioprotective effects via balancing the energy metabolism in cardiomyocytes following ischemic or hypoxic insults. The present study aimed to explore the protective effects and mechanism of exendin?4, a GLP?1 analogue, on cardiomyocyte glucose uptake using an in vitro model of hypoxia/reoxygenation (H/R) of H9c2 cardiomyocyte cells. Pre?treatment with exendin?4 (200 nM) prior to H/R increased the cell viability, decreased cell apoptosis, enhanced cardiomyocyte glucose uptake and increased the production of adenosine triphosphate. Exendin?4 also decreased the levels of lactate dehydrogenase and creatine kinase?MB in the culture medium. Furthermore, the activity of carnitine palmitoyltransferase?1 in the H9c2 cells was decreased, while the activity of phosphofructokinase?1 was increased following exendin?4 treatment. Moreover, pre?treatment with exendin?4 increased the expression of p38 mitogen?activated protein kinase (p38MAPK) ? and translocation of glucose transporter?1 in H9c2 cells subjected to H/R. However, these effects were attenuated by the p38MAPK inhibitors BIRB796 and SB203580. The results suggested that exendin?4 exerted significant cardioprotective effects against H/R?induced cell injury and restored the metabolic imbalance of cardiomyocytes by activating the p38MAPK signaling pathway in the H9c2 cell model. Importantly, p38MAPK?, one subunit of p38MAPK, may have the most important function in this process. The results of the present study may be helpful in the development of novel drugs to treat patients with coronary heart disease. PMID:25936390

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

    PubMed

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

    2011-05-01

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

  1. Upregulation of arginase activity contributes to intracellular ROS production induced by high glucose in H9c2 cells

    PubMed Central

    Zhou, Lu; Sun, Chuan-Bo; Liu, Chao; Fan, Yue; Zhu, Hong-Yi; Wu, Xiao-Wei; Hu, Liang; Li, Qing-Ping

    2015-01-01

    Arginase is upregulated in some tissues under diabetes states. Arginase can compete with nitroxide synthase (NOS) for the common substrate L-arginine and thus increases oxidative stress by NOS uncoupling. We want to analyze whether arginase is upregulated and contribute to oxidative stress in H9c2 cells during high glucose treatment. H9c2 cells were cultured in normal or high glucose DMEM. Arginase activity increased in parallel with increased cell death and oxidative stress. Arginase inhibitor N ?-hydroxy-nor-l-arginine (nor-NOHA) and NOS inhibitor N ?-nitro-l-arginine methyl ester (L-NAME) could reverse these effects. Despite of upregulated NOS activity, NO production was impaired which could be preserved by nor-NOHA, suggesting a decreased substrate availability of NOS due to increased arginase activity. L-arginine supplementation decreased superoxide production while it could not protect cells from death. Upregulated arginase activity in H9c2 treated with high glucose can cause NOS uncoupling and subsequently reactive oxygen species augmentation and cell death. These findings suggest that arginase will be a novel therapeutic target for treatment of diabetic cardiomyopathy.

  2. Upregulation of arginase activity contributes to intracellular ROS production induced by high glucose in H9c2 cells.

    PubMed

    Zhou, Lu; Sun, Chuan-Bo; Liu, Chao; Fan, Yue; Zhu, Hong-Yi; Wu, Xiao-Wei; Hu, Liang; Li, Qing-Ping

    2015-01-01

    Arginase is upregulated in some tissues under diabetes states. Arginase can compete with nitroxide synthase (NOS) for the common substrate L-arginine and thus increases oxidative stress by NOS uncoupling. We want to analyze whether arginase is upregulated and contribute to oxidative stress in H9c2 cells during high glucose treatment. H9c2 cells were cultured in normal or high glucose DMEM. Arginase activity increased in parallel with increased cell death and oxidative stress. Arginase inhibitor N ?-hydroxy-nor-l-arginine (nor-NOHA) and NOS inhibitor N ?-nitro-l-arginine methyl ester (L-NAME) could reverse these effects. Despite of upregulated NOS activity, NO production was impaired which could be preserved by nor-NOHA, suggesting a decreased substrate availability of NOS due to increased arginase activity. L-arginine supplementation decreased superoxide production while it could not protect cells from death. Upregulated arginase activity in H9c2 treated with high glucose can cause NOS uncoupling and subsequently reactive oxygen species augmentation and cell death. These findings suggest that arginase will be a novel therapeutic target for treatment of diabetic cardiomyopathy. PMID:26045778

  3. Rho-kinase inhibitor Y-27632 attenuates arsenic trioxide toxicity in H9c2 cardiomyoblastoma cells.

    PubMed

    Bessho, Marie; Aki, Toshihiko; Funakoshi, Takeshi; Unuma, Kana; Noritake, Kanako; Kato, Chizuru; Uemura, Koichi

    2013-09-01

    The purpose of this study is to examine the molecular mechanism underlying the toxicity of arsenic trioxide (ATO) in cardiac cells. H9c2 rat cardiomyoblastoma cells undergo apoptosis during exposure to the concentrations of ATO > 10 ?M for 24 h. The process is accompanied by the activation of caspases and is suppressed by the pan-caspase inhibitor z-VAD. Since ATO-induced H9c2 cell death is suppressed by Rho-kinase (ROCK) inhibitor Y-27632, but not by any antioxidants tested, apoptosis by ATO seems to be initiated through a ROCK-dependent and reactive oxygen species-independent mechanism. During the execution of apoptosis by ATO, the induction of autophagy is also observed. Importantly, autophagy is accelerated in cells treated with ATO plus Y-27632, although Y-27632 alone does not induce autophagy. The cytoprotective effect of Y-27632 against ATO toxicity is abrogated by the co-administration of an autophagy inhibitor, 3-methyladenine, suggesting that autophagy contributes to the cytoprotection by Y-27632. Taken together, the data indicate that the activation of ROCK is required for apoptotic H9c2 cardiomyoblastoma cell death by ATO, and that the ROCK inhibition not only inhibits caspase-dependent apoptotic machinery, but also causes a rise in the cytoprotective autophagy processes during ATO exposure. PMID:23532581

  4. Oxidative Stress Induces DNA Fragmentation and Caspase Activation Via the c-Jun NH 2-terminal Kinase Pathway in H9c2 Cardiac Muscle Cells

    Microsoft Academic Search

    Neil A. Turner; Fen Xia; Gohar Azhar; Xiaomin Zhang; Lixin Liu; Jeanne Y Wei

    1998-01-01

    The aim of this study was to test the hypothesis that oxidative stress induces apoptosis in the H9c2 cardiac muscle cell line, and that signaling via mitogen-activated protein kinase (MAPK) pathways is involved. Three forms of oxidative stress were utilized: the superoxide generator menadione; hydrogen peroxide; or simulated ischemia followed by reperfusion. Relatively low concentrations of menadione (10?m) or H2O2(250?m)

  5. 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. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25858861

  6. Naringin inhibits ROS-activated MAPK pathway in high glucose-induced injuries in H9c2 cardiac cells.

    PubMed

    Chen, Jingfu; Guo, Runmin; Yan, Hai; Tian, Lihong; You, Qiong; Li, Shanghai; Huang, Ruina; Wu, Keng

    2014-04-01

    Naringin, an active flavonoid isolated from citrus fruit extracts, exhibits biological and pharmacological properties, such as antioxidant activity and antidiabetic effect. Mitogen-activated protein kinase (MAPK) signalling pathway has been shown to participate in hyperglycaemia-induced injury. The present study tested the hypothesis that naringin protects against high glucose (HG)-induced injuries by inhibiting MAPK pathway in H9c2 cardiac cells. To examine this, the cells were treated with 35 mM glucose (HG) for 24 hr to establish a HG-induced cardiomyocyte injury model. The cells were pre-treated with 80 ?M naringin for 2 hr before exposure to HG. The findings of this study showed that exposure of H9c2 cells to HG for 24 hr markedly induced injuries, as evidenced by a decrease in cell viability, increases in apoptotic cells and reactive oxygen species (ROS) production, as well as dissipation of mitochondrial membrance potential (MMP). These injuries were significantly attenuated by the pre-treatment of cells with either naringin or SB203580 (a selective inhibitor of p38 MAPK) or U0126 (a selective inhibitor of extracellular signal regulated kinase 1/2, ERK1/2) or SP600125 (a selective inhibitor of c-jun N-termanal kinase, JNK) before exposure to HG, respectively. Furthermore, exposure of cells to HG increased the phosphorylation of p38 MAPK, ERK1/2 and JNK. The increased activation of MAPK pathway was ameliorated by pre-treatment with either naringin or N-acetyl-L-cysteine (NAC), a ROS scavenger, which also reduced HG-induced cytotoxicity and apoptosis, leading to increase in cell viability and decrease in apoptotic cells. In conclusion, our findings provide new evidence for the first time that naringin protects against HG-induced injuries by inhibiting the activation of MAPK (p38 MAPK, ERK1/2 and JNK) and oxidative stress in H9c2 cells. PMID:24118820

  7. Preparation and Characterization of Selenium Incorporated Guar Gum Nanoparticle and Its Interaction with H9c2 Cells

    PubMed Central

    Soumya, Rema Sreenivasan; Vineetha, Vadavanath Prabhakaran; Reshma, Premachandran Latha; Raghu, Kozhiparambil Gopalan

    2013-01-01

    This study deals with the preparation and characterization of selenium incorporated guar gum nanoparticle (SGG), and its effect on H9c2 cardiomyoblast. Herein, nanoprecipitation techniques had been employed for the preparation of SGG nanoparticle. The prepared nanoparticle had been subjected to various types of analytical techniques like transmission electron microscopy (TEM), X-ray diffraction (XRD) and particle size analysis to confirm the characteristics of nanoparticle as well as for selenium incorporation. Physical characterization of nanoparticle showed that the size of nanoparticles increase upto ?69–173 nm upon selenium incorporation from ?41–132 nm. Then the prepared nanoparticles were evaluated for its effect on H9c2 cells. In this regard, the effect of nanoparticle on various vital parameters of H9c2 cells was studied. Parameters like cell viability, uptake of selenium incorporated guar gum nanoparticle by the cells, effect of SGG on DNA integrity, apoptosis, reactive oxygen species generation, alteration in transmembrane potential of mitochondria and cytoskeletal integrity had been investigated. Viability results showed that up to 25 nM of SGG was safe (10.31%) but beyond that it induces cytotoxicity. Cellular uptake of selenium showed that cell permeability for SGG is significantly high compared to normal selenium (7.2 nM of selenium for 25 nM SGG compared with 5.2 nM selenium for 25 nM sodium selenite). There was no apoptosis with SGG and also it protects DNA from hydroxyl radical induced breakage. Likewise no adverse effect on mitochondria and cytoskeleton was observed for 25 nM of SGG. Overall results reveal that SGG is highly suitable for biomedical research application. PMID:24098647

  8. 3,3'-Diindolylmethane attenuates cardiac H9c2 cell hypertrophy through 5'-adenosine monophosphate-activated protein kinase-?.

    PubMed

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

    2015-07-01

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

  9. Group IV cytosolic phospholipase A 2 mediates arachidonic acid release in H9c2 rat cardiomyocyte cells in response to hydrogen peroxide

    Microsoft Academic Search

    Michelle V. Winstead; Karin Killermann Lucas; Edward A. Dennis

    2005-01-01

    Damaging reactive oxygen species are released during episodes of ischemia and reperfusion. Some cellular adaptive responses are triggered to protect the injured organ, while other cascades are triggered which potentiate the damage. In these studies, we demonstrate that rat cardiomyocte H9c2 cells release arachidonic acid in response to hydrogen peroxide. In H9c2 cells, arachidonic acid release is attenuated by methyl

  10. THE INHIBITORY EFFECT OF PACLITAXEL ON (KV2.1) K(+) CURRENT IN H9c2 CELLS.

    PubMed

    Kitamura, Naoko; Sakamoto, Kazuho; Ono, Tomoyuki; Kimura, Junko

    2015-07-01

    Using the whole-cell voltage clamp technique, we investigated the effect of paclitaxel, an anticancer agent which promotes microtubule formation, on K(+) current in H9c2 cells originated from rat embryonic cardiac myocytes. Paclitaxel inhibited Kv2.1 voltage-dependent K(+) current (IKur) with ultra-rapidly activating and slowly inactivating kinetics in a concentration-dependent manner. The inhibitory effect of paclitaxel on IKur was time-dependent and more marked at 200 ms after the onset than at the beginning of the depolarizing pulse. The IC50 value of paclitaxel was 1.1 µM at 200 ms. The time-dependent inhibition suggests that paclitaxel might be an open channel blocker of Kv2.1. This inhibition of Kv2.1 may be involved in the adverse effects of paclitaxel on cardiac and neuronal cells. PMID:25994081

  11. TanshinoneIIA and Cryptotanshinone Protect against Hypoxia-Induced Mitochondrial Apoptosis in H9c2 Cells

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

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

    SciTech Connect

    Witting, Paul K. [ANZAC Research Institute, Concord Hospital, Concord, NSW (Australia)]. E-mail: pwitting@anzac.edu.au; Liao Wenqiang [Apoptosis Research Group, Heart Foundation Research Centre, School of Medical Science, Griffith University, Qld (Australia); Harris, Matthew J. [ANZAC Research Institute, Concord Hospital, Concord, NSW (Australia); Neuzil, Jiri [Apoptosis Research Group, Heart Foundation Research Centre, School of Medical Science, Griffith University, Qld (Australia); Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague (Czech Republic)

    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.

  14. Effects of Downregulation of MicroRNA-181a on H2O2-Induced H9c2 Cell Apoptosis via the Mitochondrial Apoptotic Pathway

    PubMed Central

    Wang, Lei; Huang, He; Fan, Yang; Kong, Bin; Hu, He; Hu, Ke; Guo, Jun; Mei, Yang; Liu, Wan-Li

    2014-01-01

    Glutathione peroxidase-1 (GPx1) is a pivotal intracellular antioxidant enzyme that enzymatically reduces hydrogen peroxide to water to limit its harmful effects. This study aims to identify a microRNA (miRNA) that targets GPx1 to maintain redox homeostasis. Dual luciferase assays combined with mutational analysis and immunoblotting were used to validate the bioinformatically predicted miRNAs. We sought to select miRNAs that were responsive to oxidative stress induced by hydrogen peroxide (H2O2) in the H9c2 rat cardiomyocyte cell line. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-181a in H2O2-treated H9c2 cells was markedly upregulated. The downregulation of miR-181a significantly inhibited H2O2-induced cellular apoptosis, ROS production, the increase in malondialdehyde (MDA) levels, the disruption of mitochondrial structure, and the activation of key signaling proteins in the mitochondrial apoptotic pathway. Our results suggest that miR-181a plays an important role in regulating the mitochondrial apoptotic pathway in cardiomyocytes challenged with oxidative stress. MiR-181a may represent a potential therapeutic target for the treatment of oxidative stress-associated cardiovascular diseases. PMID:24683439

  15. Effect of N-n-butyl haloperidol iodide on ROS/JNK/Egr-1 signaling in H9c2 cells after hypoxia/reoxygenation

    PubMed Central

    Zhang, Yanmei; Liao, Han; Zhong, Shuping; Gao, Fenfei; Chen, Yicun; Huang, Zhanqin; Lu, Shishi; Sun, Ting; Wang, Bin; Li, Weiqiu; Xu, Han; Zheng, Fuchun; Shi, Ganggang

    2015-01-01

    Reactive oxygen species (ROS)-induced oxidative stress in cells is an important pathophysiological process during myocardial ischemia/reperfusion (I/R) injury, and the transcription factor Egr-1 is a master switch for various damage pathways during reperfusion injury. An in vitro model of myocardial I/R injury and H9c2 cardiomyoblast cells hypoxia/reoxygenation (H/R) was used to assess whether there is abnormal intracellular ROS/JNK/Egr-1 signaling. We also assessed whether N-n-butyl haloperidol (F2), which exerts protective effects during myocardial I/R injury, can modulate this pathway. H/R induced ROS generation, JNK activation, and increased the expression of Egr-1 protein in H9c2 cells. The ROS scavengers edaravone (EDA) and N-acetyl-L-cysteine (NAC) reduced ROS level, downregulated JNK activation, and Egr-1 expression in H9c2 cells after H/R. The JNK inhibitor SP600125 inhibited Egr-1 overexpression in H9c2 cells caused by H/R. F2 could downregulate H/R-induced ROS level, JNK activation, and Egr-1 expression in H9c2 cells in a dose-dependent manner. The ROS donor hypoxanthine-xanthine oxidase (XO/HX) and the JNK activator ANISO antagonized the effects of F2. Therefore, H/R activates ROS/Egr-1 signaling pathway in H9c2 cells, and JNK activation plays an important role in this pathway. F2 regulates H/R-induced ROS/JNK/Egr-1 signaling, which might be an important mechanism by which it antagonizes myocardial I/R injury. PMID:26134032

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

    PubMed

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

    2015-03-01

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

  17. M-LDH physically associated with sarcolemmal K ATP channels mediates cytoprotection in heart embryonic H9C2 cells.

    PubMed

    Jovanovi?, Sofija; Du, Qingyou; Sukhodub, Andriy; Jovanovi?, Aleksandar

    2009-11-01

    Muscle form of lactate dehydrogenase (M-LDH) physically associate with K(ATP) channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K(+) (K(ATP)) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (Delta M-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the K(ATP) channel protein complex. The K(+) current was increased in wild type cells, but not in cells expressing Delta M-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10mM). At the same time, the outcome of chemical hypoxia was much worse in Delta M-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with Delta M-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes K(ATP) channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing Delta M-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal K(ATP) channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival. PMID:19464385

  18. Antiapoptotic effect of novel compound from Herba leonuri - leonurine (SCM-198): a mechanism through inhibition of mitochondria dysfunction in H9c2 cells.

    PubMed

    Liu, Xin Hua; Pan, Li Long; Gong, Qi Hai; Zhu, Yi Zhun

    2010-12-01

    Apoptosis of cardiomyocytes induced by oxidative stress play a critical role in cardiac dysfunction associated with ventricular remodeling and heart failure. We recently reported that leonurine attenuated hypoxia-induced cardiomyocyte damage. In this study, we investigated the mechanism of leonurine (originally from Herba leonuri but we synthesized it chemically it as also called SCM-198) (H?O?)-induced rat embryonic heart-derived H9c2 cells from apoptosis. Exposing H9c2 cells to H?O? significantly decreased cell viability, and this was attenuated by pretreatment with leonurine for 4 h in a concentration-dependent manner. Meanwhile, leonurine was found to reduce intracellular reactive oxygen species (ROS) generation in H?O?-stimulated cell. Moreover, H9c2 cells stimulated by H?O? was accompanied with apparent apoptotic characteristics, including fragmentation of DNA, apoptotic body formation, release of cytochrome c, translocation of Bax to mitochondria, loss of mitochondrial membrane potential (??(m)) and activation of caspase 3. Furthermore, H?O? also induced rapid and significant phosphorylation of the c-Jun-N-terminal kinase 1/2 (JNK1/2), which was inhibited SP600125 (a JNK1/2 inhibitor). All of these events were attenuated by leonurine pretreatment. Taken together, these results demonstrated that leonurine could protect H9c2 cells from H?O?-induced apoptosis via modulation of mitochondrial dysfunction associated with blocking the activation of JNK1/2. PMID:20874679

  19. Ocimum gratissimum Aqueous Extract Protects H9c2 Myocardiac Cells from H2O2-Induced Cell Apoptosis through Akt Signalling

    PubMed Central

    Lee, Mu-Jang; Chen, Han-Min; Tzang, Bor-Show; Lin, Chiu-Wen; Wang, Chau-Jong; Liu, Jer-Yuh; Kao, Shao-Hsuan

    2011-01-01

    Increased cell death of cardiomyocyte by oxidative stress is known to cause dysfunction of the heart. O. gratissimum is one of the more well-known medicinal plants among the Ocimum species and widely used in treatment of inflammatory diseases. In this study, we hypothesized that aqueous extract of O. gratissimum leaf (OGE) may protect myocardiac cell H9c2 from oxidative injury by hydrogen peroxide (H2O2). Our results revealed that OGE pretreatment dose-dependently protects H9c2 cells from cell death when exposed to H2O2. Additionally, DNA condensation induced by H2O2 was also reduced by OGE pretreatment, suggesting that Ocimum gratissimum extract may attenuate H2O2-induced chromosome damage. Further investigation showed that OGE pretreatment inhibited H2O2-induced activation of caspase-3 and caspase-9, as well as H2O2-induced upregulation of proapoptotic Apaf-1 and the release of cytosolic cytochrome c, but has little effect on the activation of caspase-8. Additionally, OGE pretreatment significantly upregulated Bcl-2 expression and Akt phosphorylation, and slightly affected the phosphorylation of mitogen-activated protein kinases including p38 MAPK and JNK. Taken together, our findings revealed that Ocimum gratissimum extract effectively inhibited the mitochondrial pathway and upregulated Bcl-2 expression, which may be important in protecting H9c2 cells from H2O2-induced cell death. PMID:20953436

  20. 4-Guanidino-n-butyl syringate (Leonurine, SCM 198) protects H9c2 rat ventricular cells from hypoxia-induced apoptosis.

    PubMed

    Liu, Xin-hua; Chen, Pei-fang; Pan, Li-long; Silva, Ranil De; Zhu, Yi-zhun

    2009-11-01

    In the present study, we examined the ability of a chemically synthesized compound based on the structure of leonurine, a phytochemical component of Herba leonuri, to protect H9c2 rat ventricular cells from apoptosis induced by hypoxia and serum deprivation, as a model of ischemia. The results revealed a concentration-dependent increase in cell viability associated with leonurine treatment, accompanied by a consistent decline in lactate dehydrogenase leakage into the culture medium. The fraction of annexin V-fluorescein isothiocyanate-positive cells was increased by hypoxia but reduced by leonurine. These changes were associated with increased expression of the antiapoptotic gene, Bcl-2, and reduced expression of the proapoptotic gene, Bax. Leonurine also reduced the cytosolic Ca overload induced by hypoxia. These results suggest that leonurine elicits potent cardioprotective effects in H9c2 cells, and these effects may be mediated by inhibition of intracellular Ca overload and apoptosis during hypoxia. PMID:19940642

  1. Extracts of Artemisia ciniformis Protect Cytotoxicity Induced by Hydrogen Peroxide in H9c2 Cardiac Muscle Cells through the Inhibition of Reactive Oxygen Species.

    PubMed

    Mojarrab, Mahdi; Jamshidi, Maryam; Ahmadi, Farahnaz; Alizadeh, Ellahe; Hosseinzadeh, Leila

    2013-01-01

    Objective. Artemisia ciniformis (Asteraceae) and A. biennis are two of 34 Artemisia species growing naturally in Iran. In this study we investigated whether different extracts of A. ciniformis and A. biennis have protective effect against hydrogen peroxide-induced cytotoxicity in rat cardiomyoblast cells (H9c2). Method. The dried and ground aerial parts of these two species were extracted successively using petroleum ether (40-60), dichloromethane, ethyl acetate (EA), ethanol (EtOH) and ethanol?:?water (1?:?1) by maceration method. To evaluate whether different extracts of A. ciniformis and A. biennis protect cardiomyoblast H9c2 cells from H2O2 cytotoxicity, we examined the direct cytotoxic effect of H2O2 on H9c2 cells in the presence and absence of different extracts. After then, cell viability was measured by MTT assay. Results. H2O2 induced cytotoxicity in a concentration dependent manner. The IC50 value was 62.5? ? M for 24?h exposure. However, pretreatment of cells with various concentrations of EA, EtOH, and EtOH/wt extract of A. ciniformis protected cells from H2O2-induced cytotoxicity. Moreover, pretreatment with EA, EtOH and EtOH/wt extracts of A. ciniformis lead to a decrease in the reactive oxygen species (ROS) generation. Taken together our observation indicated that nontoxic concentration of different extracts of A. ciniformis has protective effect on H2O2-induced cytotoxicity in H9c2 cells. PMID:24381586

  2. Effects of metformin and insulin on h9c2 rat cardiomyocytes in cell culture with elevated glucose levels.

    PubMed

    Lee, Lishia; Tucci, Michelle A; Benghuzzi, Hamed A

    2014-01-01

    Patients with type 2 diabetes mellitus require adequate blood glucose control with insulin, metformin or other anti-hyperglycemic agents to prevent significant co-morbidities such as cardiovascular disease. Studies have determined that metformin increases the activity of AMP activated protein kinase and reduces hepatic glucose production and increases tissue sensitivity to insulin. Insulin has some vascular protective qualities; however insulin resistance of type 2 diabetes may be harmful to vascular endothelium. The objective of this study was to investigate the effects of metformin and insulin on H9c2 rat cardiomyocytes in high glucose culture media. Results of this study indicate that clinical doses both insulin and metformin are capable of reducing supernatant glucose levels in high glucose media. Metformin may offer some protection to cardiomyoctyes. Morphologically, cellular changes occurred after exposure to both insulin and metformin at both 24 and 48 hours although at 72 hours, both groups exhibit evidence of cellular damage. PMID:25405446

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

    SciTech Connect

    Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw [Department of Physiology and Pharmacology and Health Aging Research Center, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan (China); Heart Failure Center, Division of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Keelung, Keelung, Taiwan (China); Lee, I-Ta [Department of Physiology and Pharmacology and Health Aging Research Center, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan (China); Department of Anesthetics, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan (China); Hsu, Ru-Chun; Chi, Pei-Ling; Hsiao, Li-Der [Department of Physiology and Pharmacology and Health Aging Research Center, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan (China)

    2013-10-15

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

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

    PubMed Central

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

    2014-01-01

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

  5. Transforming growth factor-? type I receptor\\/ALK5 contributes to doxazosin-induced apoptosis in H9C2 cells

    Microsoft Academic Search

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

    2009-01-01

    The mechanism of doxazosin-induced apoptosis through ?1-adrenoceptor-independent pathway has been reported in various types of cell models. However, the molecular events involved\\u000a in this effect are still not fully discovered. In present study, we proposed that the transforming growth factor-? type I\\u000a receptor (T?RI\\/ALK5) may contribute to the doxazosin-induced apoptosis in H9C2 cardiomyoblasts. Via the detection of cell\\u000a viability, apoptotic

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

    PubMed

    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

    2015-09-01

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

  7. 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 TGF?, TNF-?, IFN? 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 TGF?- and TNF-?-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-? and IFN? specific networks at 6 h, followed by elicitation of IL-6 and IFN?-centered gene networks at 24h. Conclusions Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically. PMID:23249388

  8. Epigallocatechin-3-gallate and zinc provide anti-apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells

    PubMed Central

    ZENG, XING; TAN, XUERUI

    2015-01-01

    It has previously been demonstrated that phospha-tidylinositol-3-kinase (PI3K)/Akt and cleaved caspase-3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin-3-gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription-1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3-h hypoxia, followed by 1-h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti-apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R-induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice. PMID:25872640

  9. Epigallocatechin?3?gallate and zinc provide anti?apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells.

    PubMed

    Zeng, Xing; Tan, Xuerui

    2015-08-01

    It has previously been demonstrated that phosphatidylinositol?3?kinase (PI3K)/Akt and cleaved caspase?3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin?3?gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription?1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3?h hypoxia, followed by 1?h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti?apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R?induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice. PMID:25872640

  10. Studies on the cardioprotective role of gallic acid against AGE-induced cell proliferation and oxidative stress in H9C2 (2-1) cells.

    PubMed

    Umadevi, S; Gopi, V; Simna, S P; Parthasarathy, A; Yousuf, S M J; Elangovan, V

    2012-12-01

    Epidemiological studies have shown that high glucose levels and oxidative stress cause elevation of advanced glycation end products (AGEs) that are known to contribute to diabetic complications. Thus, agents that hamper reactive oxygen species (ROS) load can be used as a potential drug against AGEs-mediated complications. Hence, the present study investigated the protective role of gallic acid (GA) against the effects of AGEs in cardiac H9C2(2-1) cells. Exposure of cells to AGEs resulted in release of ROS (P < 0.05) with significant (P < 0.05) decline in antioxidant enzyme levels and increase in collagen (P < 0.01) content. In addition, the altered mitochondrial membrane potential (mmp) (P < 0.01) was also observed in cells exposed to AGEs, whereas AGEs-exposed cells pretreated with GA prevented the release of ROS, and there were no significant changes in the antioxidant status, collagen content and mmp. Thus, the results of the present study provide evidence that GA exhibits protective role against AGEs-induced cardiovascular complications probably through its free radical scavenging activity. PMID:22588841

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

    PubMed Central

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

    2015-01-01

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

  12. 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

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

    PubMed Central

    Jin, Hyou-Ju; Li, Chun-Guang

    2013-01-01

    The protective actions of tanshinones on hypoxia-induced cell damages have been reported, although the mechanisms have not been fully elucidated. Given the importance of nitric oxide (NO) and reactive oxygen species (ROS) in regulation of cell functions, the present study investigated the effects of two major tanshinones, Tanshinone IIA (TIIA) and cryptotanshinone (CT), on hypoxia-induced myocardial cell injury and its relationships with intracellular NO and ROS, calcium, and ATP levels in H9c2 cells. Chronic hypoxia significantly reduced cell viability which accompanied with LDH release, increase in mitochondrial ROS, intracellular NO and calcium levels, decrease in superoxide dismutase (SOD) activity, and cellular ATP contents. TIIA and CT significantly prevented cell injury by increasing cell viability and decreasing LDH release. The protective effects of tanshinones were associated with reduced mitochondrial superoxide production and enhanced mitochondrial SOD activity. Tanshinones significantly reduced intracellular NO and Ca2+ levels. ATP levels were also restored by TIIA. These findings suggest that the cytoprotective actions of tanshinones may involve regulation of intracellular NO, Ca2+, ATP productions, mitochondrial superoxide production, and SOD activity, which contribute to their actions against hypoxia injuries. PMID:23533503

  14. Isoform-specific induction of PKC-epsilon by high glucose protects heart-derived H9c2 cells against hypoxic injury.

    PubMed

    Kim, Min Hwa; Jung, Yi-Sook; Moon, Chang-Hyun; Jeong, Euy-Myoung; Lee, Soo Hwan; Baik, Eun Joo; Moon, Chang-Kiu

    2003-09-12

    We investigated which PKC isoforms are involved in high glucose-induced protection against hypoxic injury. Treatment for 48 h with high glucose (22 mM) markedly increased the expression of PKC- epsilon in the particulate fraction (213+/-22.1% of the control) but had no effect on other types of PKC isoforms, suggesting that the high glucose-induced increase in PKC expression is isoform-specific. The mRNA level for PKC- epsilon was also substantially increased, reaching its peak after 4h of high glucose treatment. The high glucose increased PKC-epsilon activity in the particulate fraction up to 183+/-32.2% of the control. During hypoxia, the amount of PKC-epsilon in the particulate fraction was remarkably diminished in the low glucose-treated cells, but remained at a higher level in high glucose-treated cells. The treatment with epsilon V1-2 (10 microM), a specific inhibitor of PKC epsilon, abolished the protective effect of high glucose against hypoxia. These results suggest that isoform-specific induction of PKC-epsilon is involved in high glucose-induced protection against hypoxic injury in heart-derived H9c2 cells. PMID:12943654

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Li, Kai; Cui, Yong-Chun; 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

  17. Proteome Modulation in H9c2 Cardiac Cells by microRNAs miR-378 and miR-378*

    PubMed Central

    Mallat, Youssef; Tritsch, Eva; Ladouce, Romain; Winter, Daniel Lorenz; Friguet, Bertrand; Li, Zhenlin; Mericskay, Mathias

    2014-01-01

    MicroRNAs are a novel class of powerful endogenous regulators of gene expression. MiR-378 and miR-378* are localized in the first intron of the Ppargc1b gene that codes the transcriptional co-activator PGC-1?. The latter regulates energy expenditure as well as mitochondrial biogenesis. The miR-378:miR-378* hairpin is highly expressed in cardiac cells. To better assess their role in cardiomyocytes, we identified miR-378 and miR-378* targets via a proteomic screen. We established H9c2 cellular models of overexpression of miR-378 and miR-378* and identified a total of 86 down-regulated proteins in the presence of either one of these miRs. Functional annotation clustering showed that miR-378 and miR-378* regulate related pathways in cardiomyocytes, including energy metabolism, notably glycolysis, cytoskeleton, notably actin filaments and muscle contraction. Using bioinformatics algorithms we found that 20 proteins were predicted as direct targets of the miRs. We validated eight of these targets by quantitative RT-PCR and luciferase reporter assay. We found that miR-378 targets lactate dehydrogenase A and impacts on cell proliferation and survival whereas miR-378* targets cytoskeleton proteins actin and vimentin. Proteins involved in endoplasmic reticulum stress response such as chaperone and/or calcium buffering proteins GRP78, PPIA (cyclophilin A), calumenin, and GMMPA involved in glycosylation are repressed by these miRs. Our results show that the miR-378/378* hairpin establishes a connection among energy metabolism, cytoskeleton remodeling, and endoplasmic reticulum function through post-transcriptional regulation of key proteins involved in theses pathways. PMID:24068033

  18. Impact of methylmercury exposure on mitochondrial energetics in AC16 and H9C2 cardiomyocytes.

    PubMed

    Truong, Jocelyn; Mailloux, Ryan J; Chan, Hing Man

    2015-08-01

    It has been reported that chronic low dose exposures of methylmercury (MeHg) is associated with cardiovascular diseases in many populations worldwide. The toxic mechanisms through which these adverse effects occur are currently unknown. The objective of this study was to determine the bioenergetic and cytotoxic effects of MeHg on AC16 and H9C2 cardiomyocyte cell lines. Both cell lines exhibit significantly decreased mitochondrial function, cell viability and increased reactive oxygen species (ROS) production. Decreases in maximal respiration and reserve capacity was observed in both cell lines at 1?M. Bioenergetic profile experiments were also performed in tandem with cells exposed to diamide or menadione, compounds which accumulate in mitochondria and disrupt oxidative phosphorylation. AC16 cells show MeHg dose dependant sensitivities with Stateapparent and ATP production values, but H9C2 cells do not show these trends. H9C2 cells may be more resistant to MeHg toxicity than AC16 cells as reflected in the increases of proton leak and Stateapparent. No changes in expression of respiratory complexes were observed. Results suggest that MeHg has the potential to induce cytotoxicity. Furthermore, MeHg may have differential effects on AC16 and H9C2 cells, derived from human and rat cardiac tissue respectively, suggesting that differences in MeHg toxicity may be species-dependent. PMID:25835517

  19. The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3? and ERK1/2 signaling pathway.

    PubMed

    Kim, Dae-Eun; Kim, Bokyung; Shin, Hwa-Sup; Kwon, Ho Jeong; Park, Eun-Seok

    2014-10-01

    Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3? and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3? and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3? and ERK1/2 signaling pathways. PMID:25128810

  20. Exogenous H2S protects H9c2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the activation of the NF-?B and IL-1? pathways.

    PubMed

    Xu, Wenming; Chen, Jingfu; Lin, Jianchong; Liu, Donghong; Mo, Liqiu; Pan, Wanying; Feng, Jianqiang; Wu, Wen; Zheng, Dongdan

    2015-01-01

    Hyperglycemia has been reported to activate the nuclear factor-?B (NF-?B) pathway. We have previously demonstrated that exogenous hydrogen sulfide (H2S) protects cardiomyocytes against high glucose (HG)-induced injury by inhibiting the activity of p38 mitogen-activated protein kinase (MAPK), which can activate the NF-?B pathway and induce interleukin (IL)-1? production. In the present study, we aimed to investigate the hypothesis that exogenous H2S protects cardiomyocytes against HG-induced injury and inflammation through the inhibition of the NF-?B/IL-1? pathway. H9c2 cardiac cells were treated with 35 mM glucose (HG) for 24 h to establish a model of HG-induced damage. Our results demonstrated that treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) or 100 µM pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-?B) for 30 min prior to exposure to HG markedly attenuated the HG-induced increase in the expression levels of the phosphorylated (p)-NF-?B p65 subunit. Notably, pre-treatment of the H9c2 cardiac cells with NaHS or PDTC significantly suppressed the HG-induced injury, including cytotoxicity, apoptosis, oxidative stress and mitochondrial insults, as evidenced by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). In addition, pre-treatment of the cells with NaHS or PDTC ameliorated the HG-induced inflammatory response, leading to a decrease in the levels of IL-1?, IL-6 and tumor necrosis factor-? (TNF-?). Importantly, co-treatment of the H9c2 cells with 20 ng/ml IL-1 receptor antagonist (IL-1Ra) and HG markedly reduced the HG-induced increase in p-NF-?B p65 expression, cytotoxicity, the number of apoptotic cells, as well as the production of TNF-?. In conclusion, the present study presents novel mechanistic evidence that exogenous H2S protects H9c2 cardiac cells against HG-induced inflammation and injury, including cytotoxicity, apoptosis, overproduction of ROS and the dissipation of MMP, by inhibiting the NF-?B/IL-1? pathway. We also provide new data indicating that the positive interaction between the NF-?B pathway and IL-1? is critical in HG-induced injury and inflammation in H9c2 cardiac cells. PMID:25412187

  1. Inhibition of the leptin-induced activation of the p38 MAPK pathway contributes to the protective effects of naringin against high glucose-induced injury in H9c2 cardiac cells.

    PubMed

    Chen, Jingfu; Mo, Hailiang; Guo, Runmin; You, Qiong; Huang, Ruina; Wu, Keng

    2014-03-01

    Leptin, a product of the obese gene, has been reported to contribute to the development of cardiomyocyte hypertrophy in patients with diabetes and to activate the p38 mitogen-activated protein kinase (MAPK) pathway in cardiomyocytes. In this study, we demonstrate that naringin, a citrus flavonone, protects cardiomyoblasts (H9c2 cells) against high glucose (HG)-induced apoptosis by modulating the activation of the p38 MAPK pathway. We investigated the hypothesis that naringin prevents HG-induced injury by inhibiting the leptin-induced activation of the p38 MAPK pathway in H9c2 cells. Our results demonstrated that the exposure of H9c2 cells to HG (35 mmol/l) for a 24 h markedly upregulated the expression levels of both leptin and leptin receptors. However, the increase in the expression levels of leptin and leptin receptors was greatly attenuated by treatment of the H9c2 cells with 80 µmol/l naringin 2 h prior to exposure to HG. In addition, treatment of the cells with 50 ng/ml leptin antagonist (LA) for 24 h prior to exposure to HG markedly ameliorated the increased expression of phosphorylated (p)-p38 MAPK induced by HG. Of note, pre-treatment of the cells with either 80 µmol/l naringin or 50 ng/ml LA markedly inhibited the HG-induced injury, leading to an increase in cell viability and a decrease in the total number of apoptotic cells, preventing reactive oxygen species (ROS) generation, as well as the dissipation of mitochondrial membrane potential (MMP). In conclusion, the findings of the present study provide the first evidence that the leptin-induced activation of the p38 MAPK pathway is involved in HG-induced injury, including cytotoxicity, apoptosis, ROS generation and the dissipation of MMP in H9c2 cardiac cells. Our data demonstrate that naringin protects cardiac cells against HG-induced injury by inhibiting the leptin-induced activation of the p38 MAPK pathway. PMID:24399619

  2. Exogenous hydrogen sulfide protects H9c2 cardiac cells against high glucose-induced injury by inhibiting the activities of the p38 MAPK and ERK1/2 pathways.

    PubMed

    Xu, Wenming; Wu, Wen; Chen, Jingfu; Guo, Runmin; Lin, Jiancong; Liao, Xinxue; Feng, Jianqiang

    2013-10-01

    Hyperglycemia is a risk factor for the development of diabetic cardiovascular complications, which are associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we demonstrate the inhibitory effects of exogenous hydrogen sulfide (H?S) on the activation of the MAPK pathway. The aim of the present study was to determine whether exogenous H?S prevents high glucose (HG)-induced injury by inhibiting the activation of the p38 MAPK and extracellular signal-regulated kinase (ERK)1/2 (members of MAPK) pathways in cardiomyoblasts (H9c2 cells). The findings of the present study demonstrated that the treatment of H9c2 cells with HG (35 mM glucose) for 24 h not only significantly induced injury, including cytotoxicity, apoptosis, overproduction of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP), but also upregulated the expression levels of phosphorylated (p)-p38 MAPK and p-ERK1/2. The increased expression levels of p-p38 MAPK and p-ERK1/2 were markedly reduced by pre-treatment of the H9c2 cells with 400 µM sodium hydrogen sulfide (NaHS; a donor of H2S) prior to exposure to 35 mM glucose. Importantly, pre-treatment of the cells with 400 µM NaHS or 3 µM SB203580 (a selective inhibitor of p38 MAPK) or 15 µM U0126 (a selective inhibitor of ERK1/2) attenuated the HG-induced cardiomyocyte injury, leading to an increase in cell viability and a decrease in the number of apoptotic cells, preventing ROS generation, as well as the loss of MMP. In addition, pre-treatment of the cells with 1,000 µM N?acetyl?L?cysteine (a ROS scavenger) prior to exposure to HG ameliorated the HG-induced cytotoxicity. Taken together, the data from the present study demonstrate for the first time, to our knowledge, that exogenous H2S exerts a protective effect against HG?induced injury by inhibiting the activation of the p38 MAPK and ERK1/2 pathways and preventing oxidative stress in H9c2 cells. PMID:23912965

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

    PubMed

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

    2015-08-01

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

  4. Mitochondrial membrane potential measurement of H9c2 cells grown in high-glucose and galactose-containing media does not provide additional predictivity towards mitochondrial assessment.

    PubMed

    Rana, Payal; Nadanaciva, Sashi; Will, Yvonne

    2011-03-01

    Drug-induced mitochondrial toxicity is a contributing factor to many organ toxicities. The fact that some, but not all members of a particular drug class can induce mitochondrial dysfunction has necessitated the need for predictive screens within the drug development process. One of these screens is a cell viability assay done in two types of media, one containing high-glucose, the other, galactose. Since galactose-grown cells are more susceptible to mitochondrial toxicants than high-glucose-grown cells, this assay distinguishes compounds that cause toxicity primarily through mitochondrial targets from those that cause multifactorial toxicity. However, the assay does not show if compounds that cause multifactorial toxicity cause impairment on mitochondria. To address this problem, we investigated if multiplexing the assay with mitochondrial membrane potential measurements using the fluorescent dye, JC-1, could provide further information. We tested 28 drugs in the multiplexed assay and found that, although mitochondrial toxicants could be detected, no additional information was revealed about compounds that caused multifactorial toxicity. Hence, measurements with JC-1 did not provide additional information beyond what was detected using the cell viability assay. We conclude that even though the multiplexed assay is useful for HTS applications, it provides no additional value over the high-glucose-galactose cell viability assay. PMID:21126567

  5. Confinement of ?1- and ?2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    PubMed Central

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, ?1- and ?2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that ?ARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated ?1- and ?2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and ?2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in ?2AR confinement. For both ?1- and ?2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing ?1- or ?2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  6. MicroRNA-34a regulates high glucose-induced apoptosis in H9c2 cardiomyocytes.

    PubMed

    Zhao, Fang; Li, Bo; Wei, Yin-zhi; Zhou, Bin; Wang, Han; Chen, Ming; Gan, Xue-dong; Wang, Zhao-hui; Xiong, Shi-xi

    2013-12-01

    Hyperglycemia is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. The purpose of the present study was to investigate whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9c2 through microRNA-mediated Bcl-2 signaling pathway. The expression of miR-34a and Bcl-2 mRNA was detected by using real-time PCR. Western blotting was used to examine the changes in apoptosis-associated protein Bcl-2. Apoptosis of H9c2 cells was tested by using flow cytometry. The results showed that the expression of miR-34a was significantly elevated and that of Bcl-2 was strongly reduced, and apoptosis of cardiomyocytes was apparently increased in the high-glucose-treated H9c2 cells as compared with normal-glucose-treated controls. In addition, we identified Bcl-2 gene was the target of miR-34a. miR-34a mimics reduced the expression of Bcl-2 and increased glucose-induced apoptosis, but miR-34a inhibitor acted as the opposite mediator. Our data demonstrate that miR-34a contributes to high glucose-induced decreases in Bcl-2 expression and subsequent cardiomyocyte apoptosis. PMID:24337844

  7. Sodium Fluoride Induces Apoptosis in H9c2 Cardiomyocytes by Altering Mitochondrial Membrane Potential and Intracellular ROS Level.

    PubMed

    Yan, Xiaoyan; Yang, Xia; Hao, Xianhui; Ren, Qiurong; Gao, Jiping; Wang, Yu; Chang, Na; Qiu, Yulan; Song, Guohua

    2015-08-01

    Chronic excessive fluoride intake is known to be toxic, and effects of long-term fluorosis on different organ systems have been examined. However, there are few studies about the effects of fluorosis on cardiovascular systems. Here, we studied the fluoride-induced apoptosis in H9c2 cells and determined the underlying molecular mechanisms including the cell viability, intracellular reactive oxygen species (ROS) level, the changes of mitochondrial membrane potential (??m), and the cell apoptosis. Sodium fluoride (NaF) at concentrations of 0, 2, 4, 8, and 16 mg/L was administered to cultured H9c2 cells for up to 48 h. After the treatment, H9c2 cells were collected and the associated parameters were measured by flow cytometry. Our study found that fluoride not only inhibited H9c2 cell proliferation but also induced cell apoptosis. With the increment of NaF concentration, the apoptotic rates and ROS generation were increased, while the ??m was decreased. In summary, these data suggested that NaF-induced H9c2 cell apoptosis is mediated by direct increased intracellular ROS and downregulated ??m. PMID:25707396

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

    PubMed

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

    2015-06-01

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

  9. Cytotoxic effects of polystyrene-titanium-arsenate composite in cultured H9c2 cardiomyoblasts.

    PubMed

    Mohammad, Faruq; Arfin, Tanvir

    2013-12-01

    In the present report, we explored the toxicological behaviour of engineered polystyrene-titanium-arsenate (PS-Ti-As) composite using cultured H9c2 cardiomyoblasts in vitro. From in vitro cytotoxicity studies, it appears that the composite can be toxic to the cardiac cells and the value of IC50 investigated to be the highest concentration of 500 ?g mL(-1), during 16-24 h of incubation period. The cell morphological studies based on dual staining with acridine orange and ethidium bromide indicates that apoptosis is the dominating pathway of cell death. Furthermore, an enhanced DNA fragmentation, increased reactive oxygen species production and caspase release demonstrates the potential risks associated with the exposure of PS-Ti-As composite to the cardiac cells. PMID:24154614

  10. Polyunsaturated fatty acids incorporation into cardiolipin in H9c2 cardiac myoblast.

    PubMed

    Ting, Hsiu-Chi; Chao, Yu-Jen; Hsu, Yuan-Hao Howard

    2015-07-01

    Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), known as ?-3 polyunsaturated fatty acid (PUFA), are common nutrients in daily food intake and have been shown to prevent cardiovascular disease and improve cardiac functions. Cardiolipin is a mitochondrial phospholipid necessary for maintaining physiological function of mitochondria. Several studies have indicated that the cardiolipin acyl chain compositions affect the function of cardiolipin and mitochondria. Here, we investigated the structural changes of cardiolipin after DHA and EPA supplementation and compared them to arachidonic acid (AA) treatment. H9c2 cardiac myoblast was used as a cell model, and cardiolipin species was monitored and identified via LC-MS and MS/MS. Our results showed distinct mass envelopes of cardiolipin with the same carbon number but different double bonds in mass spectrum. There were 116 cardiolipin species with 36 distinct mass in 6 mass envelopes identified by MS/MS. Three days of PUFA treatment resulted in decreases of low-molecular-weight cardiolipin and increases of high-molecular-weight cardiolipin, suggesting the incorporation of exogenous DHA, EPA and AA into mitochondrial cardiolipin. PUFA incorporation was further verified by MS/MS analysis. More importantly, we found that DHA supplementation elevated the percent content of less unsaturated cardiolipin species and highly unsaturated cardiolipin species, containing ?-3 fatty acyl chains, indicating a ?-3 fatty acid incorporation mechanism with peroxidation protection. Our results indicate that PUFA supplementation differentially perturbed the fatty acyl chain compositions in the mitochondrial cardiolipin in the H9c2 cardiac myoblast, suggesting that mitochondrial membrane and the function of mitochondria are susceptible to exogenous lipid species. PMID:25866137

  11. Cardioprotective and cardiotoxic effects of quercetin and two of its in vivo metabolites on differentiated h9c2 cardiomyocytes.

    PubMed

    Daubney, James; Bonner, Philip L; Hargreaves, Alan J; Dickenson, John M

    2015-02-01

    Whilst mitotic rat embryonic cardiomyoblast-derived H9c2 cells have been widely used as a model system to study the protective mechanisms associated with flavonoids, they are not fully differentiated cardiac cells. Hence, the aim of this study was to investigate the cardioprotective and cardiotoxic actions of quercetin and two of its major in vivo metabolites, quercetin 3-glucuronide and 3'-O-methyl quercetin, using differentiated H9c2 cells. The differentiated cardiomyocyte-like phenotype was confirmed by monitoring expression of cardiac troponin 1 after 7 days of culture in reduced serum medium containing 10 nM all-trans retinoic acid. Quercetin-induced cardiotoxicity was assessed by monitoring MTT reduction, lactate dehydrogenase (LDH) release, caspase 3 activity and reactive oxygen species production after prolonged flavonoid exposure (72 hr). Cardiotoxicity was observed with quercetin and 3'-O-methyl quercetin, but not quercetin 3-glucuronide. Cardioprotection was assessed by pre-treating differentiated H9c2 cells with quercetin or its metabolites for 24 hr prior to 2-hr exposure to 600 ?M H2 O2, after which oxidative stress-induced cell damage was assessed by measuring MTT reduction and LDH release. Cardioprotection was observed with quercetin and 3'-O-methyl quercetin, but not with quercetin 3-glucuronide. Quercetin attenuated H2 O2 -induced activation of ERK1/2, PKB, p38 MAPK and JNK, but inhibitors of these kinases did not modulate quercetin-induced protection or H2 O2 -induced cell death. In summary, quercetin triggers cardioprotection against oxidative stress-induced cell death and cardiotoxicity after prolonged exposure. Further studies are required to investigate the complex interplay between the numerous signalling pathways that are modulated by quercetin and which may contribute to the cardioprotective and cardiotoxic effects of this important flavonoid. PMID:25203460

  12. Expression of microRNA-122 contributes to apoptosis in H9C2 myocytes

    PubMed Central

    Huang, Xiaoyan; Huang, Fang; Yang, Deye; Dong, Fengquan; Shi, Xiangxiang; Wang, Hongyu; Zhou, Xi; Wang, Suyun; Dai, Shengchuan

    2012-01-01

    The microRNAs (miRNAs) can post-transcriptionally regulate gene expression and heart development. The Pax-8 gene knockout mice have apparent heart abnormalities. This study investigated the role of miRNAs in regulation of cardiac apoptosis and development in the knockout mice. MicroRNA microarrays demonstrated differential expression of microRNAs between Pax-8?/? and Pax-8+/? mice, confirmed by real-time PCR. The miR-122 was up-regulated by 1.92 folds in Pax-8?/? mice. There were ventricular septum defects in Pax-8?/? mice, and increased numbers of apoptotic cells in the left ventricular wall and interventricular septum in Pax-8?/? mice. In H9C2 myocytes, treatment with miR-122 mimics or miR-122 inhibitor affects the expression of CCK-8 and activity of Caspase-3. The miR-122 is up-regulated in the myocytes of Pax-8?/? mice and may participate in the apoptotic gene expression and pathogenesis of heart development defect. PMID:22453009

  13. Hydrogen sulfide protects against apoptosis under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes.

    PubMed

    Wu, Dan; Hu, Qingxun; Liu, Xinhua; Pan, Lilong; Xiong, Qinghui; Zhu, Yi Zhun

    2015-04-30

    Oxidative stress plays a great role in the pathogenesis of heart failure (HF). Oxidative stress results in apoptosis, which can cause the damage of cardiomyocytes. Hydrogen sulfide (H2S), the third gasotransmitter, is a good reactive oxygen species (ROS) scavenger, which has protective effect against HF. Sirtuin-1 (SIRT1) is a highly conserved nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylase that plays a critical role in promoting cell survival under oxidative stress. The purpose of this article is to investigate the interaction between H2S and SIRT1 under oxidative stress in H9c2 cardiomyocytes. Oxidative stress was induced by hydrogen peroxide (H2O2). Treatment with NaSH (25-100 µmol/L) dose-dependently increased the cell viability and improved the cell apoptosis induced by H2O2 in H9c2 cardiomyocytes. The protective effect of NaSH against the apoptosis could be attenuated by SIRT1 inhibitor Ex 527 (10 µmol/L). Treatment with NaSH (100 µmol/L) could increase the expression of SIRT1 in time dependent manner, which decreased by different concentration of H2O2. NaSH (100 µmol/L) increased the cellular ATP level and the expression of ATPase. These effects were attenuated by Ex 527 (10 µmol/L). After NaSH (100 µmol/L) treatment, the decrease in ROS production and the enhancement in SOD, GPx and GST expression were observed. Ex 527 (10 µmol/L) reversed these effects. In conclusion, for the first time, this article can identify antioxidative effects of H2S under oxidative stress through SIRT1 pathway in H9c2 cardiomyocytes. PMID:25461268

  14. Icariin attenuates angiotensin II-induced hypertrophy and apoptosis in H9c2 cardiomyocytes by inhibiting reactive oxygen species-dependent JNK and p38 pathways.

    PubMed

    Zhou, Heng; Yuan, Yuan; Liu, Yuan; Deng, Wei; Zong, Jing; Bian, Zhou-Yan; Dai, Jia; Tang, Qi-Zhu

    2014-05-01

    Icariin, the major active component isolated from plants of the Epimedium family, has been reported to have potential protective effects on the cardiovascular system. However, it is not known whether icariin has a direct effect on angiotensin II (Ang II)-induced cardiomyocyte enlargement and apoptosis. In the present study, embryonic rat heart-derived H9c2 cells were stimulated by Ang II, with or without icariin administration. Icariin treatment was found to attenuate the Ang II-induced increase in mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide and B-type natriuretic peptide, in a concentration-dependent manner. The cell surface area of Ang II-treated H9c2 cells also decreased with icariin administration. Furthermore, icariin repressed Ang II-induced cell apoptosis and protein expression levels of Bax and cleaved-caspase 3, while the expression of Bcl-2 was increased by icariin. In addition, 2',7'-dichlorofluorescein diacetate incubation revealed that icariin inhibited the production of intracellular reactive oxygen species (ROS), which were stimulated by Ang II. Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in Ang II-treated H9c2 cells was blocked by icariin. Therefore, the results of the present study indicated that icariin protected H9c2 cardiomyocytes from Ang II-induced hypertrophy and apoptosis by inhibiting the ROS-dependent JNK and p38 pathways. PMID:24940396

  15. Mitochondrial disruption occurs downstream from ?-adrenergic overactivation by isoproterenol in differentiated, but not undifferentiated H9c2 cardiomyoblasts: differential activation of stress and survival pathways.

    PubMed

    Branco, Ana F; Sampaio, Susana F; Wieckowski, Mariusz R; Sardăo, Vilma A; Oliveira, Paulo J

    2013-11-01

    ?-Adrenergic receptor stimulation plays an important role in cardiomyocyte stress responses, which may result in apoptosis and cardiovascular degeneration. We previously demonstrated that toxicity of the ?-adrenergic agonist isoproterenol on H9c2 cardiomyoblasts depends on the stage of cell differentiation. We now investigate ?-adrenergic receptor downstream signaling pathways and stress responses that explain the impact of muscle cell differentiation on hyper-?-adrenergic stimulation-induced cytotoxicity. When incubated with isoproterenol, differentiated H9c2 muscle cells have increased cytosolic calcium, cyclic-adenosine monophosphate content and oxidative stress, as well as mitochondrial depolarization, increased superoxide anion, loss of subunits from the mitochondrial respiratory chain, decreased Bcl-xL content, increased p53 and phosphorylated-p66Shc as well as activated caspase-3. Undifferentiated H9c2 cells incubated with isoproterenol showed increased Bcl-xL protein and increased superoxide dismutase 2 which may act as protective mechanisms. We conclude that the differentiation of H9c2 is associated with differential regulation of stress responses, which impact the toxicity of several agents, namely those acting through ?-adrenergic receptors and resulting in mitochondrial disruption in differentiated cells only. PMID:23958426

  16. Cytosolic renin is targeted to mitochondria and induces apoptosis in H9c2 rat cardiomyoblasts.

    PubMed

    Wanka, Heike; Kessler, Nicole; Ellmer, Janett; Endlich, Nicole; Peters, Barbara S; Clausmeyer, Susanne; Peters, Jörg

    2009-09-01

    One important goal in cardiology is to prevent necrotic cell death in the heart. Necrotic cell death attracts neutrophils and monocytes into the injured myocardium. The consequences are fibrosis, remodelling and cardiac failure. The renin-angiotensin system promotes the development of cardiac failure. Recently, alternative renin transcripts have been identified lacking the signal sequence for a cotranslational transport to the endoplasmatic reticulum. These transcripts encode for a cytosolic renin with unknown functions. The expression of this alternative transcript increases after myocardial infarction. We hypothesized that cytosolic renin plays a role in survival and death of cardiomyocytes. To test this hypothesis, we overexpressed secretory or cytosolic renin in H9c2 cardiomyblasts and determined the rate of proliferation, necrosis and apoptosis. Proliferation rate, as indicated by BrdU incorporation into DNA, was reduced by secretory and cytosolic renin (cells transfected with control vector: 0.33 +/- 0.06; secretory renin: 0.12 +/- 0.02; P < 0.05; cytosolic renin: 0.15 +/- 0.03; P < 0.05). Necrosis was increased by secretory renin but decreased by cytosolic renin (LDH release after 10 days from cells transfected with control vector: 68.5 +/- 14.9; secretory renin: 100.0 +/- 0; cytosolic renin: 25.5 +/- 5.3% of content, each P < 0.05). Mitochondrial apoptosis, as indicated by phosphatidylserin translocation to the outer membrane, was unaffected by secretory renin but increased by cytosolic renin (controls: 23.8 +/- 3.9%; secretory renin: 22.1 +/- 4.7%; cytoplasmatic renin: 41.2 +/- 3.8%; P < 0.05). The data demonstrate that a cytosolic renin exists in cardiomyocytes, which in contradiction to secretory renin protects from necrosis but increases apoptosis. Non-secretory cytosolic renin can be considered as a new target for cardiac failure. PMID:18671756

  17. Activin receptor-like kinase 7 mediates high glucose-induced H9c2 cardiomyoblast apoptosis through activation of Smad2/3.

    PubMed

    Liu, Lin; Ding, Wen-yuan; Zhao, Jing; Wang, Zhi-hao; Zhong, Ming; Zhang, Wei; Chen, Yu-guo; Zhang, Yun; Li, Li; Tang, Meng-xiong

    2013-09-01

    Cardiomyocyte apoptosis is an important pathological change of diabetic cardiomyopathy. How the elevated glucose levels cause cell apoptosis remains unknown. The aim of our study was to investigate whether activin receptor-like kinase 7 (ALK7)-Smad2/3 signaling pathway plays an important role in high glucose-induced cardiomyocyte apoptosis. H9c2 cardiomyoblasts and neonatal rat cardiomyocytes were treated with 33mmol/l glucose. The expression of ALK7, Smad2 and Smad3 were inhibited by small interfering RNA respectively. The level of ALK7, total Smad2/3, phosphorylated Smad2/3, B-cell lymphoma-2 (Bcl-2) and cleaved Caspase3 were evaluated using western blot. The apoptosis rate was detected by flow cytometer. High glucose treatment caused the apoptosis of H9c2 cardiomyocyte and the inhibition of Smad2 or Smad3 attenuated this apoptosis. ALK7 existed in both H9c2 cardiomyoblasts and neonatal rat cardiomyocytes and high ambient glucose upregulated its expression. The increased expression level of cleaved Caspase3 and apoptosis rate and decreased expression of Bcl-2 were reversed after ALK7 was inhibited. The expression of phosphorylated Smad2/3 also decreased after the knockdown of ALK7. Our findings suggest that ALK7 mediates high ambient glucose-induced H9c2 cardiomyoblasts apoptosis through the activation of Smad2/3. PMID:23830891

  18. BNC Protects H9c2 Cardiomyoblasts from H2O2-Induced Oxidative Injury through ERK1/2 Signaling Pathway

    PubMed Central

    Huang, Bin; Zhao, Ye; Tang, Shihuan; Wang, Lan; Liang, Rixin

    2013-01-01

    Buchang naoxintong capsule (BNC) is a traditional Chinese medicine approved for the treatment of cerebrovascular and cardiovascular diseases. However, little is known about the specific protective function or mechanism by which BNC protects against myocardial injury. This research was designed to investigate the cardioprotective effects of BNC in vitro model of hydrogen peroxide (H2O2)-induced H9c2 rat cardiomyoblasts. BNC intestinal absorption liquid was used in this study instead of drug-containing serum or extracting solution. Our study revealed that BNC preconditioning enhanced antioxidant function by increasing the activities of total-antioxygen capacity, total-superoxide dismutase, and catalase and by decreasing the production of reactive oxygen species and malondialdehyde. BNC preconditioning also activated extracellular signal-regulated kinases (ERK1/2) and inhibited apoptosis-related proteins such as poly ADP-ribose polymerase (PARP) and caspase-3. Additionally, preincubation with BNC reduced intracellular Ca2+ concentration, improved mitochondrial membrane potential, and decreased the apoptosis rate of H9c2 cells in a dose-dependent manner. These data demonstrated that BNC protects H9c2 cardiomyoblasts from H2O2-induced oxidative injury by increasing antioxidant abilities, activating ERK1/2, and blocking Ca2+-dependent and mitochondria-mediated apoptosis. Based on our results, the potency of BNC for protecting H9c2 cells from oxidative damage is comparable to that of trimetazidine. PMID:24223618

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

    PubMed Central

    Zhao, Li; Qin, Yuan

    2015-01-01

    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

  20. 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

  1. Cerium Oxide Nanoparticles Inhibits Oxidative Stress and Nuclear Factor-?B Activation in H9c2 Cardiomyocytes Exposed to Cigarette Smoke Extract

    PubMed Central

    Wang, Kangkai; Kolattukudy, Pappachan E.

    2011-01-01

    Cigarette smoke contains and generates a large amount of reactive oxygen species (ROS) that affect normal cellular function and have pathogenic consequences in the cardiovascular system. Increased oxidative stress and inflammation are considered to be an important mechanism of cardiovascular injury induced by cigarette smoke. Antioxidants may serve as effective therapeutic agents against smoke-related cardiovascular disease. Because of the presence of oxygen vacancies on its surface and self-regenerative cycle of its dual oxidation states, Ce3+ and Ce4+, cerium oxide (CeO2) nanoparticles offer a potential to quench ROS in biological systems. In this study, we determined the ability of CeO2 nanoparticles to protect against cigarette smoke extract (CSE)-induced oxidative stress and inflammation in cultured rat H9c2 cardiomyocytes. CeO2 nanoparticles pretreatment of H9c2 cells resulted in significant inhibition of CSE-induced ROS production and cell death. Pretreatment of H9c2 cells with CeO2 nanoparticles suppressed CSE-induced phosphorylation of I?B?, nuclear translocation of p65 subunit of nuclear factor-?B (NF-?B), and NF-?B reporter activity in H9c2 cells. CeO2 nanoparticles pretreatment also resulted in a significant down-regulation of NF-?B-regulated inflammatory genes tumor necrosis factor-?, interleukin (IL)-1?, IL-6, and inducible nitric-oxide synthase and further inhibited CSE-induced depletion of antioxidant enzymes, such as copper zinc superoxide dismutase, manganese superoxide dismutase, and intracellular glutathione content. These results indicate that CeO2 nanoparticles can inhibit CSE-induced cell damage via inhibition of ROS generation, NF-?B activation, inflammatory gene expression, and antioxidant depletion and may have a great potential for treatment of smoking-related diseases. PMID:21464334

  2. A physiologically relevant hyperthermia selectively activates constitutive hsp70 in H9c2 cardiac myoblasts and confers oxidative protection.

    PubMed

    Su, C Y; Chong, K Y; Chen, J; Ryter, S; Khardori, R; Lai, C C

    1999-04-01

    Whole body hyperthermia (42-43 degrees C for 15-20 min) elicits the formation of heat shock proteins (hsps) and improves cardiac recovery from subsequent ischemia/reperfusion. However, the beneficial effects of this response are compromised by initial tissue injury, which limits its clinical applicability. Using a simplified myocardial model (rat heart-derived H9c2 myoblasts) a hypothesis was tested that chronic, mild hyperthermia is as effective as acute heat shock in inducing the heat shock response. Our results indicate that 39 degrees C pre-conditioning evoked thermotolerance and oxidative resistance, but caused no detectable adverse effects. An improved survival after hydrogen peroxide (H2O2) exposure (40-54 microm for 3 h) was first observed in cells pre-conditioned at 39 degrees C for 24 h. As the duration of thermal pre-incubation increased, cells became more resistant than the control (37 degrees C) to a greater toxicity of H2O2(68 microm). An optimal oxidative protection developed by;4 days at 39 degrees C and this persisted for as long as the cells were incubated at this temperature. Three hsps are known to modulate cellular antioxidant defenses: the constitutive-hsp70 (hsc70), its inducible counterpart (hsp70), and hsp27. The authors found that mild hyperthermia selectively induced only hsc70, which demonstrates a lower temperature threshold for activation of hsc70. The initial protection against the milder H2O2challenge correlated with a homogenous distribution of pre-existing hsc70. The subsequent optimal protection was associated with an identical distribution pattern and a moderate increase of hsc70. These observations suggest that mild hyperthermia induces a beneficial adaptive response, in which hsc70 plays a critical role. PMID:10329212

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

    PubMed Central

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

    2014-01-01

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

  4. Insulin-like growth factor-1 protects H9c2 cardiac myoblasts from oxidative stress-induced apoptosis via phosphatidylinositol 3-kinase and extracellular signal-regulated kinase pathways

    Microsoft Academic Search

    Feng Hong; Si Joong Kwon; Bong Sook Jhun; Sung Soo Kim; Joohun Ha; Soo-Ja Kim; Nak Won Sohn; Chulhun Kang; Insug Kang

    2001-01-01

    Oxidative stress plays a critical role in cardiac injuries during ischemia\\/reperfusion. Insulin-like growth factor-1 (IGF-1) promotes cell survival in a number of cell types, but the effect of IGF-1 on the oxidative stress has not been elucidated in cardiac muscle cells. Therefore, we examined the role of IGF-1 signaling pathway in cell survival against H2O2-induced apoptosis in H9c2 cardiac myoblasts.

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

    SciTech Connect

    Park, Eun-Seok; Kang, Jun Chul; Kang, Do-Hyun; Jang, Yong Chang [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Yi, Kyu Yang [Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Daejeon, Chungnam, 305-600 (Korea, Republic of); Chung, Hun-Jong [Industrial Medicine Department, Chungju Hospital, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Park, Jong Seok [Department of Biomedical Laboratory Science, Taegu Health College, Taegu 702-722 (Korea, Republic of); Kim, Bokyung [Department of Physiology, Konkuk Medical School, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of); Feng, Zhong-Ping [Department of Physiology, College of Medicine, University of Toronto, Toronto, Ont., Canada M5S 1A8 (Canada); Shin, Hwa-Sup, E-mail: hsshin@kku.ac.kr [Department of Applied Biochemistry, Konkuk University, Chungju, Chungbuk, 380-701 (Korea, Republic of)

    2013-04-01

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

  6. Cardioprotective effect of Danshensu against myocardial ischemia/reperfusion injury and inhibits apoptosis of H9c2 cardiomyocytes via Akt and ERK1/2 phosphorylation.

    PubMed

    Yin, Ying; Guan, Yue; Duan, Jialin; Wei, Guo; Zhu, Yanrong; Quan, Wei; Guo, Chao; Zhou, Dan; Wang, Yanhua; Xi, Miaomiao; Wen, Aidong

    2013-01-15

    Danshensu, as the effective component of Salvia miltiorrhiza (Danshen), has been widely used in clinic for treatment of cardiovascular diseases in China. In the present study, we aimed to confirm the cardioprotective effect of Danshensu from myocardial ischemia/reperfusion (MI/R) injury in vivo, and investigate the potential molecular mechanisms against simulated ischemia/reperfusion (SI/R) induced cardiomyocytes apoptosis in vitro. The rat model of MI/R injury was induced by a transient vessel occlusion for 30 min and reperfusion for 3h, Danshensu significantly reduced myocardium infarct size and the production of creatine kinase-MB (CK-MB), cardiac troponin (cTnI) in serum. In vitro experiment, H9c2 cardiomyocytes were incubated with vehicle or ischemic buffer during hypoxia, and then it was reoxygenated with or without Danshensu. Danshensu markedly improved cell viability and decreased lactate dehydrogenase (LDH) release. We confirmed anti-apoptotic effect of Danshensu both by flow-cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and this effect was associated with the increase of Bcl-2/Bax ratio and the decrease of active caspase-3 expression. Western blot analysis also showed that Danshensu increased phosphorylation of Akt and extracellular signal-related kinase 1/2 (ERK1/2) in H9c2 cells, and the protective effects of Danshensu were partially inhibited by phosphatidylinositol 3'-kinase (PI3K) specific inhibitor wortmannin or ERK specific inhibitor U0126. Our results suggested that Danshensu could provide significant cardioprotection against MI/R injury, and the potential mechanisms might to suppression of cardiomyocytes apoptosis through activating the PI3K/Akt and ERK1/2 signaling pathways. PMID:23200898

  7. Icariin protects H9c2 cardiomyocytes from lipopolysaccharide?induced injury via inhibition of the reactive oxygen species?dependent c?Jun N?terminal kinases/nuclear factor-?B pathway.

    PubMed

    Zhou, Heng; Yuan, Yuan; Liu, Yuan; Ni, Jian; Deng, Wei; Bian, Zhou-Yan; Dai, Jia; Tang, Qi-Zhu

    2015-06-01

    The inflammatory response is involved in the pathogenesis of the most common forms of heart disease. Icariin has a number of pharmacological actions, including anti?inflammatory, anti?oxidative and anti?apoptotic effects. However, the role of icariin in cardiac inflammation has remained elusive. In the present study, H9c2 rat cardiomyocytes were stimulated by lipopolysaccharide (LPS) and treated with icariin. The results showed that icariin significantly reduced the increase in the mRNA expression of tumor necrosis factor ?, interleukin (IL)?1? and IL?6 that occurred in response to LPS. Furthermore, icariin regulated the expression of B-cell lymphoma 2 and B-cell lymphoma 2-associated X, and rescued H9c2 cells from apoptosis. Incubation with 2',7'?dichlorofluorescein diacetate demonstrated that icariin inhibited the production of intracellular reactive oxygen species (ROS). In addition, the phosphorylation of c?Jun N?terminal kinases (JNK), the degradation of inhibitor of ?B and the nuclear translocation of nuclear factor??B (NF??B) p65 in LPS?treated H9c2 cells were blocked by icariin treatment. These results suggested that icariin prevented cardiomyocytes from inflammatory response and apoptosis, and that this effect may be mediated by inhibition of the ROS?dependent JNK/NF??B pathway. PMID:25647547

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

    PubMed

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

    2015-08-01

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

  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 ?-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. 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

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

    PubMed Central

    ZHONG, LEI; ZHOU, XING-LU; LIU, YAN-SONG; WANG, YI-MIN; MA, FEI; GUO, BAO-LIANG; YAN, ZHAO-QI; ZHANG, QING-YUAN

    2015-01-01

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

  12. Differential gene expression profiles of two excitable rat cell lines after over-expression of WT- and C121W-?1 sodium channel subunits.

    PubMed

    Baroni, D; Moran, O

    2015-06-25

    Voltage-dependent sodium channels are membrane proteins essential for cell excitability. They are composed by a pore-forming ?-subunit, encoded in mammals by up to nine different genes, and four different ancillary ?-subunits. The expression pattern of the ? subunit isoforms confers the distinctive functional and pharmacological properties to different excitable tissues. ?-Subunits are important modulators of channel function and expression. Mutation C121W of the ?1-subunit causes an autosomal dominant epileptic syndrome without cardiac symptoms. In neuroectoderm GH3 and cardiac H9C2 cells, the over-expression of ?1 subunit augments ? subunit mRNA and protein levels as well as sodium current density. Interestingly, the introduction of the epileptogenic C121W-?1 subunit produces additional changes in the ?-subunit expression pattern of H9C2 cells, leaving unaltered the sodium channel isoform composition of GH3 cells. The challenge of the present work was to identify those genes that were differentially expressed in response to WT- or C121W-?1 subunit over-expression in the two rat cell lines under analysis. Hence, we analyzed the total mRNA extracted from control-untransfected and from WT- and C121W-?1-transfected GH3 and H9C2 cells by DNA-microarray. We found that, in agreement with their different embryonal origin, the over-expression of WT- and C121W-?1 subunits modifies the expression of different gene sets in GH3 and H9C2 cells. Focusing on the effects of the C121W mutation, we found that it causes the modification of 214 genes, most of them were down-regulated (202) in GH3 cells; on the contrary, it determined the up-regulation of only five genes in H9C2 cells. Interestingly, most genes modified by the C121W ?1 subunit are involved in pivotal processes of the cell such as cellular communication and protein expression. Our results confirm the important role of the sodium channel ?1 subunit in the control of NaCh gene expression, and highlight once more the tissue-specific effect of the C121W mutation. PMID:25827112

  13. Danshen-Gegen decoction protects against hypoxia/reoxygenation-induced apoptosis by inhibiting mitochondrial permeability transition via the redox-sensitive ERK/Nrf2 and PKC?/mKATP pathways in H9c2 cardiomyocytes.

    PubMed

    Chiu, Po Yee; Leung, Hoi Yan; Leong, Pou Kuan; Chen, Na; Zhou, Limin; Zuo, Zhong; Lam, Philip Y; Ko, Kam Ming

    2012-01-15

    Danshen-Gegen (DG) Decoction, an herbal formulation containing Radix Salviae miltiorrhizae and Radix Puerariae lobatae, has been used for the treatment of coronary artery disease in Chinese medicine. In the present study, the involvement of ERK- and PKC?-mediated pathways in the cytoprotection against apoptosis afforded by DG pretreatment was investigated in H9c2 cardiomyocytes. Pretreatment with a methanol extract of aqueous DG decoction protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated the enhancement of cellular reduced glutathione and a reduced sensitivity to Ca(2+)-induced mitochondrial permeability transition. DG extract increased the production of cytochrome P-450 (CYP)-dependent reactive oxygen species (ROS) in H9c2 cardiomyocytes, which was accompanied by the concomitant activation of ERK1/2 and PKC?. The DG-induced ERK1/2 activation was followed by the translocation of Nrf2 from the cytosol to the mitochondria accompanied by an increase in the expression of glutathione-related antioxidant proteins. In addition, the increased expression of hemeoxygenase-1 was associated with the activation of Akt and BAD, indicative of anti-apoptotic activity. In conclusion, DG treatment activated both ERK/Nrf2 and PKC? pathways, presumably by ROS arising from CYP-catalyzed processes, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment. PMID:21899994

  14. Effect of the Multitargeted Tyrosine Kinase Inhibitors Imatinib, Dasatinib, Sunitinib, and Sorafenib on Mitochondrial Function in Isolated Rat Heart Mitochondria and H9c2 Cells

    Microsoft Academic Search

    Yvonne Will; James A. Dykens; Sashi Nadanaciva; Brad Hirakawa; Joseph Jamieson; Lisa D. Marroquin; James Hynes; Shem Patyna; Bart A. Jessen

    2008-01-01

    Cardiovascular disease has recently been suggested to be a significant complication of cancer treatment with several kinase inhibitors. In some cases, the mechanisms leading to cardiotox- icity are postulated to include mitochondrial dysfunction, either as a primary or secondary effect. Detecting direct effects on mitochondrial function, such as uncoupling of oxidative phos- phorylation or inhibition of electron transport chain components,

  15. SIH—a novel lipophilic iron chelator—protects H9c2 cardiomyoblasts from oxidative stress-induced mitochondrial injury and cell death

    Microsoft Academic Search

    Tomáš Šim?nek; Christa Boer; R. Arthur Bouwman; Ronald Vlasblom; Amanda M. G. Versteilen; Martin Št?rba; Vladimír Geršl; Radomír Hrdina; P?emysl Po?ka; Jaap J. de Lange; Walter J. Paulus; René J. P. Musters

    2005-01-01

    Recent evidence suggests that oxidative stress is a common denominator in many aspects of cardiovascular pathogenesis. Free cellular iron plays a crucial catalytic role in the formation of highly toxic hydroxyl radicals, and thereby it may aggravate the contribution of oxidative stress to cardiovascular disease. Therefore, iron chelation may be an effective therapeutic approach, but the progress in this area

  16. Characterization of Cell-Matrix Adhesion Requirements for the Formation of Fascin Microspikes

    Microsoft Academic Search

    Josephine C. Adams

    1997-01-01

    Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell-substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix require- ments for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent

  17. Cell line characterization and authentication.

    PubMed

    Kaplan, J; Hukku, B

    1998-01-01

    Research and development involving the use of cell lines require precise knowledge of the purity and species of origin of the cell lines used. This can only be assured by periodic monitoring of cultured cell lines for possible contamination by other cells and for characteristics that authenticate the cell line identity. In the absence of such monitoring, inter- and intraspecies cell line contaminations are likely to occur in the laboratories of unsuspecting investigators and can result in the generation of mistaken conclusions with an attendant loss of investigators' time, effort, and resources. This chapter provides a history and an overview of the methods that have been developed for cell line authentication, the type of information each of these different methods provides, and how synthesis of that information can be used to characterize a cell line and confirm its identity. An effective cell line monitoring strategy is described that involves testing for a combination of genetic markers, including cell membrane species antigens, isoenzymes, chromosomes, and DNA fingerprints, and use of databases for each marker system to compare the results obtained with a test cell culture with results from an extensive panel of previously tested cell lines. PMID:9648106

  18. 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

  19. Plant cell lines in cell morphogenesis research.

    PubMed

    Seifertová, Daniela; Klíma, Petr; Pa?ezová, Markéta; Petrášek, Jan; Zažímalová, Eva; Opatrný, Zden?k

    2014-01-01

    Plant organs and tissues consist of many various cell types, often in different phases of their development. Such complex structures do not allow direct studies on behavior of individual cells. In contrast, populations of in vitro-cultured plant cells represent valuable tool for studying processes on a single-cell level, including cell morphogenesis. Here we describe characteristics of well-established model tobacco and Arabidopsis cell lines and provide detailed protocol on their cultivation, characterization, and genetic transformation. PMID:24132432

  20. Comparative Analysis of ?B-Crystallin Expression in Heat-Stressed Myocardial Cells In Vivo and In Vitro

    PubMed Central

    Chen, Hongbo; Adam, Abdelnasir; Cheng, Yanfen; Hartung, Jörg; Bao, Endong

    2014-01-01

    Relationships between ?B-crystallin expression patterns and pathological changes of myocardial cells after heat stress were examined in vitro and in vivo in this study using the H9C2 cell line and Sprague-Dawley rats, respectively. Histopathological lesions, characterized by acute degeneration, karyopyknosis and loss of a defined nucleus, became more severe in rat hearts over the course of heat stress treatment from 20 min to 100 min. The expression of ?B-crystallin in rat hearts showed a significant decrease (P<0.05) throughout the heat stress treatment period, except at the 40 min time point. Likewise, decreased ?B-crystallin expression was also observed in the H9C2 cell line exposed to a high temperature in vitro, although its expression recovered to normal levels at later time points (80 and 100 min) and the cellular damage was less severe. The results suggest that ?B-crystallin is mobilized early after exposure to a high temperature to interact with damaged proteins but that the myocardial cells cannot produce sufficient ?B-crystallin for protection against heat stress. Lower ?B-crystallin expression levels were accompanied by obvious cell/tissue damage, suggesting that the abundance of this protein is associated with protective effects in myocardial cells in vitro and in vivo. Thus, ?B-crystallin is a potential biomarker of heat stress. PMID:24466295

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

    PubMed

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

    2013-06-01

    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

  2. Development and characterization of insect cell lines

    Microsoft Academic Search

    Dwight E. Lynn

    1996-01-01

    With the wide availability of insect cell culture media, it can generally be considered a routine process to develop new cell lines. Exceptions to this statement do exist, of course. Difficulties may arise when attempting to culture a specific cell type. For example, while there are a few cell lines from insect fat body and at least one from the

  3. How Embryonic Stem Cell Lines are Made

    NSDL National Science Digital Library

    Use of embryonic stem cells in research has been hotly debated for several years. This animation presents the basics on how stem cell lines are established. This animation from Cold Spring Harbor Laboratory's Dolan DNA Learning Center presents how embryonic stem cell lines are made through a series of illustrations of the processes involved.

  4. Review article Immortal porcine lymphoblastoid cell lines

    E-print Network

    Paris-Sud XI, Université de

    Review article Immortal porcine lymphoblastoid cell lines: interest for veterinary and medical (Received 18 January 1994; accepted 22 April 1994) Summary ― Immortal lymphoblastoid cell lines of B porcine breeds. The lymphoblast immortalization has been putatively attributed to an oncogenic virus

  5. Infectious mononucleosis: immunoglobulin synthesis by cell lines

    PubMed Central

    Glade, Philip R.; Chessin, Lawrence N.

    1968-01-01

    Immunoglobulin synthesis by 16 long-term suspension cultures of mononuclear cells derived from peripheral blood of nine patients with heterophile-positive infectious mononucleosis (IM) has been demonstrated by radioimmunoelectrophoretic techniques. All cell lines synthesized molecules with IgG (?) heavy chain specificity. 14 cell lines produced molecules with IgM (?) heavy chain specificity and 11 cell lines produced molecules with IgA (?) heavy chain specificity. No detectable synthesis of molecules with IgD (?) heavy chain specificity was observed by these cell lines derived from peripheral blood of patients with IM. 13 cell lines produced molecules with type K (?) light chain specificity and 6 cell lines produced molecules with type L (?) light chain specificity. Of interest, 9 of 16 lines produced IgG (?), IgA (?), and IgM (?) heavy chain molecules and 5 of these cell lines produced molecules with type K (?) and type L (?) light chain specificity as well. Further characterization by combined polyacrylamide gel filtration, immunodiffusion, and radioautography indicated the presence of newly synthesized immunoglobulin molecules with both heavy and light polypeptide chains in close association as well as free light polypeptide chain synthesis. Investigation of the localization of immunoglobulin in single cells by immunofluorescent techniques revealed that 5-22% of cells in these lines were strongly reactive with a fluorescein isothiocyanate-conjugated rabbit antisera directed against the antigenic determinants of human IgG and cross-reactive with the determinants common to IgA and IgM. No heterophile antibody, heteroagglutinin, or hemolytic antibody could be demonstrated in these cell lines derived from peripheral blood of patients with heterophile-positive infectious mononucleosis. Images PMID:4175543

  6. 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

  7. Induced pluripotent stem (iPS) cells repair and regenerate infarcted myocardium.

    PubMed

    Singla, Dinender K; Long, Xilin; Glass, Carley; Singla, Reetu D; Yan, Binbin

    2011-10-01

    Cardiac myocyte differentiation reported thus far is from iPS cells generated from mouse and human fibroblasts. However, there is no article on the generation of iPS cells from cardiac ventricular specific cell types such as H9c2 cells. Therefore, whether transduced H9c2 cells, originally isolated from embryonic cardiac ventricular tissue, will be able to generate iPS cells and have the potential to repair and regenerate infarcted myocardium remains completely elusive. We transduced H9c2 cells with four stemness factors, Oct3/4, Sox2, Klf4, and c-Myc, and successfully reprogrammed them into iPS cells. These iPS cells were able to differentiate into beating cardiac myocytes and positively stained for cardiac specific sarcomeric ?-actin and myosin heavy chain proteins. Following transplantation in the infarcted myocardium, there were newly differentiated cardiac myocytes and formation of gap junction proteins at 2 weeks post-myocardial infarction (MI), suggesting newly formed cardiac myocytes were integrated into the native myocardium. Furthermore, transplanted iPS cells significantly (p < 0.05) inhibited apoptosis and fibrosis and improved cardiac function compared with MI and MI+H9c2 cell groups. Moreover, our iPS cell derived cardiac myocyte differentiation in vitro and in vivo was comparable to embryonic stem cells in the present study. In conclusion we report for the first time that we have H9c2 cell-derived iPS cells which contain the potential to differentiate into cardiac myocytes in the cell culture system and repair and regenerate infarcted myocardium with improved cardiac function in vivo. PMID:21542647

  8. Cell assessment by at-line microscopy.

    PubMed

    Babitzky, Alexander; Lindner, Patrick; Scheper, Thomas

    2014-01-01

    This protocol regards a microscopic application and software for image-guided monitoring of mammalian cells which grow in suspension cultures. It has been developed in order to establish an automated microscopic application for in situ and at-line cell monitoring in bioreactors (Akin et al., Biosens Bioelectron 26:4532-4537, 2011; Babitzky et al., At-line microscopic analysis of suspension cell cultures. In: ECCE/ECAB, the first joint European Congress of chemical engineering and applied biotechnology, September 25-29, 2011, Berlin, Germany, 2011. http://www.tci.uni-hannover.de, Poster). The application aims to assess the analysis of an appropriated sample volume of mammalian cell cultivation medium. The sample is injected into a microfluidic slide which is suitable for transmitted light microscopy and is attached to an automated microscope device, the at-line microscope. The major attribute of microscope automation ascribes to the camera software, which enables sequential image capturing and storing. Image analysis and cell detection are performed by the software that is based on the edge detection algorithm developed by Canny (IEEE Trans Pattern Anal Mach Intell 8:679-698, 1986; Finding edges and lines in images.Technical Report 720, MIT Artificial Intelligence Laboratory, 1983). The analysis results are cell count, morphological characteristics, and grayscale values of the detected cells. The presented setup can be applied to low-volume cultivations and has been successfully tested for monitoring CHO-K1 cell cultivation processes. PMID:24297425

  9. Mast cell and basophil cell lines: a compendium.

    PubMed

    Passante, Egle

    2014-01-01

    Mast cells and basophils play a crucial role during type I hypersensitivity reactions. However, despite efforts to elucidate their role in the pathogenesis of allergy and inflammation, our understanding of mast cell and basophil biology is still relatively scarce. The practical difficulty in obtaining a sufficient number of purified primary cells from biological samples has slowed down the process of reaching a full understanding of the physiological role of these functionally similar cell types. The establishment of several immortalized cell lines has been a useful tool to establish and perform sophisticated laboratory protocols that are impractical using primary cells. Continuous cell lines have been extensively used to investigate the allergen/IgE-mediated cell activation, to elucidate the degranulation dynamics, to investigate structural and functional properties of the high-affinity receptor (Fc?RI), and to test cell-stabilizing compounds. In this chapter we review the most widely used and better characterized mast cell and basophil cell lines, highlighting their advantages and drawbacks. It must be pointed out, however, that while cell lines represent a useful in vitro tool due to their easy manipulability and reduced culture costs, they often show aberrant characteristics which are not fully representative of primary cell physiology; results obtained with such cells therefore must be interpreted with due care. PMID:25149487

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

    SciTech Connect

    Felthaus, O. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany) [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Ettl, T.; Gosau, M.; Driemel, O. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Brockhoff, G. [Department of Gynecology and Obstetrics, University of Regensburg (Germany)] [Department of Gynecology and Obstetrics, University of Regensburg (Germany); Reck, A. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Zeitler, K. [Institute of Pathology, University of Regensburg (Germany)] [Institute of Pathology, University of Regensburg (Germany); Hautmann, M. [Department of Radiotherapy, University of Regensburg (Germany)] [Department of Radiotherapy, University of Regensburg (Germany); Reichert, T.E. [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany)] [Department of Oral and Maxillofacial Surgery, University of Regensburg (Germany); Schmalz, G. [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)] [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany); Morsczeck, C., E-mail: christian.morsczeck@klinik.uni-regensburg.de [Department of Operative Dentistry and Periodontology, University of Regensburg (Germany)

    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.

  11. Transitional cell cancer: establishment and characterization of cell lines.

    PubMed

    Elliott, A Y; Bronson, D L; Fraley, E E

    1978-12-01

    Eleven long-term (in culture more than 1 yr) cell lines were established from surgical specimens of human TCC. Characterization studies performed on the individual cell lines showed that each 1) demonstrated an abnormal human karyotype, 2) grew in soft agar, 3) exhibited rapid growth and multilayering 4) was free from microbial and HeLa cell contamination, 5) produced tumors in cheek pouches of immunosuppressed Syrian golden hamsters, 6) contained ultrastructural features consistently found in epithelial cells in culture, and 7) could be grown to high cell densities in roller-bottle cultures. PMID:748774

  12. Cancer stem cells from colorectal cancer-derived cell lines

    PubMed Central

    Yeung, Trevor M.; Gandhi, Shaan C.; Wilding, Jennifer L.; Muschel, Ruth; Bodmer, Walter F.

    2010-01-01

    Cancer stem cells (CSCs) are the subpopulation of cells within a tumor that can self-renew, differentiate into multiple lineages, and drive tumor growth. Here we describe a two-pronged approach for the identification and characterization of CSCs from colorectal cancer cell lines, using a Matrigel-based differentiation assay, and cell surface markers CD44 and CD24. About 20 to 30% of cells from the SW1222 cell line form megacolonies in Matrigel that have complex 3D structures resembling colonic crypts. The megacolonies’ capacity to self-renew in vitro is direct evidence that they contain the CSCs. Furthermore, just 200 cells from SW1222 megacolonies initiate tumors in NOD/SCID mice. We also showed that CD44+CD24+ cells enriched for colorectal CSCs in the HT29 and SW1222 cell lines, which can self-renew and reform all four CD44/CD24 subpopulations, are the most clonogenic in vitro and can initiate tumors in vivo. A single SW1222 CD44+CD24+ CSC, when grown in Matrigel, can form large megacolonies that differentiate into enterocyte, enteroendocrine, and goblet cell lineages. The HCT116 line does not differentiate or express CDX1, nor does it contain subpopulations of cells with greater tumor-forming capacity, suggesting that HCT116 contains mainly CSCs. However, forced expression of CDX1 in HCT116 leads to reduced clonogenicity and production of differentiating crypt-containing colonies, which can explain the selection for reduced CDX1 expression in many colorectal cancers. In summary, colorectal cancer cell lines contain subpopulations of CSCs, characterized by their cell surface markers and colony morphology, which can self-renew and differentiate into multiple lineages. PMID:20133591

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

    PubMed Central

    Kumar, Arvind; Selvakumar, S.

    2015-01-01

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

  14. TRANSFECTION OF INSECT CELL LINES USING POLYETHYLENIMINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Investigating citrullinated proteins in tumour cell lines

    PubMed Central

    2013-01-01

    Background The conversion of arginine into citrulline, termed citrullination, has important consequences for the structure and function of proteins. Studies have found PADI4, an enzyme performing citrullination, to be highly expressed in a variety of malignant tumours and have shown that PADI4 participates in the process of tumorigenesis. However, as citrullinated proteins have not been systematically investigated in tumours, the present study aimed to identify novel citrullinated proteins in tumours by 2-D western blotting (2-D WB). Methods Two identical two-dimensional electrophoresis (2-DE) gels were prepared using extracts from ECA, H292, HeLa, HEPG2, Lovo, MCF-7, PANC-1, SGC, and SKOV3 tumour cell lines. The expression profiles on a 2-DE gel were trans-blotted to PVDF membranes, and the blots were then probed with an anti-citrulline antibody. By comparing the 2-DE profile with the parallel 2-D WB profile at a global level, protein spots with immuno-signals were collected from the second 2-DE gel and identified using mass spectrometry. Immunoprecipitation was used to verify the expression and citrullination of the targeted proteins in tumour cell lines. Results 2-D WB and mass spectrometry identified citrullinated ?-enolase (ENO1), heat shock protein 60 (HSP60), keratin 8 (KRT8), tubulin beta (TUBB), T cell receptor chain and vimentin in these cell lines. Immunoprecipitation analyses verified the expression and citrullination of ENO1, HSP60, KRT8, and TUBB in the total protein lysates of the tumour cell lines. Conclusions The citrullination of these proteins suggests a new mechanism in the tumorigenic process. PMID:24099319

  16. Dual pathways of p53 mediated glucolipotoxicity-induced apoptosis of rat cardiomyoblast cell: activation of p53 proapoptosis and inhibition of Nrf2-NQO1 antiapoptosis.

    PubMed

    Wang, Hye Jin; Lee, Eun Young; Han, Seung Jin; Kim, Soo Hyun; Lee, Byung-Wan; Ahn, Chul Woo; Cha, Bong Soo; Lee, Hyun Chul

    2012-04-01

    Reactive oxygen species (ROS), driven by excessive levels of glucose and free fatty acids, appears to induce cell apoptosis. However, the underlying molecular mechanism of this process remains unclear in cardiac myocytes. We investigated the glucolipotoxicity effects of high glucose and palmitic acid (C16:0) on the rat cardiomyoblast cell line (H9c2) focusing on tumor suppressor p53. Cultured H9c2 rat cardiomyoblasts were exposed to palmitate and /or to an elevated glucose concentration for 18 hours. Only the glucolipotoxic condition of 30 mM glucose in combination with 250 ?M palmitate resulted in significant generation of ROS and upregulation of p53 which caused to an increased cleavage of caspase-3. On the other hand, the expression of NF-E2-related factor 2 (Nrf2) showed increased tendency while the expression of NAD(P)H: quinone oxidoreductase-1 (NQO1) was decreased. N-acetyl L cysteins and pifithrin-?, an inhibitor of p53 abrogated glucolipotoxicity-induced ROS generation and p53 expression. Chromatin immunoprecipitation analysis revealed that p53 interacted antioxidant responsive elements (ARE)-containing promoter of NQO1. Upregulated p53 counteracted the Nrf2-induced transcription of ARE-containing promoter of NQO1 gene and leaded to decrease in NQO1 expression. We demonstrated that the elevated p53 mediated glucolipotoxicity-induced apoptosis of rat cardiomyoblast cell through dual pathways: stimulating pro-apoptosis signaling as well as suppressing anti-apoptosis pathway of Nrf2-NQO1 signaling. PMID:22154326

  17. On the ontology based representation of cell lines.

    PubMed

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

    2012-01-01

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

  18. 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

  19. Metabolic profiling of insect cell lines: Unveiling cell line determinants behind system's productivity.

    PubMed

    Monteiro, Francisca; Bernal, Vicente; Saelens, Xavier; Lozano, Ana B; Bernal, Cristina; Sevilla, Angel; Carrondo, Manuel J T; Alves, Paula M

    2014-04-01

    Baculovirus infection boosts the host biosynthetic activity towards the production of viral components and the recombinant protein of interest, hyper-productive phenotypes being the result of a successful adaptation of the cellular network to that scenario. Spodoptera frugiperda derived Sf9 and Trichoplusia ni derived High Five cell lines have a major track record for the production of recombinant proteins, with High Five cells presenting higher productivities. A metabolic profiling of the two insect cell lines was pursued to underpin specific cellular traits behind productive phenotypes. Multivariate analysis identified cell-line dependent metabolic signatures linked to productivity. Pathway analysis highlighted cellular pathways of paramount importance in supporting infection and protein production. Moreover, better producer phenotypes proved to be correlated with the capacity of cells to shift their metabolism in favor of energy-generating pathways to fuel biosynthesis, a scenario observed in the High Five cell line. Metabolomic profiling allowed us to identify metabolic pathways involved in infection and recombinant protein production, which can be selected as targets for further improvement of the system. PMID:24258249

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

    PubMed Central

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

    2013-01-01

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

  1. 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.

  2. Characterization of a transformed rat retinal ganglion cell line

    Microsoft Academic Search

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

    2001-01-01

    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 ?2 E1A virus. In order to isolate retinal ganglion cells (RGC), single cell clones were chosen at random from the transformed cells.

  3. Characterization and Properties of Nine Human Ovarian Adenocarcinoma Cell Lines

    Microsoft Academic Search

    Simon P. Langdon; Sandra S. Lawrie; Frances G. Hay; Mary M. Hawkes; Amanda McDonald; Ian P. Hayward; Dick J. Schol; Jo Hilgers; Robert C. F. Leonard; John F. Smyth

    Four series of cell lines have been derived from patients with ovarian adenocarcinoma. Nine cell lines have been established at different stages of treatment: eight from malignant effusions and one from a solid metas tasis. Six lines were derived from the ascites or pleural effusion of patients with poorly differentiated adenocarcinoma: PEO1, PEO4, and PEO6 from one patient, PEA1 and

  4. A circulating factor(s) mediates cell depolarization in hemorrhagic shock.

    PubMed

    Evans, J A; Darlington, D N; Gann, D S

    1991-06-01

    Cell depolarization in hemorrhagic shock has been attributed to hypoperfusion, but the mechanism remains unclear. Suspensions of single cell lines loaded with the potential-sensitive fluorescent dye bis-(1,3-dibutylbarbiturioc acid) trimethine oxonal (DIBAC) and exposed for 30 minutes to rat plasma drawn either before or after hemorrhagic shock (bled 20 mL/kg: mean arterial blood pressure less than 40 mmHg) were studied. Plasma drawn after, but not before, hemorrhage led to partial depolarization regardless of cell type (rat H9C2 skeletal muscle, A-10 smooth muscle, C-9 liver, adrenal, kidney, red blood cell [RBC], white blood cell [WBC]) or species (cat, dog, pig RBC; cat WBC; mouse C2C12 skeletal muscle; and human intestinal smooth muscle [HISM]). Dialysis did not remove the factor(s), suggesting a molecular weight of more than 10,000 daltons. The factor appeared within 5 minutes of shock. The depolarization amplitude increased as a function of plasma concentration and demonstrated saturation kinetics indicating specific receptor binding. Cells were equivalently oxygenated, excluding hypoperfusion as a necessary condition for depolarization. Tumor necrosis factor or platelet activating factor alone or in combination were not effective in this system. Stable measurements can be obtained with this noninvasive system that avoids cell injury consequent to cell impalement with electrodes. This system provides a sensitive in vitro bioassay that should permit identification of the plasma factors mediating cell depolarization, as well as definition of the responsible intracellular mechanisms. PMID:2039285

  5. The pursuit of ES cell lines of domesticated ungulates

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R. (Oakland, CA)

    1989-01-01

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

  7. Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

    PubMed Central

    Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

    2013-01-01

    Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

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

    SciTech Connect

    Humphrey, Glen W.; Mekhedov, Elena; Blank, Paul S. [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Morree, Antoine de [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands)] [Center for Human Genetics, Leiden University Medical Center, Leiden (Netherlands); Pekkurnaz, Gulcin [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States); Nagaraju, Kanneboyina [Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010 (United States)] [Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC 20010 (United States); Zimmerberg, Joshua, E-mail: zimmerbj@mail.nih.gov [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)] [Program in Physical Biology, Eunice Kennedy Schriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 (United States)

    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.

  9. Polyamine synthesis in maize cell lines

    SciTech Connect

    Hiatt, A. (Scripps Clinic and Research Foundation, La Jolla, CA (USA))

    1989-08-01

    Uptake of ({sup 14}C)putrescine, ({sup 14}C)arginine, and ({sup 14}C)ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.

  10. Proteomics of cancer cell lines resistant to microtubule stabilizing agents

    PubMed Central

    Albrethsen, Jakob; Angeletti, Ruth H.; Horwitz, Susan Band; Yang, Chia-Ping Huang

    2013-01-01

    In spite of the clinical success of microtubule interacting agents (MIAs), a significant challenge for oncologists is the inability to predict the response of individual cancer patients to these drugs. In the present study, six cell lines were compared by 2D DIGE proteomics to investigate cellular resistance to the class of MIAs known as microtubule stabilizing agents (MSAs). The human lung cancer cell line A549 was compared to two drug-resistant daughter cell lines, a Taxol resistant cell line (AT12) and an epothilone B (EpoB) resistant cell line (EpoB40). The ovarian cancer cell line Hey was compared to two drug-resistant daughter cell lines, an EpoB resistant cell line (EpoB8) and an ixabepilone resistant cell line (Ixab80). All 2D DIGE results were validated by Western blot analyses. A variety of cytoskeletal and cytoskeleton-associated proteins were differentially expressed in drug resistant cells. Differential abundance of 14-3-3?, galectin-1 and phosphorylation of stathmin are worthy of further studies as candidate predictive biomarkers for MSAs. This is especially true for galectin-1, a ?-galactose-binding lectin that mediates tumor invasion and metastasis. Galectin-1 was greatly increased in EpoB- and ixabepilone-resistant cells and its suppression caused an increase in drug sensitivity in both drug-sensitive and -resistant Hey cells. Furthermore, the growth medium from resistant Hey cells contained higher levels of galectin-1, suggesting that galectin-1 could play a role in resistance to microtubule stabilizing agents. PMID:24252851

  11. Morphometric Subtyping for a Panel of Breast Cancer Cell Lines

    Microsoft Academic Search

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

    2009-01-01

    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

  12. The effects of oncolytic reovirus in canine lymphoma cell lines.

    PubMed

    Hwang, C C; Umeki, S; Igase, M; Coffey, M; Noguchi, S; Okuda, M; Mizuno, T

    2014-10-15

    Reovirus is a potent oncolytic virus in many human neoplasms that has reached phase II and III clinical trials. Our laboratory has previously reported the oncolytic effects of reovirus in canine mast cell tumour (MCT). In order to further explore the potential of reovirus in veterinary oncology, we tested the susceptibility of reovirus in 10 canine lymphoma cell lines. Reovirus-induced cell death, virus replication and infectivity were confirmed in four cell lines with variable levels of susceptibility. The level of Ras activation varied among the cell lines with no correlation with reovirus susceptibility. Reovirus-susceptible cell lines underwent apoptosis as proven by propidium iodide (PI) staining, Annexin V-FITC/PI assay, cleavage of PARP and inhibition of cell death by caspase inhibitor. A single intratumoral injection of reovirus suppressed the growth of canine lymphoma subcutaneous tumour in NOD/SCID mice. Unlike canine MCT, canine lymphoma is less susceptible to reovirus. PMID:25319493

  13. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines

    PubMed Central

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

    2014-01-01

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

  14. T-cell and mast cell lines respond to B-cell stimulatory factor 1.

    PubMed

    Mosmann, T R; Bond, M W; Coffman, R L; Ohara, J; Paul, W E

    1986-08-01

    The murine lymphokine B-cell stimulatory factor 1 (BSF-1) has been described previously in terms of its action on B lymphocytes. We now provide evidence that BSF-1 is also responsible for two additional biological activities. The first of these is the stimulation or maintenance of a state of activation in mouse T-cell lines. The second activity is the increase in the proliferative rate of certain mast cell lines costimulated with interleukin 3. The T-cell and mast cell activities are mediated by purified BSF-1 and copurify with BSF-1 from supernatants of certain T-cell lines. Each of these activities is inhibited by monoclonal anti-BSF-1 but not by monoclonal anti-interleukin 2 antibody. The antibody inhibition results also indicate that BSF-1 is the major or only source of these two activities in the activated T-cell supernatants that we have tested. PMID:3090545

  15. Studies on tumor-cell-induced platelet aggregation in human lung cancer cell lines

    Microsoft Academic Search

    Ernst Heinmöller; Rolf J. Weinel; Hans H. Heidtmann; Ursula Salge; Reiner Seitz; Inge Schmitz; Klaus M. Müller; Hubert Zirngibl

    1996-01-01

    We investigated the ability of human lung cancer cells of different histological subtypes to cause platelet aggregation. Tumor-cell-induced platelet aggregation (TCIPA) was studied in vitro in 13 human lung cancer cell lines [small-cell lung cancer (SCLC), squamous-cell lung cancer, large-cell lung cancer, adenocarcinoma and alveolar-cell lung cancer]. Three tumor cell lines failed to aggregate platelets in plateletrich plasma, whereas platelet

  16. Identification of a Novel Rhabdovirus in Spodoptera frugiperda Cell Lines

    PubMed Central

    Ma, Hailun; Galvin, Teresa A.; Glasner, Dustin R.; Shaheduzzaman, Syed

    2014-01-01

    ABSTRACT The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. IMPORTANCE The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell line. This paper reports on the identification and characterization of a novel rhabdovirus in Sf9 cells. This was accomplished through the use of next-generation sequencing platforms, de novo assembly tools, and extensive bioinformatics analysis. Rhabdovirus identification was further confirmed by transmission electron microscopy. Infectivity studies showed the lack of replication of Sf-rhabdovirus in human cell lines. The overall study highlights the use of a combinatorial testing approach including conventional methods and new technologies for evaluation of cell lines for unexpected viruses and use of comprehensive bioinformatics strategies for obtaining confident next-generation sequencing results. PMID:24672045

  17. Respiratory epithelial cell lines exposed to anoxia produced inflammatory mediator.

    PubMed

    Shahriary, Cyrus M; Chin, Terry W; Nussbaum, Eliezer

    2012-12-01

    Human epithelial cell lines were utilized to examine the effects of anoxia on cellular growth and metabolism. Three normal human epithelial cells lines (A549, NHBE, and BEAS-2B) as well as a cystic fibrosis cell line (IB3-1) and its mutation corrected cell line (C38) were grown in the presence and absence of oxygen for varying periods of time. Interleukin-8 (IL-8) levels were measured by enzyme-linked immunosorbent assay technique. Cellular metabolism and proliferation were assayed by determining mitochondrial oxidative burst activity by tetrazolium compound reduction. The viability of cells was indirectly measured by lactate dehydrogenase release. A549, NHBE, and BEAS-2B cells cultured in the absence of oxygen showed a progressive decrease in metabolic activity and cell proliferation after one to three days. There was a concomitant increase in IL-8 production. Cell lines from cystic fibrosis (CF) patients did not show a similar detrimental effect of anoxia. However, the IL-8 level was significantly increased only in IB3-1 cells exposed to anoxia after two days. Anoxia appears to affect certain airway epithelial cell lines uniquely with decreased cellular proliferation and a concomitant increased production of a cytokine with neutrophilic chemotactic activity. The increased ability of the CF cell line to respond to anoxia with increased secretion of inflammatory cytokines may contribute to the inflammatory damage seen in CF bronchial airway. This study indicates the need to use different cell lines in in vitro studies investigating the role of epithelial cells in airway inflammation and the effects of environmental influences. PMID:23301190

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

    PubMed Central

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

    2014-01-01

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

  19. Establishment and characterization of a bovine rectal myxoma cell line.

    PubMed

    Sahoo, Aditya P; Tiwari, Ashok K; Ravi Kumar, G; Chaturvedi, U; Veer Singh, Lakshya; Saxena, Shikha; Palia, S K; Jadon, N S; Singh, R; Singh, K P; Brahmaprakash, B S; Maiti, S K; Das, A K

    2015-02-01

    A new bovine cell line was developed from tumor biopsy material of rectum obtained from clinical case of 7 years old cattle with tumor mass obliterating the rectal opening. Histopathology of tumor revealed scattered stellate cells arranged singly or in clusters in loose mucinous ground substance, simulating myxoma. The cells obtained from tumor mass have been cultured for more than 36 months in DMEM supplemented with 10% fetal bovine serum (FBS). The population doubling time of this cell line was about 20.64 h. The cytogenetic analysis revealed several chromosomal abnormalities with bizarre karyotype. The origin of the cell line was confirmed by PCR amplification of 1086 bp fragment of 16s rRNA using bovine species specific primers. The new cell line would act as in vitro model to study many aspect of cancer biology such as tumor development, differentiation and therapeutics regimen to combat cancer. PMID:25441618

  20. Epithelial mesenchymal transition traits in human breast cancer cell lines

    Microsoft Academic Search

    T. Blick; E. Widodo; H. Hugo; M. Waltham; M. E. Lenburg; R. M. Neve; E. W. Thompson

    2008-01-01

    Epithelial mesenchymal transition (EMT) has long been associated with breast cancer cell invasiveness and evidence of EMT\\u000a processes in clinical samples is growing rapidly. Genome-wide transcriptional profiling of increasingly larger numbers of\\u000a human breast cancer (HBC) cell lines have confirmed the existence of a subgroup of cell lines (termed Basal B\\/Mesenchymal)\\u000a with enhanced invasive properties and a predominantly mesenchymal gene

  1. Coitinuous cell lines from embryonic tissues of ticks (Acari: Ixodidae)

    Microsoft Academic Search

    C. E. Yunker; J. Cory; H. Meibos

    1981-01-01

    Summary  Six new cell lines were established in continous culture from embryonic tissues of ixodid ticks. Four were fromDermacentor variabilis and two fromD. parumapertus. The cells are mostly fibroblastic and diploid. Mosquito-borne viruses (Chikungunya, O'nyong, yellow fever, and St. Louis\\u000a encephalitis) as well as tick-borne ones (Langat, Powassan, Colorado tick fever, Kemerovo, and Sawgrass) replicated in certain\\u000a of these cell lines,

  2. Pretreatment of therapeutic cells with poly(ADP-ribose) polymerase inhibitor enhances their efficacy in an in vitro model of cell-based therapy in myocardial infarct

    PubMed Central

    SZEPES, MÓNIKA; JANICSEK, ZSÓFIA; BENK?, ZSOLT; CSELENYÁK, ATTILA; KISS, LEVENTE

    2012-01-01

    The potential of cell-based therapies in diseases involving ischemia-reperfusion is greatly hampered by the excessive loss of administered cells in the harsh and oxidative environment where these cells are supposed to act. Therefore, we investigated if inhibition of poly(ADP-ribose) polymerase (PARP) in the therapeutically added cells would lead to their increased viability and, subsequently, to an enhanced effect in an in vitro simulated ischemia-reperfusion (I-R) setting. Ischemic conditions were simulated by oxygen and glucose deprivation for 160 min using H9c2 rat cardiomyoblast cells. After 30 min of reperfusion, these cells received 4 types of treatments: no added cells (I-R model), fluorescently labeled (Vybrant DiD) therapeutic H9c2 cells with vehicle (H9c2) or PARP inhibitor (10 ?M or 100 ?M PJ34) pretreatment. We assessed viability (live, apoptotic and necrotic) of both ‘postischemic’ and therapeutic cells with flow cytometric analysis using calcein-AM/ethidium homodimer-2 fluorescent staining after 24 h of co-culture. Further measurements on necrosis and metabolic activity were performed using lactate dehydrogenase (LDH) release and resazurin based assays. The percentage of surviving therapeutic cells increased significantly with PARP inhibition (untreated, 52.02±5.01%; 10 ?M PJ34, 63.38±4.50%; 100 ?M PJ34, 64.99±3.47%). The percentage of necrotic cells decreased in a similar manner (untreated, 37.23±4.40%; 10 ?M PJ34, 26.83±3.49%; 100 ?M PJ34, 24.96±2.43%). Notably, the survival of the cells that suffered I-R injury was also significantly higher when treated with PARP-inhibited therapeutic cells (I-R model, 36.44±5.05%; H9c2, 42.81±5.11%; 10 ?M PJ34, 52.07±5.80%; 100 ?M PJ34, 54.95±5.55%), while necrosis was inhibited (I-R model, 43.64±4.00%; H9c2, 37.29±4.55%; 10 ?M PJ34, 30.18±4.60%; 100 ?M PJ34, 25.52±3.47%). In subsequent experiments, PARP inhibition decreased LDH-release of the observed combined cell population and enhanced the metabolic activity. Thus, our results suggest that pretreating the therapeutically added cells with a PARP inhibitor could be beneficial in the setting of cell-based therapies. PMID:23165319

  3. [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

  4. 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

  5. Clonal cell lines from the rat central nervous system

    Microsoft Academic Search

    D. Schubert; S. Heinemann; W. Carlisle; H. Tarikas; B. Kimes; J. Patrick; J. H. Steinbach; W. Culp; B. L. Brandt

    1974-01-01

    Five neuronal and a large collection of putative glial cell lines from the rat central nervous system have been established in clonal cell culture and partially characterised. These cells shed new light on the distribution of neurotransmitter synthesis and brain-specific antigens among nerve and glia.

  6. A cell line (HBL-100) established from human breast milk

    Microsoft Academic Search

    Edwin V. Gaffney

    1982-01-01

    A continuous cell line (HBL-100) was obtained from primary cultures of cells derived from an early lactation sample of human milk. There was no evidence of a breast lesion in the milk donor. Karyotype analysis showed that all metaphases contained human chromosomes including a large acrocentric marker chromosome. Both desmosomes and cytoplasmic tonofibrils were observed during early passage. HBL-100 cells

  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. Role of cyclooxygenase-2 in Trypanosoma cruzi survival in the early stages of parasite host-cell interaction

    PubMed Central

    Moraes, Karen CM; Diniz, Lívia F; Bahia, Maria Terezinha

    2015-01-01

    Chagas disease, caused by the intracellular protozoan Trypanosoma cruzi, is a serious health problem in Latin America. During this parasitic infection, the heart is one of the major organs affected. The pathogenesis of tissue remodelling, particularly regarding cardiomyocyte behaviour after parasite infection and the molecular mechanisms that occur immediately following parasite entry into host cells are not yet completely understood. When cells are infected with T. cruzi, they develop an inflammatory response, in which cyclooxygenase-2 (COX-2) catalyses rate-limiting steps in the arachidonic acid pathway. However, how the parasite interaction modulates COX-2 activity is poorly understood. In this study, the H9c2 cell line was used as our model and we investigated cellular and biochemical aspects during the initial 48 h of parasitic infection. Oscillatory activity of COX-2 was observed, which correlated with the control of the pro-inflammatory environment in infected cells. Interestingly, subcellular trafficking was also verified, correlated with the control of Cox-2 mRNA or the activated COX-2 protein in cells, which is directly connected with the assemble of stress granules structures. Our collective findings suggest that in the very early stage of the T. cruzi-host cell interaction, the parasite is able to modulate the cellular metabolism in order to survives. PMID:25946241

  9. The effect of desferrioxamine on cell proliferation in human tumour cell lines.

    PubMed

    Siegers, C P; Bumann, D; Baretton, G

    1991-01-01

    Iron is known to have a stimulatory effect on cell proliferation whereas the iron-complexing agent desferrioxamine (DFO) has an inhibitory influence on the growth of cultured cells. The effect of iron salts and DFO on cell proliferation and DNA synthesis was studied on two established human tumour cell lines, a colon carcinoma (Caco-2) and a hepatoma-derived cell line (Hep.G2). Cell proliferation was estimated by the neutral red method, DNA synthesis by measuring the [(3)H]thymidine incorporation into the cells. For the analysis of the cell cycle the cells were marked with bromodeoxyuridine for S-phase cells and the monoclonal antibody Ki-67 for all proliferating cells. Ferric chloride stimulated cell proliferation in the Caco-2 line whereas there was no effect in the Hep.G2 line. DFO inhibited cell proliferation and DNA synthesis in both cell lines. Cell cycle analysis revealed a prolongation of the cell cycle by DFO, thereby reducing the number of cells entering the proliferating phases of the cell cycle in both cell lines. The data support the essential role of iron in cell proliferation and tumour growth. PMID:20732049

  10. A Stable Cranial Neural Crest Cell Line from Mouse

    PubMed Central

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

    2012-01-01

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

  11. DEVELOPMENT OF A BRAIN METASTATIC CANINE PROSTATE CANCER CELL LINE

    PubMed Central

    Thudi, Nanda K.; Shu, Sherry T.; Martin, Chelsea K.; Lanigan, Lisa G.; Nadella, Murali V.P.; Van Bokhoven, Adrie; Werbeck, Jillian L.; Simmons, Jessica K.; Murahari, Sridhar; Kisseberth, William C.; Breen, Matthew; Williams, Christina; Chen, Ching-Shih; McCauley, Laurie K.; Keller, Evan T.; Rosol, Thomas J.

    2010-01-01

    Background Prostate cancer in men has a high mortality and morbidity due to metastatic disease. The pathobiology of prostate cancer metastasis is not well understood and cell lines and animal models that recapitulate the complex nature of the disease are needed. Therefore, the goal of the study was to establish and characterize a new prostate cancer line derived from a dog with spontaneous prostate cancer. Methods A new cell line (Leo) was derived from a dog with spontaneous prostate cancer. Immunohistochemistry and PCR were used to characterize the primary prostate cancer and xenografts in nude mice. Subcutaneous tumor growth and metastases in nude mice were evaluated by bioluminescent imaging, radiography and histopathology. In vitro chemosensitivity of Leo cells to therapeutic agents was measured. Results Leo cells expressed the secretory epithelial cytokeratins (CK) 8, 18 and ductal cell marker, CK7. The cell line grew in vitro (over 75 passages) and was tumorigenic in the subcutis of nude mice. Following intracardiac injection, Leo cells metastasized to the brain, spinal cord, bone, and adrenal gland. The incidence of metastases was greatest to the central nervous system (80%) with a lower incidence to bone (20%) and the adrenal glands (16%). In vitro chemosensitivity assays demonstrated that Leo cells were sensitive to velcade and an HDAC-42 inhibitor with IC50 concentrations of 1.9 nM and 0.95 ?M respectively. Conclusion The new prostate cancer cell line (Leo) will be a valuable model to investigate the mechanisms of the brain and bone metastases. PMID:21321976

  12. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines

    Microsoft Academic Search

    Allan Bradley; Martin Evans; Matthew H. Kaufman; Elizabeth Robertson

    1984-01-01

    The recent availability in culture of embryo-derived pluripotential cells which exhibit both a normal karyotype and a high differentiative ability1-3 has encouraged us to assess the potential of these cells to form functional germ cells following their incorporation into chimaeric mice. We report here the results of blastocyst injection studies using three independently isolated XY embryo-derived cell lines (EK.CP1, EK.CC1.1

  13. Transport of paraquat by a renal epithelial cell line, MDCK.

    PubMed

    Chan, B S; Lazzaro, V A; Seale, J P; Duggin, G G

    1997-11-01

    Transport of paraquat (PQ), a herbicidal cation, was previously investigated in a proximal (LLC-PK1), renal epithelial cell line using permeable collagen-coated filters. PQ was actively transported from the basolateral side via a cation transport system by the LLC-PK1 cells. In the present study, the transport of PQ was investigated in a distal renal epithelial cell line, MDCK. PQ was predominantly transported from the basolateral to apical (B to A) side. The basolateral transport of PQ in MDCK cells was not saturable with increasing concentrations and not energy dependent. The flux and uptake of PQ was much lower in the MDCK than LLC-PK1 cells. It is concluded that MDCK, a distal renal tubular cell line, does not have an active transport system for PQ. PMID:9415931

  14. Neurectoderm markers retained in phenotypical skeletal muscle cells arising from a glial cell line

    Microsoft Academic Search

    Vanda A. Lennon; Susan Peterson; David Schubert

    1979-01-01

    Differentiation in vitro of striated muscle from apparently non-muscle precursor cells has been reported in thymus reticulum1, a fibroblast-like mouse embryo line2 and in a neuronelike cell line derived from a rat brain tumour3. Also Tomozawa and Sueko reported the differentiation of a peripheral neurotumour clonal stem cell line into separate neuronal and glial cell types4. We report here the

  15. Establishment of the DU.528 human lymphohemopoietic stem cell line

    PubMed Central

    1985-01-01

    We have established the DU.528 cell line from the pretreatment leukemia cells of a patient who underwent a T lymphoblastic-to-promyelocytic phenotype conversion during treatment with the adenosine deaminase inhibitor, deoxycoformycin. The cell line and clones obtained from it by limiting dilution have the same karyotype previously found in the patient's pretreatment T lymphoblasts and post-deoxycoformycin treatment promyelocytes. DU.528 cells in continuous culture for greater than 2 yr display a predominant undifferentiated T lymphoblastoid phenotype. These cells spontaneously generate progeny of at least three lineages, T lymphoid, granulocytic/monocytic, and erythroid. The surface marker most consistently expressed by DU.528 cells in the undifferentiated state is the 3A1 antigen, which has been found on prothymocytes in the embryonic thymus. Some undifferentiated DU.528 cells also expressed the IL-2 receptor, but no other T cell differentiation antigens. Exposure of DU.528 cells to a variety of agents induced myeloid maturation; adenosine and deoxyadenosine, in the presence of deoxycoformycin, induced expression of myeloid differentiation antigens. Our results suggest that DU.528 is a lymphohematopoietic stem cell line and support the hypothesis that differentiation of pluripotent stem cells may be altered by genetic deficiency of adenosine deaminase. DU.528 cells may provide a useful model for examining factors that regulate stem cell proliferation and differentiation. PMID:4056659

  16. Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

    Microsoft Academic Search

    Cristian M Ortiz; Tetsuo Ito; Yosuke Hashimoto; Satoshi Nagayama; Akira Iwai; Shigeru Tsunoda; Fumiaki Sato; Miguel Martorell; Jose Garcia; Ana Perez; Yutaka Shimada

    2010-01-01

    BACKGROUND: Fascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC). Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation. METHODS: We examined whether fascin is a potential target in ESCC

  17. A sub-40-ns chain FRAM architecture with 7-ns cell-plate-line drive

    Microsoft Academic Search

    Daisaburo Takashima; Susumu Shuto; Iwao Kunishima; Hiroyuki Takenaka; Yukihito Oowaki; Shin-ichi Tanaka

    1999-01-01

    A nonvolatile chain FRAM adopting a new cell-plate-line drive technique was demonstrated. Two key circuit techniques, a two-way metal cell-plate line and a cell-plate line shared with 16 cells, reduce cell-plate-line delay to 7 ns and reduce plate drive area to 1\\/5. The total cell-plate-line delay, including cell transistor delay due to eight cells in series, is reduced to 15

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

    PubMed

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

    2013-10-01

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

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

    Microsoft Academic Search

    Mariana Fernandez-Cobo; James F Holland; Beatriz GT Pogo

    2006-01-01

    BACKGROUND: Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related

  20. Variation in Hematopoietic Potential of Induced Pluripotent Stem Cell Lines

    Microsoft Academic Search

    Kasem Kulkeaw; Yuka Horio; Chiyo Mizuochi; Minetaro Ogawa; Daisuke Sugiyama

    2010-01-01

    Induced pluripotent stem (iPS) cells were originally generated from somatic cells by ectopic expression of four transcription\\u000a factor genes: Oct3\\/4, Sox2, Klf4 and c-Myc. Currently, iPS cell lines differ in tissue origin, the combination of factors used to construct them, the method of gene\\u000a delivery and expression of pluripotency markers. Thus to evaluate iPS cells for haematotherapy, the hematopoietic potential

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

    SciTech Connect

    Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A. (Tohoku Univ., Sendai (Japan))

    1991-01-01

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

  2. Modulation of melphalan cytotoxic activity in human melanoma cell lines.

    PubMed

    Supino, R; Caserini, C; Orlandi, L; Zaffaroni, N; Silvestrini, R; Vaglini, M; Zunino, F

    1996-07-01

    The aim of the present study was to potentiate the cytotoxic effects of melphalan through pharmacological and physical modulators. The combination of the cytotoxic agent with ethacrynic acid, a glutathione-S-transferase pi (GST pi) inhibitor, or topotecan, a topoisomerase I inhibitor, or mild hyperthermia was investigated. The selected cell lines exhibited variable levels of expression of GST pi, DNA topoisomerase I and heat-shock proteins. Mild hyperthermia (42 degrees C) alone potentiated melphalan cytotoxicity, especially in the two cell lines exhibiting low basal levels of HSP70 expression. The combination of the GST inhibitor with melphalan resulted in a potentiation of drug cytotoxicity only in JR8 cells, one of the two cell lines which expressed high levels of GST pi mRNA and which were the less responsive to ethacrinic acid alone. A synergistic interaction between topotecan and melphalan was observed only in the cell lines expressing low levels of topoisomerase I even if all cell lines exhibited a comparable sensitivity to this agent. The results support an involvement of GST and DNA topoisomerase in cell defense and response to the alkylating agent. However, the variable potentiation of the cytotoxic effects of melphalan achieved in different cell systems suggests that factors other than the level of expression of the modulation target are responsible of such potentiation. PMID:8862730

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

    Cancer.gov

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

  4. PI Control of Gene Expression in Tumorous Cell Lines 

    E-print Network

    Mendonca, Rouella J.

    2010-01-16

    PI CONTROL OF GENE EXPRESSION IN TUMOROUS CELL LINES A Thesis by ROUELLA JOAN MENDONCA Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of MASTER... OF SCIENCE May 2009 Major Subject: Electrical Engineering PI CONTROL OF GENE EXPRESSION IN TUMOROUS CELL LINES A Thesis by ROUELLA JOAN MENDONCA Submitted to the Office of Graduate Studies of Texas A&M University in partial...

  5. Baculovirus studies in new, indigenous lepidopteran cell lines.

    PubMed

    Pant, U; Sudeep, A B; Athawale, S S; Vipat, V C

    2002-01-01

    Eight lepidopteran cell lines were established recently and their susceptibility to different insect viruses was studied. Two Spodoptera litura cell lines from the larval and pupal ovaries, were found highly susceptible to S. litura nuclear polyhedrosis virus (SLNPV, 5-6 x 10(6) NPV/ml). The Helicoverpa armigera cell line from the embryonic tissue was highly susceptible to H. armigera NPV (HaNPV, 6.3 x 10(6) NPV/ml). These in vitro grown SLNPV and HaNPV caused 100% mortality to respective 2nd instar larvae. The susceptibility of the cryo-preserved cell lines to respective baculoviruses (SLNPV/HaNPV) was studied and no significant difference in their susceptibility status was observed. The cultures could grow as suspension culture on shakers and may find application for in vitro production of wild type/recombinant baculoviruses as bio-insecticides. S. litura and Bombyx mori cell lines from larval ovaries, were highly susceptible to Autographa californica NPV (5.5 x 10(6) NPV/ml) and Bombyx mori NPV (BmNPV, 6.1 x 10(6) NPV/ml) respectively. These cell lines may find application in baculovirus expression vector studies for the production of recombinant proteins, useful in the development of diagnostic kits or as vaccines. PMID:12561971

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

    PubMed Central

    Fernandez-Cobo, Mariana; Holland, James F; Pogo, Beatriz GT

    2006-01-01

    Background Searches for differentially expressed genes in tumours have made extensive use of array technology. Most samples have been obtained from tumour biopsies or from established tumour-derived cell lines. Here we compare cultures of non-immortalized breast cancer cells, normal non-immortalized breast cells and immortalized normal and breast cancer cells to identify which elements of a defined set of well-known cancer-related genes are differentially expressed. Methods Cultures of cells from pleural effusions or ascitic fluids from breast cancer patients (MSSMs) were used in addition to commercially-available normal breast epithelial cells (HMECs), established breast cancer cell lines (T-est) and established normal breast cells (N-est). The Atlas Human Cancer 1.2 cDNA expression array was employed. The data obtained were analysed using widely-available statistical and clustering software and further validated through real-time PCR. Results According to Significance Analysis of Microarray (SAM) and AtlasImage software, 48 genes differed at least 2-fold in adjusted intensities between HMECs and MSSMs (p < 0.01). Some of these genes have already been directly linked with breast cancer, metastasis and malignant progression, whilst others encode receptors linked to signal transduction pathways or are otherwise related to cell proliferation. Fifty genes showed at least a 2.5-fold difference between MSSMs and T-est cells according to AtlasImage, 2-fold according to SAM. Most of these classified as genes related to metabolism and cell communication. Conclusion The expression profiles of 1176 genes were determined in finite life-span cultures of metastatic breast cancer cells and of normal breast cells. Significant differences were detected between the finite life-span breast cancer cell cultures and the established breast cancer cell lines. These data suggest caution in extrapolating information from established lines for application to clinical cancer research. PMID:16626496

  7. Hedgehog signaling pathway is inactive in colorectal cancer cell lines.

    PubMed

    Chatel, Guillaume; Ganeff, Corine; Boussif, Naima; Delacroix, Laurence; Briquet, Alexandra; Nolens, Gregory; Winkler, Rosita

    2007-12-15

    The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines. PMID:17683069

  8. Rabeprazole exhibits antiproliferative effects on human gastric cancer cell lines.

    PubMed

    Gu, Mengli; Zhang, Yan; Zhou, Xinxin; Ma, Han; Yao, Hangping; Ji, Feng

    2014-10-01

    Intracellular proton extrusion in gastric cancer cells has been reported to promote cancer cell survival under acidic conditions via hydrogen/potassium adenosine triphosphatase (H(+)/K(+)-ATPase). Rabeprazole is a frequently used second-generation proton pump inhibitor (PPI) that irreversibly inactivates gastric H(+)/K(+)-ATPase. Therefore, we hypothesized that rabeprazole could reduce the viability of gastric cancer cells. In the present study, four human gastric cancer cell lines and one non-cancer gastric cell line were cultured. Cell viability, the ?- and ?-subunits of H(+)/K(+)-ATPase and cellular apoptosis were analyzed by dye exclusion assay, reverse transcription-polymerase chain reaction and annexin V-fluorescein isothiocyanate/propidium iodide staining, respectively. The expression level of total extracellular signal-regulated protein kinase 1/2 (ERK 1/2) and phosphorylated-ERK protein was detected by western blot analysis. Gastric cancer cell lines were more tolerant of the acidic culture media than non-cancer cells. Administration of rabeprazole led to a marked decrease in the viability of MKN-28 cells. Exposure to rabeprazole induced significant apoptosis in AGS cells. Rabeprazole completely inhibited the phosphorylation of ERK 1/2 in the MKN-28 cells, whereas the same effect was not observed in either the KATO III or MKN-45 cells. The ERK 1/2 inhibitor, PD98059, attenuated the viability of the AGS cells. A similar antiproliferative effect was observed in the rabeprazole treatment group. In addition, PD98059 and rabeprazole were able to efficaciously inhibit the phosphorylation of ERK 1/2 in the gastric cancer cells. Therefore, it was concluded that rabeprazole can attenuate the cell viability of human gastric cancer cells through inactivation of the ERK1/2 signaling pathway. The results of the present study demonstrate that rabeprazole inhibits the viability of gastric cancer cells in vitro and may serve as a novel antineoplastic agent. PMID:25202402

  9. Establishment and Characterization of Four New Human Non-Small Cell Lung Cancer Cell Lines1

    Microsoft Academic Search

    Pao-Min Loh; Gerald H. Clamori; Robert A. Robinson; Mark L. White; Bharati Hukku; Nicholas P. Rossi; Ward D. Peterson

    1984-01-01

    Four new human non-small cell lung cancer cell lines have been established in vitro. These cell lines have been character ized by (a) growth of a tumor in nude mice with histopathology similar to that of the primary, (b) isoenzyme patterns phenotypi- cally human and distinct from each other, (c) distinguishing karyotypic findings, (d) growth rate determinations, and (e) pres

  10. 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).

  11. p53 is frequently mutated in Burkitt's lymphoma cell lines.

    PubMed Central

    Farrell, P J; Allan, G J; Shanahan, F; Vousden, K H; Crook, T

    1991-01-01

    A panel of 12 Burkitt's lymphoma cell lines and four other B cell lines were tested for the presence of mutations in p53. Protein analysis using a mutant-specific antibody and sequencing of both cDNA and genomic DNA revealed changes relative to the standard p53 protein sequence in 12 of the 16 lines studied, including 10 of the BL lines. Mutation of p53 in the BL lines was usually accompanied by loss of the other allele of p53. Testing of the mutated p53 cDNAs for gain of transforming activity or loss of growth suppression activity showed that several of the BL mutants were functionally altered from wild-type p53. Images PMID:1915267

  12. Novel cell lines established from pediatric brain tumors

    PubMed Central

    Xu, Jingying; Erdreich-Epstein, Anat; Gonzalez-Gomez, Ignacio; Melendez, Elizabeth Y.; Smbatyan, Goar; Moats, Rex A.; Rosol, Michael; Biegel, Jaclyn A.

    2012-01-01

    The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat “fingerprints” matching that of the patient’s tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors. PMID:22120608

  13. Novel cell lines established from pediatric brain tumors.

    PubMed

    Xu, Jingying; Erdreich-Epstein, Anat; Gonzalez-Gomez, Ignacio; Melendez, Elizabeth Y; Smbatyan, Goar; Moats, Rex A; Rosol, Michael; Biegel, Jaclyn A; Reynolds, C Patrick

    2012-04-01

    The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat "fingerprints" matching that of the patient's tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors. PMID:22120608

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

    Microsoft Academic Search

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

    2006-01-01

    Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line

  15. Retinal ganglion cell line apoptosis induced by hydrostatic pressure

    Microsoft Academic Search

    Ashish Agar; Shaojuan Li; Neeraj Agarwal; Minas T. Coroneo; Mark A. Hill

    2006-01-01

    Cellular responses to changes in pressure are implicated in numerous disease processes. In glaucoma apoptosis of retinal ganglion cells (RGCs) is associated with elevated intra-ocular pressure, however, the exact cellular mechanisms remain unclear. We have previously shown that pressure can induce apoptosis in B35 and PC12 neuronal cell lines, using an in vitro model for pressure elevation. A novel RGC

  16. MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

    EPA Science Inventory

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

  17. Drug resistance in malignant rhabdoid tumor cell lines

    Microsoft Academic Search

    Gary B. Rosson; Timothy S. Vincent; Betty W. Oswald; Cynthia F. Wright

    2002-01-01

    Purpose: We evaluated the in vitro sensitivity of four malignant rhabdoid tumor (MRT) cell lines to six chemotherapeutic agents: 5-fluororuacil, vincristine, carboplatin, doxorubicin, etoposide, and paclitaxel. We also sought to determine whether a defect in the p53 signaling pathway may contribute to the pronounced drug resistance of MRT. Methods: MRT cells were treated with various concentrations of each drug and

  18. Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells

    Microsoft Academic Search

    Junying Yu; Maxim A. Vodyanik; Kim Smuga-Otto; Jessica Antosiewicz-Bourget; Jennifer L. Frane; Shulan Tian; Jeff Nie; Gudrun A. Jonsdottir; Victor Ruotti; Ron Stewart; Igor I. Slukvin; James A. Thomson

    2007-01-01

    Somatic cell nuclear transfer allows trans-acting factors present in the mammalian oocyte to reprogram somatic cell nuclei to an undifferentiated state. We show that four factors (OCT4, SOX2, NANOG, and LIN28) are sufficient to reprogram human somatic cells to pluripotent stem cells that exhibit the essential characteristics of embryonic stem (ES) cells. These induced pluripotent human stem cells have normal

  19. Genotypic and phenotypic characterization of two newly established renal cell carcinoma cell lines

    Microsoft Academic Search

    Ulf S. R. Bergerheim; Mats Söderhäll; Eugene Zabarovsky; Bo Franzén; Agneta Manneborg-Sandlund; Chunde Li; Stefan H. Jacobson; Gert Auer; Georg Klein; V. Peter Collins

    1996-01-01

    Two new cell lines from human renal cell carcinoma are reported. Primary cell cultures from 75 consecutive cases of nephrectomy and metastatic surgery due to different stages of RCC during 4 years were studied. Two cell cultures could be propagated for more than 50 passages in vitro. HN4 was derived from a grade III clear cell carcinoma. HN51 originated from

  20. Differential effects of monastrol in two human cell lines.

    PubMed

    Leizerman, I; Avunie-Masala, R; Elkabets, M; Fich, A; Gheber, L

    2004-08-01

    The kinesin-related protein HsEg5 plays essential roles in mitotic spindle dynamics. Although inhibition of HsEg5 has been suggested as an aid in cancer treatment, the effects of such inhibition on human cells have not been characterized. Here we studied the effects of monastrol, an allosteric HsEg5 inhibitor, on AGS and HT29 cell lines and compared them to those of taxol. While both cell lines were similarly sensitive to taxol, AGS cells were more sensitive to monastrol. The differences in sensitivity were determined by the degree of inhibitory effect on cell proliferation, reversibility of monastrol-induced G2/M arrest, intracellular phenotypes and induction of apoptosis. In both cell lines, monastrol-induced apoptosis was accompanied by mitochondrial membrane depolarization and poly-ADP-ribose polymerase 1 cleavage. In AGS, but not HT29 cells, monastrol-induced apoptosis involved a prominent cleavage of procaspases 8 and 3. While in AGS cells, monastrol induced the formation of symmetric microtubule asters only, in HT29 cells, asymmetric asters were also formed, which may be related to specific HsEg5 functions in HT29 cells. PMID:15316655

  1. Genetic design of an optimized packaging cell line for gene vectors transducing human B cells

    Microsoft Academic Search

    E Hettich; A Janz; R Zeidler; D Pich; E Hellebrand; B Weissflog; A Moosmann; W Hammerschmidt

    2006-01-01

    Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein–Barr virus (EBV), a human ?-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification

  2. 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

  3. Generation of islet-like cell aggregates from human non-pancreatic cancer cell lines.

    PubMed

    Kanafi, Mohammad Mahboob; Mamidi, Murali Krishna; Sureshbabu, Shalini Kashipathi; Shahani, Pradnya; Bhawna, Chandravanshi; Warrier, Sudha R; Bhonde, Ramesh

    2015-01-01

    To explore a novel source for the derivation of islets, we examined the differentiation potential of human non-pancreatic cancer cell lines, HeLa (cervical carcinoma cell line) and MCF-7 (breast cancer cell line). These cells were subjected to a serum-free, three-step sequential differentiation protocol which gave two distinct cell populations: single cells and cellular aggregates. Subsequent analysis confirmed their identity as pancreatic acinar cells and islet-like cell aggregates (ICAs), as evidenced by amylase secretion and diphenylthiocarbazone staining respectively. Reverse transcriptase-PCR and immunocytochemistry assessment of the ICAs revealed the expression of pancreatic specific markers Ngn-3, Glut-2, Pax-6 and Isl-1. These ICAs secreted insulin in response to glucose challenge, confirming their functionality. We propose that ICAs generated from HeLa and MCF-7 cell lines could form a promising in vitro platform of human islet equivalents (hIEQs) for diabetes research. PMID:25257585

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2009-01-01

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

  6. Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes

    Microsoft Academic Search

    Denise K. Walters; Valerie L. Goss; Eric P. Stoffregen; Ting-Lei Gu; Kimberly Lee; Julie Nardone; Laura McGreevey; Michael C. Heinrich; Michael W. Deininger; Roberto Polakiewicz; Brian J. Druker

    2006-01-01

    STAT5 is constitutively phosphorylated in leukemic cells in approximately 70% of acute myeloid leukemia (AML) patients. To identify kinase candidates potentially responsible for STAT5 phosphorylation, we used liquid chromatography–tandem mass spectrometry (LC–MS\\/MS) mass spectrometry to detect phosphoproteins in AML cell lines. We established TEL-ARG and BCR-ABL fusion proteins as the mechanism underlying STAT5 phosphorylation in HT-93 and KBM-3 cells, respectively.

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

    Microsoft Academic Search

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

    2007-01-01

    Parthenogenesis is one of the main, and most useful, methods to derive embryonic stem cells (ESCs), which may be an important source of histocompatible cells and tissues for cell therapy. Here we describe the derivation and characterization of two ESC lines (hPES-1 and hPES-2) from in vitro developed blastocysts following parthenogenetic activation of human oocytes. Typical ESC morphology was seen,

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

    PubMed Central

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

    2015-01-01

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

  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. 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.

  11. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

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

  12. Epstein-Barr Virus Infection of Human Astrocyte Cell Lines

    PubMed Central

    Menet, Anne; Speth, Cornelia; Larcher, Clara; Prodinger, Wolfgang M.; Schwendinger, Michael G.; Chan, Philippe; Jäger, Michael; Schwarzmann, Fritz; Recheis, Heidrun; Fontaine, Marc; Dierich, Manfred P.

    1999-01-01

    Epstein-Barr virus (EBV) is implicated in different central nervous system syndromes. The major cellular receptor for EBV, complement receptor type 2 (CR2) (CD21), is expressed by different astrocyte cell lines and human fetal astrocytes, suggesting their susceptibility to EBV infection. We demonstrated the infection of two astrocyte cell lines, T98 and CB193, at low levels. As infection was mediated by CR2, we used two stable CR2 transfectant astrocyte cell lines (T98CR2 and CB193CR2) to achieve a more efficient infection. We have monitored EBV gene expression for 2 months and observed the transient infection of T98 and T98CR2 cells and persistent infection of CB193 and CB193CR2 cells. The detection of BZLF1, BALF2, and BcLF1 mRNA expression suggests that the lytic cycle is initiated at early time points postinfection. At later time points the pattern of mRNA expressed (EBER1, EBNA1, EBNA2, and LMP1) differs from latency type III in the absence of LMP2A transcription and in the expression of BALF2 and BcLF1 but not BZLF1. A reactivation of the lytic cycle was achieved in CB193CR2 cells by the addition of phorbol esters. These studies identify astrocyte cell lines as targets for EBV infection and suggest that this infection might play a role in the pathology of EBV in the brain. PMID:10438862

  13. Modeling Adenovirus Latency in Human Lymphocyte Cell Lines ? †

    PubMed Central

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

    2010-01-01

    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

  14. The effects of 20-hydroxyecdysone on cell surface proteins and cell interactions in Drosophila melanogaster cell lines 

    E-print Network

    Stachowiak, Janice Ann

    1986-01-01

    THE EFFECTS OF 20-HYDROXYECDYSONE ON CELL SURFACE PROTEINS AND CELL INTERACTIONS IN DROSOPHILA MEIANOGASTER CELL LINES A Thesrs JANICE ANN STACHOWIAK Submitted to the Graduate College of Texas ASM Unrversz. ty rn partial fulfillment... (20-HOE), causes changes in intercellular adhesion resulting in evagination in Drosophila imaginal discs and ir. aggregation in Drosophila tissue culture cells by affecting the synthesis of cell surface proteins. The DrosophDa cell lines, L3 and S3...

  15. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

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

    2012-05-01

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

  16. ScanningElectronMicroscopicObservationof Two RetinoblastomaCell Lines

    Microsoft Academic Search

    Rosemary C. McFall; Rose Marie Nagy; Barbara T. Nagle; Loily M. McGreevy

    Two continuousretinoblastomacell lines were ob served by scanning electron microscopy. Both cell lines spontaneously grow as a suspensionof roundcells in clusters,chains, and unique ring (rosette)formations. Scanningelectronmicroscopy ofsuspension cellsreveals somevariationin the numberand frequencyof surface adornmentssuch as blebs, lamellipodia,and microvibli. Although thecelllinesare nonadherent to substratum and thereforeassumea sphericalform,highlyvillouscellsare notcharacteristic of the entirecellpopulations. WhenWERI-Rbl and Y79 are seededontoa polyorni thine-treatedsubstrate,aftachmentandgrowthas adher ent culturesare

  17. Molecular cytogenetic analysis of breast cancer cell lines

    PubMed Central

    Davidson, J M; Gorringe, K L; Chin, S-F; Orsetti, B; Besret, C; Courtay-Cahen, C; Roberts, I; Theillet, C; Caldas, C; Edwards, P A W

    2000-01-01

    The extensive chromosome rearrangements of breast carcinomas must contribute to tumour development, but have been largely intractable to classical cytogenetic banding. We report here the analysis by 24-colour karyotyping and comparative genomic hybridization (CGH) of 19 breast carcinoma cell lines and one normal breast epithelial cell line, which provide model examples of karyotype patterns and translocations present in breast carcinomas. The CGH was compared with CGH of 106 primary breast cancers. The lines varied from perfectly diploid to highly aneuploid. Translocations were very varied and over 98% were unbalanced. The most frequent in the carcinomas were 8;11 in five lines; and 8;17, 1;4 and 1;10 in four lines. The most frequently involved chromosome was 8. Several lines showed complex multiply-translocated chromosomes. The very aneuploid karyotypes appeared to fall into two groups that evolved by different routes: one that steadily lost chromosomes and at one point doubled their entire karyotype; and another that steadily gained chromosomes, together with abnormalities. All karyotypes fell within the range seen in fresh material and CGH confirmed that the lines were broadly representative of fresh tumours. The karyotypes provide a resource for the cataloguing and analysis of translocations in these tumours, accessible at http://www.path.cam.ac.uk/~pawefish. © 2000 Cancer Research Campaign PMID:11044355

  18. The role of calcium in differentiation of leukemic cell lines.

    PubMed

    Rephaeli, A; Aviram, A; Rabizadeh, E; Englender, T; Shaklai, M

    1990-04-01

    Increased calcium influx associated with differentiation of four human myeloid leukemic cell lines: HL-60, KG-1, U-937 and K-562, to either monocytic or granulocytic direction was demonstrated. Calcium influx was measured employing two methods; measurement of radioactive calcium influx rate at 4 degrees C and employing the fluorescent probe, fura-2 acetoxymethyl ester. The increase in Ca2+ influx was demonstrated with three chemically unrelated differentiation inducers: retinoic acid, 1 alpha, 25 dihydroxy vitamin D3 and dimethyl sulfoxide. Inhibitors of calcium uptake such as verapamil diltiazem and cromolyn, partially reduced differentiation, suggesting that differentiation of myeloid leukemic cell lines is dependent on the availability of extracellular calcium. PMID:2383856

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

    E-print Network

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

    2015-01-01

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

  20. 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

  1. 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

  2. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

    Ljungkvist, Anna S.E. [Department of Radiation Oncology, Radboud University Medical Center Nijmegen, Nijmegen (Netherlands) and Department of Radiation Sciences, Umeaa University, Umeaa (Sweden)]. E-mail: a.ljungkvist@rther.umcn.nl; Bussink, Johan [Department of Radiation Oncology, Radboud University Medical Center Nijmegen, Nijmegen (Netherlands); Kaanders, Johannes H.A.M. [Department of Radiation Oncology, Radboud University Medical Center Nijmegen, Nijmegen (Netherlands); Rijken, Paulus F.J.W. [Department of Radiation Oncology, Radboud University Medical Center Nijmegen, Nijmegen (Netherlands); Begg, Adrian C. [Division of Experimental Therapy, Netherlands Cancer Institute, Amsterdam (Netherlands); Raleigh, James A. [Department of Radiation Oncology, University of North Carolina School of Medicine, Chapel Hill, NC (United States); Kogel, Albert J. van der [Department of Radiation Oncology, Radboud University Medical Center Nijmegen, Nijmegen (Netherlands)

    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.

  3. Cytolytic cells induce HMGB1 release from melanoma cell lines.

    PubMed

    Ito, Norimasa; DeMarco, Richard A; Mailliard, Robbie B; Han, Jie; Rabinowich, Hannah; Kalinski, Pawel; Stolz, Donna Beer; Zeh, Herbert J; Lotze, Michael T

    2007-01-01

    High mobility group box 1 (HMGB1) is one of the recently defined damage-associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis. We induced melanoma cell death with cytolytic lymphokine-activated killing (LAK) cells, tumor-specific cytolytic T lymphocytes, TRAIL, or granzyme B delivery and assessed intracellular HMGB1 retention or release to investigate the mechanism of HMGB1 release by cytolytic cells. HMGB1 release from melanoma cells (451Lu, WM9) was detected within 4 h and 24 h following incubation with IL-2-activated PBMC (LAK activity). HLA-A2 and MART1 or gp100-specific cytolytic T lymphocytes induced HMGB1 release from HLA-A2-positive and MART1-positive melanoma cells (FEM X) or T2 cell-loaded, gp100-specific peptides. TRAIL treatment, however, induced HMGB1 release, and it is interesting that this extrinsic pathway-mediated cell death was blocked with the pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Conversely, granzyme B delivery did not induce HMGB1 release. HMGB1, along with other intracellular factors released from tumor cells induced by cytolysis, may be important components of the disordered tumor microenvironment. This has important implications for the immunotherapy of patients with cancer. Specifically, HMGB1 may promote healing or immune reactivity, depending on the nature of the local inflammatory response and the presence (or absence) of immune effectors. PMID:16968820

  4. The role of mitochondria in direct cell-to-cell connection dependent rescue of postischemic cardiomyoblasts.

    PubMed

    Pankotai, Eszter; Cselenyák, Attila; Rátosi, Orsolya; Lörincz, Judit; Kiss, Levente; Lacza, Zsombor

    2012-03-01

    In this in vitro study we induced ischemic injury on H9c2 rat cardiomyoblasts using the oxygen-glucose deprivation model (OGD). We monitored if the addition of healthy or mitochondria-depleted cells can save OGD treated cells from post-ischemic injury. We were able to significantly improve the surviving cell number of oxidatively damaged H9c2 cells by the addition of healthy cells to the culture. On the contrary, cells with disturbed mitochondria did not increase the number of surviving cells. High-resolution confocal time-lapse imaging also proved that mitochondria are drifting from cell-to-cell through tunneling membrane bridges, however, they do not get into the cytoplasm of the other cell. We conclude that addition of healthy cells to severly injured post-ischemic cardiomyoblasts can rescue them from death during the first 24h after reoxigenation. Grafted cells must maintain their mitochondria in an actively respiring state, and although cell contact is required for the mechanism, neither cell fusion nor organelle transfer occurs. This novel mechanism opens a new possiblity for cell-based cardiac repair in ischemic heart disease. PMID:21983690

  5. Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines.

    PubMed

    Lopez-Campistrous, Ana; Song, Xiaohua; Schrier, Adam J; Wender, Paul A; Dower, Nancy A; Stone, James C

    2012-08-01

    The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim(-) mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim(+) cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim(-)) and Daudi (Bim(+)), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases. PMID:22465296

  6. Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines

    PubMed Central

    Lopez-Campistrous, Ana; Song, Xiaohua; Schrier, Adam J.; Wender, Paul A.; Dower, Nancy A.; Stone, James C.

    2014-01-01

    The anti-cancer effects of bryostatin-1, a potent diacylglycerol analogue, have traditionally been attributed to its action on protein kinase C. However, we previously documented apoptosis in a B non-Hodgkin lymphoma cell line involving diacylglycerol analogue stimulation of Ras guanyl-releasing protein, a Ras activator, and Bim, a proapoptotic Bcl-2 family protein. To further explore the role of Bim, we examined several Bim-deficient B non-Hodgkin lymphoma cells for their responses to pico, a synthetic bryostatin-1-like compound. The Bim? mantle cell lymphoma cell lines Jeko-1, Mino, Sp53, UPN1, and Z138 and the Bim+ cell line Rec-1, as well as the Burkitt lymphoma cells lines BL2 (Bim?) and Daudi (Bim+), were examined for their response to pico using assays for proliferation and apoptosis as well as biochemical methods for Ras guanyl-releasing proteins and Bcl-2 family members. With the exception of UPN1, mantle cell lymphoma cell lines underwent pico-induced apoptosis, as did BL2. In some cases, hallmarks of apoptosis were substantially diminished in the presence of mitogen-activated protein kinase kinase inhibitors. Pico treatment generally led to increased expression of proapoptotic Bik, although the absolute levels of Bik varied considerably between cell lines. A pico-resistant variant of Z138 exhibited decreased Bik induction compared to parental Z138 cells. Pico also generally decreased expression of anti-apoptotic Bcl-XL and Mcl1. Although, these changes in Bcl-2 family members seem unlikely to fully account for the differential behavior of the cell lines, our demonstration of a potent apoptotic process in most cell lines derived from mantle cell lymphoma encourages a re-examination of diacylglycerol analogues in the treatment of this subset of B non-Hodgkin lymphoma cases. PMID:22465296

  7. THP-1 cell line: an in vitro cell model for immune modulation approach.

    PubMed

    Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

    2014-11-01

    THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. PMID:25130606

  8. Continuous porcine cell lines developed from alveolar macrophages

    Microsoft Academic Search

    H. M Weingartl; M Sabara; J Pasick; E van Moorlehem; L Babiuk

    2002-01-01

    Porcine monomyeloid cell lines were established following transfection of primary porcine alveolar macrophage cultures with plasmid pSV3neo, carrying genes for neomycin resistance and SV40 large T antigen. The parental clone 3D4 exhibited a relatively rapid doubling time (25.5 h), high plating efficiency and mixed phenotype with respect to growth on a solid support. Single cell cloning of the 3D4 parent

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  10. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

    ...ASN-0002 Authentication of Human Cell Lines: Standardization...Development Organization Workgroup. Human cell line samples are cells taken from a human being that can be grown in...can be used for scientific experiments, as examples of the...

  11. What is the nature of the RGC-5 cell line?

    PubMed

    Sippl, C; Tamm, E R

    2014-01-01

    The immortalized RGC-5 cell line has been widely used as a cell culture model to study the neurobiology of retinal ganglion cells (RGCs). The cells were originally introduced as derived from rat RGC showing expression of various neuronal markers, in particular the RGC-characteristic proteins Brn3 and Thy1. Recent data gave rise to concerns regarding the origin and nature of the cells. RGC-5 cells were identified to be of mouse origin and their expression of RGC characteristics was questioned by some laboratories. This article summarizes the available data on the properties of RGC-5, discusses common protocols for their differentiation and is aimed at providing researchers some guidelines on the benefits and limitations of RGC-5 for research. PMID:24664692

  12. Studies on BrdU labeling of hematopoietic cells: stem cells and cell lines.

    PubMed

    Pang, Lizhen; Reddy, Prem Veer; McAuliffe, Christina I; Colvin, Gerald; Quesenberry, Peter J

    2003-11-01

    Studies using chronic in vivo BrdU exposure, isolating primitive stem cells, and determining BrdU labeling, indicate that stem cells cycle. BrdU is also incorporated into DNA during damage/repair. DNA, which has incorporated BrdU due to cycle transit is heavier than normal, while the density of DNA with damage/repair incorporation is intermediate. DNA density of purified lineage-rhodamine low (rho(low)) Hoechst low (Ho(low)) stem cells or FDC-P1 cell line cells-was assessed in vitro, after exposure to cytokines and BrdU (cycling model) or cytokines and BrdU with bleomycin to induce strand breaks and hydroxyurea to halt cycle progression (damage/repair model). We determined DNA density using cesium chloride (CsCl) gradients and either fluorometry or dot blot chemiluminesence. DNA from BrdU labeled cycling Lin-rho(lo)Ho(lo) or FDC-P1 cells was heavier than normal DNA, while damage repair DNA had an intermediate density. We then assessed BrdU labeling of Lin-rho(lo)Ho(lo) cells in vivo. We found that 70.9% of lin-rho(lo)Ho(lo) cells labeled at 5 weeks. DNA density of these cells was low, in the damage/repair range, but similar results were obtained with stem cells, which had proliferated in vivo. Dilution of BrdU in in vitro culture of proliferating FDC-P1 cells also resulted in damage/repair density. We conclude that in vitro BrdU labeling models can distinguish between proliferation and damage/repair, but that we cannot obtain high enough in vivo levels to address this issue. All together, while we cannot absolutely exclude damage/repair as contributing to stem cell BrdU labeling, the data indicate that primitive bone marrow stem cells are probably a cycling population. PMID:14502565

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

    PubMed

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

    2013-10-01

    Cellular immortality happens upon impairment of cell-cycle checkpoint pathways (p53/p16/pRb), reactivation or up-regulation of telomerase enzyme, or upregulation of some oncogenes or oncoproteins leading to a higher rate of cell division.There are also some other factors and mechanisms involved in immortalisation, which need to be discovered. Immortalisation of cells derived from different sources and establishment of immortal cell lines has proven useful in understanding the molecular pathways governing cell developmental cascades in eukaryotic, especially human, cells. After the breakthrough of achieving the immortal cells and understanding their critical importance in the field of molecular biology, intense efforts have been dedicated to establish cell lines useful for elucidating the functions of telomerase, developmental lineage of progenitors, self-renewal potency, cellular transformation, differentiation patterns and some bioprocesses, like odontogenesis. Meanwhile, discovering the exact mechanisms of immortality, a major challenge for science yet, is believed to open new gateways toward understanding and treatment of cancer in the long term. This review summarises the methods involved in establishing immortality, its advantages and the challenges still being faced in this field. PMID:23723166

  14. Induced pluripotent stem cell lines derived from equine fibroblasts.

    PubMed

    Nagy, Kristina; Sung, Hoon-Ki; Zhang, Puzheng; Laflamme, Simon; Vincent, Patrick; Agha-Mohammadi, Siamak; Woltjen, Knut; Monetti, Claudio; Michael, Iacovos Prodromos; Smith, Lawrence Charles; Nagy, Andras

    2011-09-01

    The domesticated horse represents substantial value for the related sports and recreational fields, and holds enormous potential as a model for a range of medical conditions commonly found in humans. Most notable of these are injuries to muscles, tendons, ligaments and joints. Induced pluripotent stem (iPS) cells have sparked tremendous hopes for future regenerative therapies of conditions that today are not possible to cure. Equine iPS (EiPS) cells, in addition to bringing promises to the veterinary field, open up the opportunity to utilize horses for the validation of stem cell based therapies before moving into the human clinical setting. In this study, we report the generation of iPS cells from equine fibroblasts using a piggyBac (PB) transposon-based method to deliver transgenes containing the reprogramming factors Oct4, Sox2, Klf4 and c-Myc, expressed in a temporally regulated fashion. The established iPS cell lines express hallmark pluripotency markers, display a stable karyotype even during long-term culture, and readily form complex teratomas containing all three embryonic germ layer derived tissues upon in vivo grafting into immunocompromised mice. Our EiPS cell lines hold the promise to enable the development of a whole new range of stem cell-based regenerative therapies in veterinary medicine, as well as aid the development of preclinical models for human applications. EiPS cell could also potentially be used to revive recently extinct or currently threatened equine species. PMID:21347602

  15. A cell line model for the differentiation of human dendritic cells

    Microsoft Academic Search

    Carsten Berges; Cord Naujokat; Sarah Tinapp; Hubert Wieczorek; Alexandra Höh; Mahmoud Sadeghi; Gerhard Opelz; Volker Daniel

    2005-01-01

    We have identified human monocytic (THP-1) and myelogenous CD34+ (KG-1) leukemia cell lines that can be differentiated rapidly into mature dendritic cells (DCs) when cultured in serum-free medium containing GM-CSF, TNF-?, and ionomycin. These hematopoietic cell line-derived DCs are highly pure and monotypic, and display the morphologic, phenotypic, molecular, and functional properties of DCs generated from human donor-derived monocytes or

  16. Human cystic fibrosis embryonic stem cell lines derived on placental mesenchymal stromal cells

    Microsoft Academic Search

    S Deleu; E Gonzalez-Merino; N Gaspard; TMU Nguyen; P Vanderhaeghen; L Lagneaux; M Toungouz; Y Englert; F Devreker

    2009-01-01

    This study describes the production of two new human embryonic stem cell (hESC) lines affected by cystic fibrosis. These cell lines are heterozygous compounds, each a carrier of the DF508 mutations associated either with E585X or with 3849+10 kb C?T. The derivation process was performed on irradiated human placental mesenchymal stromal cells and designed to minimize contact with xeno-components. This

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

    Microsoft Academic Search

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

    2005-01-01

    In this paper we would like to address the key role of fabrication in the performance and lifetime of organic photovoltaics. The realization of a complete process line for the construction of large area organic photovoltaics (250 x 400 mm) is described. Among many of the factors that influence organic solar cell lifetime, oxygen and water exposure is the most

  18. 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...

  19. USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY

    EPA Science Inventory

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

  20. Differential Sensitivity in the Survival of Oligodendrocyte Cell Lines to

    E-print Network

    Bongarzone, Ernesto R.

    Differential Sensitivity in the Survival of Oligodendrocyte Cell Lines to Overexpression of Myelin in oligodendrocyte survival by overexpression studies in vitro and in vivo. The classic and sr proteolipids are targeted to different cellular com- partments in the oligodendrocyte, suggesting different cellular

  1. Selection of endosulfan-tolerant gram cell line

    Microsoft Academic Search

    R. P. Saxena; M. U. Beg

    1988-01-01

    Three alternate exposures of Cicer arietinum (gram) root callus to lethal (1 ppm) endosulfan-containing selective medium (SM) and non-selective medium (NSM) yielded a tolerant cell line (1 ESR). Such an induced resistance was retained during subculture on NSM for 15 generations tested so far. Higher soluble proteins and peroxidase activity were found in 1 ESR compared to mother tissue.

  2. Expression of melatoninergic receptors in human placental choriocarcinoma cell lines

    Microsoft Academic Search

    Dave Lanoix; Rodney Ouellette; Cathy Vaillancourt

    2006-01-01

    BACKGROUND: Melatonin crosses the placenta and enters the fetal circulation. Moreover, experimental data sug- gest a possible influence of melatonin on placental function and fetal development in humans. To date, the expression and role of melatonin receptors in human placenta choriocarcinoma cell lines and in human term placental tissues remain to be elucidated. METHODS AND RESULTS: Results from RT-PCR, western

  3. Radiosensitization of colorectal carcinoma cell lines by histone deacetylase inhibition

    PubMed Central

    Flatmark, Kjersti; Nome, Ragnhild V; Folkvord, Sigurd; Bratland, Ĺse; Rasmussen, Heidi; Ellefsen, Mali Strand; Fodstad, Řystein; Ree, Anne Hansen

    2006-01-01

    Background The tumor response to preoperative radiotherapy of locally advanced rectal cancer varies greatly, warranting the use of experimental models to assay the efficacy of molecular targeting agents in rectal cancer radiosensitization. Histone deacetylase (HDAC) inhibitors, agents that cause hyperacetylation of histone proteins and thereby remodeling of chromatin structure, may override cell cycle checkpoint responses to DNA damage and amplify radiation-induced tumor cell death. Methods Human colorectal carcinoma cell lines were exposed to ionizing radiation and HDAC inhibitors, and cell cycle profiles and regulatory factors, as well as clonogenicity, were analyzed. Results In addition to G2/M phase arrest following irradiation, the cell lines displayed cell cycle responses typical for either intact or defective p53 function (the presence or absence, respectively, of radiation-induced expression of the cell cycle inhibitor p21 and subsequent accumulation of G1 phase cells). In contrast, histone acetylation was associated with complete depletion of the G1 population of cells with functional p53 but accumulation of both G1 and G2/M populations of cells with defective p53. The cellular phenotypes upon HDAC inhibition were consistent with the observed repression of Polo-like kinase-1, a regulatory G2/M phase kinase. Following pre-treatment with HDAC inhibitors currently undergoing clinical investigation, the inhibitory effect of ionizing radiation on clonogenicity was significantly amplified. Conclusion In these experimental models, HDAC inhibition sensitized the tumor cells to ionizing radiation, which is in accordance with the concept of increased probability of tumor cell death when chromatin structure is modified. PMID:16887021

  4. Endogenously produced nitric oxide inhibits endothelial cell growth as demonstrated using novel antisense cell lines

    PubMed Central

    Cartwright, Judith E; Johnstone, Alan P; Whitley, Guy St J

    2000-01-01

    Proliferation of endothelial cells is a vital component of vascular repair and angiogenesis. The endothelial cell mediator, nitric oxide (NO) has been reported both to inhibit and to promote endothelial cell proliferation. In this study we have generated cell lines which constitutively express antisense RNA to a region of inducible nitric oxide synthase (iNOS) from a murine endothelial cell line, sEnd-1. In response to stimulation with lipopolysaccharide (LPS) and interferon-? (IFN-?) these antisense cells had no detectable RNA for endogenous iNOS, barely detectable iNOS protein and produced 82% less NO than did the control transfected line. Stimulation of the control transfected line caused significant NO production and inhibition of cell growth whereas for the antisense line, producing little NO in response to stimulation, proliferation remained the same as for unstimulated cells. No differences in cell death were observed between unstimulated and LPS/IFN-? stimulated cells. The data presented in this study directly demonstrate that NO derived endogenously from iNOS inhibits proliferation of endothelial cells. This approach overcomes problems in other studies where NO donors or non-isoform specific inhibitors of NO synthase have been used. PMID:10960079

  5. Metal mutagenesis in transgenic Chinese hamster cell lines.

    PubMed

    Klein, C B; Kargacin, B; Su, L; Cosentino, S; Snow, E T; Costa, M

    1994-09-01

    Metals are toxic agents for which genotoxic effects are often difficult to demonstrate. To study metal mutagenesis, we have used two stable hprt/gpt+ transgenic cell lines that were derived from Chinese hamster V79 cells. Both the G12 and G10 cell lines are known to be very sensitive to clastogens such as X-rays and bleomycin, with the mutagenic response of the integrated xanthine guanine phosphoribosyl transferase (gpt) gene in G10 usually exceeding that of the same gene in the transgenic G12 cells. In studies with carcinogenic insoluble nickel compounds, a high level of mutagenesis was found at the gpt locus of G12 cells but not at the endogenous hypoxanthine phosphoribosyl transferase (hprt) locus of V79 cells. We have since demonstrated the similar recovery of a high frequency of viable G12 mutants with other insoluble nickel salts including nickel oxides (black and green). The relative mutant yield for the insoluble nickel compounds (G12 > G10) is the opposite of that obtained with nonmetal clastogens (G10 > G12). In the G12 cells, nickel mutagenesis may be related to the integration of the gpt sequence into a heterochromatic region of the genome. For some of the insoluble nickel compounds, significant inhibition of both cytotoxicity and mutant yield resulted when the G12 cells were pretreated with vitamin E. In comparison with the nickel studies, the mutagenic responses to chromium compounds in these cell lines were not as dramatic. Mutagenesis of the gpt target could not be demonstrated with other metals such as mercury or vanadium. PMID:7843139

  6. Cytotoxicity and genotoxicity of phenazine in two human cell lines.

    PubMed

    McGuigan, Claire F; Li, Xing-Fang

    2014-06-01

    Phenazine was recently identified as a drinking water disinfection byproduct (DBP), but little is known of its toxic effects. We examined in vitro cytotoxicity and genotoxicity of phenazine (1.9-123 ?M) in HepG2 and T24 cell lines. Cytotoxicity was determined by an impedance-based real-time cell analysis instrument. The BrdU (5-bromo-2'-deoxyuridine) proliferation and MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) viability assays were used to examine mechanisms of cytotoxicity. Genotoxicity was determined using the alkaline comet assay. Concentration-dependent cytotoxicity was observed in HepG2 cells, primarily due to an antiproliferative effect (BrdU 24 h IC50: 11 ?M; 48 h IC50: 7.8 ?M) observed as low as 1.9 ?M. T24 cells experienced a minor antiproliferative effect (BrdU 24 h IC50: 47 ?M; 48 h IC50: 17 ?M). IC50 values for HepG2 proliferation and viability were 54-77% lower compared to T24 cells. In both cell lines, IC50 values for proliferation were 66-90% lower than those for viability. At phenazine concentrations producing equivalent cytotoxicity, HepG2 cells (1.9-30.8 ?M) experienced no significant genotoxic effects, while T24 cells (7.7-123 ?M) experienced significant genotoxicity at ?61.5 ?M. While these effects were seen at phenazine concentrations above those found in disinfected water, the persistence of the antiproliferative effect and the differential toxicity in each cell line deserves further study. PMID:24380821

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

    PubMed Central

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

    2015-01-01

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

  8. Establishment and characterization of two divergent cell lines derived from a human chromophobe renal cell carcinoma.

    PubMed Central

    Gerharz, C. D.; Moll, R.; Störkel, S.; Ramp, U.; Hildebrandt, B.; Molsberger, G.; Koldovsky, P.; Gabbert, H. E.

    1995-01-01

    The chromophobe renal cell carcinoma is a distinct type of renal cancer presumably derived from the intercalated cell of the collecting duct system and exhibiting a better prognosis than other types of renal cell carcinoma. Chromophobe carcinomas can be separated from other types of renal cell carcinoma by their characteristic cytomorphology, ultrastructural appearance, cytoskeletal architecture, and cytogenetic aberrations. As no permanent cell line of the chromophobe tumor type has previously been described, we are the first to report on the successful establishment and characterization of two divergent permanent cell lines, ie, chrompho-A and chrompho-B, derived from the same chromophobe renal cell carcinoma. With immunocytochemistry, two-dimensional gel electrophoresis, and Western blot, chrompho-A and chrompho-B exclusively exhibited cytokeratins (Nos. 7, 8, 18, and 19) but not vimentin. Ultrastructural studies revealed numerous cytoplasmic microvesicles as well as coated vesicles that are known to be characteristic features of the intercalated cell. Chrompho-B cells exhibited a shorter mean population doubling time (tD = 43 hours) than chrompho-A cells (tD = 51 hours). Both cell lines failed to produce tumors in nude mice with the subrenal capsule assay. Cytogenetic analyses revealed hyperdiploid chromosome numbers in both cell lines with telomeric associations as well as numeric aberrations known from chromophobe renal cell carcinomas in vivo. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 7 PMID:7717462

  9. Off-line test of the KISS gas cell

    NASA Astrophysics Data System (ADS)

    Hirayama, Yoshikazu; Watanabe, Yutaka; Imai, Nobuaki; Ishiyama, Hironobu; Jeong, Sun-Chan; Miyatake, Hiroari; Oyaizu, Michihiro; Kim, Yung Hee; Mukai, Momo; Matsuo, Yukari; Sonoda, Tetsu; Wada, Michiharu; Huyse, Mark; Kudryavtsev, Yuri; Van Duppen, Piet

    2013-12-01

    The KEK Isotope Separation System (KISS) has been constructed at RIKEN to study the ?-decay properties of neutron-rich isotopes with neutron numbers around N = 126 for application to astrophysics. A key component of KISS is a gas cell filled with argon gas at a pressure of 50 kPa to stop and collect the unstable nuclei, where the isotopes of interest will be selectively ionized using laser resonance ionization. We have performed off-line tests to study the basic properties of the gas cell and of KISS using nickel and iron filaments placed in the gas cell.

  10. Isolation and Characterization of Spheroid Cells from Human Malignant Melanoma Cell Line WM266-4

    Microsoft Academic Search

    Y. R. Na; S. H. Seok; D. J. Kim; J. H. Han; T. H. Kim; H. Jung; B. H. Lee; J. H. Park

    2009-01-01

    Background\\/Aims: Spheroid cells which can grow as nonattached spheroids in vitro culture condition are considered as tumor-initiating cells that have properties similar to those of stem cells. However, the existence of spheroid cells in WM-266-4, a human malignant metastatic melanoma cell line, has not yet been reported. Methods: Accordingly, we investigated whether WM-266-4 can form spheroids, and characterized these spheroids

  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-?H2 to set up experimental systems for cell cycle studies. We derived 2 populations from EndoC-?H2 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. Efficacy of all-trans retinoid acid in preventing nickel induced cardiotoxicity in myocardial cells of rats.

    PubMed

    Lou, Siyue; Zhong, Like; Yang, Xiaochun; Xue, Tao; Gai, Renhua; Zhu, Difeng; Zhao, Yuqin; Yang, Bo; Ying, Meidan; He, Qiaojun

    2013-01-01

    Nickel, a metal commonly found in battery plants and welding factories, has potential cardiotoxicity, while all-trans retinoid acid (atRA) can promote cardiovascular repair and myocardial recovery. The purpose of this study was to investigate whether atRA could prevent cardiotoxicity induced by nickel both in vitro and in vivo. In the study, a rat myocardial cell line (H9c2) exposed to different concentrations of nickel chloride (NiCl(2)) displayed apoptotic features accompanied by reactive oxygen species generation. In addition, NiCl(2) also caused obvious apoptosis and systolic dysfunction in primary myocardial cells. Treatment with atRA efficiently attenuated the cytotoxicities triggered by NiCl(2) as it significantly mitigated ROS generation and decreased MAP kinases activity in NiCl(2)-treated cardiomyocytes. Additionally, NiCl(2) exposure caused obvious arrhythmia in Sprague-Dawley rats with the maximum tolerance dose of NiCl(2) between 2 and 3mg/kg. A combinational intragastric administration of 40mg/kg atRA can partially reverse NiCl(2)-induced arrhythmia in rats. Our results suggested that atRA might have therapeutic potential in alleviating the adverse effects of nickel on the cardiovascular system. PMID:22989704

  13. Cetuximab enhances the efficacy of bortezomib in squamous cell carcinoma cell lines

    Microsoft Academic Search

    Jens Wagenblast; Mehran Baghi; Christoph Arnoldner; Sotirios Bisdas; Wolfgang Gstöttner; Hanns Ackermann; Angelika May; Markus Hambek; Rainald Knecht

    2009-01-01

    Purpose  Proteasome inhibition has been shown to be effective in multiple myeloma and solid tumor models. In this in vitro study, we\\u000a investigated the antitumor effect of bortezomib (Velcade®) in combination with cetuximab in squamous cell carcinoma cell lines (SCC).\\u000a \\u000a \\u000a \\u000a Methods  Dose-escalation studies were performed in five squamous cell carcinoma cell lines using bortezomib or cetuximab alone or in\\u000a combination. Cell survival

  14. 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.

  15. 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 ?-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. PMID:24863237

  16. Ecdysone and the cell cycle: investigations in a mosquito cell line.

    PubMed

    Fallon, Ann M; Gerenday, Anna

    2010-10-01

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

  17. Identification of cell surface antigen expression in canine hepatocellular carcinoma cell lines.

    PubMed

    Fujimoto, Ayumi; Neo, Sakurako; Ishizuka, Chinatsu; Kato, Takashi; Segawa, Kazuhito; Kawarai, Shinpei; Ogihara, Kikumi; Hisasue, Masaharu; Tsuchiya, Ryo

    2013-01-01

    The characteristics of surface antigens in canine hepatocellular carcinoma (cHCC) have not been clarified. The objective of this study was to investigate surface antigens, which are considered as stem/progenitor or cancer cell markers, in cHCC cell lines. Expression of various antigens including CD29, CD34, CD44, CD90, CD133 and Dlk-1 was assessed in four cHCC cell lines by flow cytometry. CD44, CD133 and Dlk-1 expression was detectable in all cell lines, and three cell lines expressed CD29. These results indicate that CD29, CD44, CD133 and Dlk-1 have potential as suitable markers in cHCC identification, suggesting that these findings will contribute to the establishment of an early diagnostic tool for the identification of hepatocellular maturation processes. PMID:23412833

  18. Establishment and characterization of four new human non-small cell lung cancer cell lines.

    PubMed

    Loh, P M; Clamon, G H; Robinson, R A; White, M L; Hukku, B; Rossi, N P; Peterson, W D

    1984-08-01

    Four new human non-small cell lung cancer cell lines have been established in vitro. These cell lines have been characterized by (a) growth of a tumor in nude mice with histopathology similar to that of the primary, (b) isoenzyme patterns phenotypically human and distinct from each other, (c) distinguishing karyotypic findings, (d) growth rate determinations, and (e) presence of epidermal growth factor receptors. Each of the cell lines will form colonies when directly seeded into a flask without soft agar. The development and availability of the four cell lines may facilitate in vitro studies of the biology of this common cancer. Their clonogenic potential may be of value in the study of sensitivity to antineoplastic agents. Their low passage level may mean that their antigens still resemble those of the primary tumor. PMID:6744280

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

    E-print Network

    Collins, Gary S.

    Fluorescence Assay References 1. 2. http://www.tgrbio.com/cancer-cell-lines-primary-cell- cultures-therapies in cancer patients. This makes the study of both agonist and antagonist ligands important as the knowledge kidney cells (HEK-293) using cDNA prepared from plasmid cDNA expressed in E.coli The pcDNA then underwent

  20. Bilirubin and light induced cell death in a murine lymphoma cell line

    Microsoft Academic Search

    Terje Christensen; Ellen B. Roll; Alicja Jaworska; Gunnar Kinn

    2000-01-01

    Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 ?M bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by

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

    SciTech Connect

    Aki, Toshihiko, E-mail: aki.legm@tmd.ac.jp [Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)] [Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi [Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)] [Section of Forensic Medicine, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)

    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. Aberrant autophosphorylation of c-Kit receptor in canine mast cell tumor cell lines.

    PubMed

    Takeuchi, Yoshinori; Fujino, Yasuhito; Watanabe, Manabu; Nakagawa, Takayuki; Ohno, Koichi; Sasaki, Nobuo; Sugano, Sumio; Tsujimoto, Hajime

    2010-10-15

    Several studies indicated that KIT mutation could cause ligand-independent activation of c-Kit receptor in canine mast cell tumor (MCT). The objective of this study was to investigate mechanisms of c-Kit receptor activation in various canine MCT cell lines. Four cell lines, HRMC (derived from cutaneous MCT), VIMC1 (visceral MCT), CoMS1 (visceral MCT) and CMMC1 (cutaneous MCT), were cultured in stem cell factor (SCF, a ligand of c-Kit receptor)-free medium and subjected to analyses of KIT mutation, c-Kit receptor phosphorylation, SCF expression and the effects of SCF stimulation. In addition, the SCF/c-Kit receptor autocrine mechanism was verified in HRMC cells. HRMC cells expressed wild type c-Kit receptor. Both VIMC1 and CoMS1 cells had the same one amino acid (AA) substitution in the extracellular domain of c-Kit receptor. CMMC1 cells had at least three variants of c-Kit receptor such as one AA deletion in the extracellular domain (variant A), one AA substitution in the extracellular domain as well as an internal tandem duplication in the juxtamembrane domain (variant B), and a nonsense mutation (variant C). Both mature and immature forms of c-Kit receptor were observed and the c-Kit receptors were phosphorylated in all cell lines. While both mature and immature forms of c-Kit receptor were substantially phosphorylated in CMMC1 cells, the immature form was slightly phosphorylated in other cell lines. Phosphorylation of c-Kit receptor in HRMC, VIMC1 and CoMS1 cells were enhanced by SCF stimulation whereas no enhancement was observed in CMMC1 cells. There was no effect of SCF stimulation on proliferation of all the cell lines. SCF protein was detectable in only HRMC cells although mRNA expression of SCF was detected in all the cell lines. A tyrosine kinase inhibitor Dasatinib (internal inhibitor) inhibited c-Kit receptor phosphorylation in HRMC cells whereas anti-canine SCF antibody (external inhibitor) had no inhibitory effect. Thus there could be no external SCF/c-Kit receptor autocrine mechanism whereas there could be an internal autocrine mechanism within HRMC cells. The results indicated that consistent c-Kit receptor phosphorylation could be caused by the stimulation with autocrine SCF in HRMC cells while it could be caused by functional mutations of KIT in VIMC1, CoMS1 and CMMC1 cells. As the four canine MCT cell lines had various aberrations associated with c-Kit receptor phosphorylation, KIT mutation and SCF expression, such molecular biological diversity might reflect the different biological behavior in canine MCT. PMID:20591500

  3. Comparative proteomic profiling of pancreatic ductal adenocarcinoma cell lines.

    PubMed

    Kim, Yikwon; Han, Dohyun; Min, Hophil; Jin, Jonghwa; Yi, Eugene C; Kim, Youngsoo

    2014-12-31

    Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines. PMID:25518923

  4. 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. PMID:25842107

  5. 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

  6. Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines

    E-print Network

    Dennis, Robert G.

    Excitability and contractility of skeletal muscle engineered from primary cultures and cell lines of skeletal muscle engineered from primary cultures and cell lines. Am J Physiol Cell Physiol 280: C288­C295-dimensional skeletal muscle constructs, termed myooids, engineered from C2C12 myoblast and 10T1/2 fibroblast cell lines

  7. Detection of circulating tumour cells on mRNA levels with established breast cancer cell lines.

    PubMed

    Zebisch, Michael; Kölbl, Alexandra C; Andergassen, Ulrich; Hutter, Stephan; Neugebauer, Julia; Engelstädter, Verena; Günthner-Biller, Maria; Jeschke, Udo; Friese, Klaus; Rack, Brigitte

    2013-03-01

    Circulating tumour cells were detected and quantified by real-time polymerase chain reaction (PCR) in peripheral blood, based on the fact that the expression of certain genes is upregulated in tumour tissues in comparison to surrounding blood cells. Calibration curves showing gene expression as functions of the number of tumour cells within a blood sample were prepared. Blood samples were therefore spiked with cells of breast cancer cell lines, RNA was extracted, transcribed to complementary DNA (cDNA) and used in real-time PCR reaction on the Cytokeratins (CK) 8, 18 and 19. Calibration curves were generated by Microsoft™ Excel®. Relative quantification curves of gene expression in different breast cancer cell lines showed no unitary tendencies. The oscillations in the relative quantification curves of gene expression suggested an occurrence of immunological effects, leading to an apparent agglutination of added tumour cells together with the blood cells of the sample. Thus, strategies to obtain evaluable results should be considered. PMID:24648925

  8. 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

  9. Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

    PubMed

    Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

    2012-04-01

    Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (?2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was significantly increased, as indicated by the large reduction of non-producing and low-producing cells after 25?µM L-MSX selection, and resulted in a six-fold efficiency improvement in identifying similar numbers of high-productive cell lines for a given recombinant monoclonal antibody. The potential impact of GS-knockout cells on recombinant protein quality is also discussed. PMID:22068567

  10. 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. PMID:25998836

  11. Characterization of a human stromal cell line supporting hematopoietic progenitor cell proliferation: Effect of HIV expression

    Microsoft Academic Search

    Joseph D. Mosca; Sumesh Kaushal; Suzanne Gartner; Stephen W. Kessler; Vince F. La Russa; Ernest F. Terwilliger; Jerome H. Kim; Richard G. Carroll; Eric R. Hall; Liyanage P. Perera; Zhipeng Yu; David W. Ritchey; Jin Xu; Daniel C. St. Louis; Douglas L. Mayers

    1995-01-01

    Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from

  12. Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro

    Microsoft Academic Search

    Pierre J. Guillou; Peter C. Sedman; Carol W. Ramsden

    1989-01-01

    The co-culture of human peripheral blood mononuclear cells (PBMC) with high concentrations of interleukin 2 normally generates lymphokine-activated killer (LAK) cells capable of indiscriminate lysis of tumor targets. However, the addition of certain cell-line-derived tumor cells to the LAK generation cultures within the first 48 h of culture initiation resulted in the suppression of the LAK cytotoxicity measured after 3–4

  13. Destabilization of Akt Promotes the Death of Myeloma Cell Lines

    PubMed Central

    Zhang, Yanan; Fu, Yunfeng; Zhang, Fan; Liu, Jing

    2014-01-01

    Constitutive activation of Akt is believed to be an oncogenic signal in multiple myeloma and is associated with poor patient prognosis and resistance to available treatment. The stability of Akt proteins is regulated by phosphorylating the highly conserved turn motif (TM) of these proteins and the chaperone protein HSP90. In this study we investigate the antitumor effects of inhibiting mTORC2 plus HSP90 in myeloma cell lines. We show that chronic exposure of cells to rapamycin can inhibit mTORC2 pathway, and AKT will be destabilized by administration of the HSP90 inhibitor 17-allylamino-geldanamycin (17-AAG). Finally, we show that the rapamycin synergizes with 17-AAG and inhibits myeloma cells growth and promotes cell death to a greater extent than either drug alone. Our studies provide a clinical rationale of use mTOR inhibitors and chaperone protein inhibitors in combination regimens for the treatment of human blood cancers. PMID:25243120

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

    PubMed

    Gerhardt, Daniéli; Horn, Ana Paula; Gaelzer, Mariana Maier; Frozza, Rudimar Luiz; Delgado-Cańedo, 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

  15. High glucose protects embryonic cardiac cells against simulated ischemia.

    PubMed

    Malliopoulou, Vassiliki; Xinaris, Christodoulos; Mourouzis, Iordanis; Cokkinos, Alexandros D; Katsilambros, Nikolaos; Pantos, Constantinos; Kardami, Elissavet; Cokkinos, Dennis V

    2006-03-01

    In the present study we investigated whether acute glucose administration could be protective against hypoxic stress. H9c2 cells were exposed to either 4.5 mM or 22 mM of glucose for 15,min and then were submitted to simulated ischemia. Cell death was microscopically assessed by combined staining with propidium iodide (PI) and Hoeschst 33358. Intracellular content of glucose was measured by enzymatic analysis. Clucose content of H9c2 cells was 48.24+/- 7.94 micromol/L in the 22 mM vs 23.86+/- 4.8 micromol/L in the 4.5 mM group (p < 0.05). PKCepsilon expression was increased 1.6 fold in the membrane fraction after pretreatment with high glucose (p < 0.05), while was decreased 1.6 fold in the cytosol (p < 0.05). In addition, no difference to PKCdelta translocation was observed after pretreatment with low glucose. After hypoxia, in the 22 mM group, cell death was found to be 17.36+/- 2.66% vs 38.2+/- 5.4% in the 4.5 mM group (p < 0.05). In the presence of iodoacetic acid, a glycolytic inhibitor, cell death was not different between the two groups (23.54+/- 3.2% in 22 mM vs 22.06+/- 5.3% in 4.5 mM). Addition of chelerythrine did not change the protective effect of high glucose (13.4+/- 1.7% cell death in 22 mM vs 27.5+/- 5.5% in 4.5 mM, p < 0.05). In conclusion, short pretreatment with high glucose protects H9c2 cells against hypoxia. Although this protective effect is associated with translocation of PKCepsilon and increased glucose uptake, it was abrogated only by inhibition of glycolysis. PMID:16541202

  16. Choosing the right cell line for breast cancer research.

    PubMed

    Holliday, Deborah L; Speirs, Valerie

    2011-01-01

    Breast cancer is a complex and heterogeneous disease. Gene expression profiling has contributed significantly to our understanding of this heterogeneity at a molecular level, refining taxonomy based on simple measures such as histological type, tumour grade, lymph node status and the presence of predictive markers like oestrogen receptor and human epidermal growth factor receptor 2 (HER2) to a more sophisticated classification comprising luminal A, luminal B, basal-like, HER2-positive and normal subgroups. In the laboratory, breast cancer is often modelled using established cell lines. In the present review we discuss some of the issues surrounding the use of breast cancer cell lines as experimental models, in light of these revised clinical classifications, and put forward suggestions for improving their use in translational breast cancer research. PMID:21884641

  17. 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.

  18. Choosing the right cell line for breast cancer research

    PubMed Central

    2011-01-01

    Breast cancer is a complex and heterogeneous disease. Gene expression profiling has contributed significantly to our understanding of this heterogeneity at a molecular level, refining taxonomy based on simple measures such as histological type, tumour grade, lymph node status and the presence of predictive markers like oestrogen receptor and human epidermal growth factor receptor 2 (HER2) to a more sophisticated classification comprising luminal A, luminal B, basal-like, HER2-positive and normal subgroups. In the laboratory, breast cancer is often modelled using established cell lines. In the present review we discuss some of the issues surrounding the use of breast cancer cell lines as experimental models, in light of these revised clinical classifications, and put forward suggestions for improving their use in translational breast cancer research. PMID:21884641

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

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

    2014-01-01

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

  1. Characteristics of rhabdomyosarcoma cell lines derived from uterine carcinosarcomas

    Microsoft Academic Search

    M. Emoto; H. Iwasaki; K. Oshima; M. Kikuchi; Y. Kaneko; T. Kawarabayashi

    1997-01-01

    Rhabdomyosarcoma (RMS) is occasionally found in the female genital tract, and mostly appears as one of the heterologous mesenchymal\\u000a components in uterine carcinosarcoma designated as malignant mixed müllerian tumour (MMMT). We examined the biological properties\\u000a of a pure rhabdomyosarcoma (RMS) cell line designated FU-MMT-3, which was newly established from a surgical specimen taken\\u000a from a patient with uterine MMMT. We

  2. Cytotoxic effects of curcumin on osteosarcoma cell lines

    Microsoft Academic Search

    Denise K. Walters; Roman Muff; Bettina Langsam; Walter Born; Bruno Fuchs

    2008-01-01

    Summary  Curcumin (diferuloylmethane), one of the main components of the Indian spice turmeric, is known to possess potent anti-inflammatory\\u000a and anti-oxidant properties. In addition, curcumin has also been shown to have in vitro and in vivo efficacy against a variety of malignancies. In the current study we examined the cytotoxic effect of curcumin on seven osteosarcoma\\u000a (OS) cell lines with varying

  3. Gypsy moth cell lines divergent in viral susceptibility

    Microsoft Academic Search

    R. H. Goodwin; G. J. Tompkins; P. McCawley

    1978-01-01

    Summary  A series of cell lines unique in insect virus susceptibility pattern have been isolated from the ovaries of the gypsy moth\\u000a (Lymantria dispar: Lepidoptera: Lymantriidae) on a synthetic medium with mammalian and avian serum supplementation. Growth curves showed the\\u000a poorest growth occurring on peptone-based media with somewhat better growth on amino-acid-based media. The best growth was\\u000a obtained with combined media.

  4. Role of the p53 Tumor Suppressor Gene in Cell Cycle Arrest and Radiosensitivity of Burkitt's Lymphoma Cell Lines

    Microsoft Academic Search

    Patrick M. O'Connor; Joany Jackman; Daniel Jondle; Kishor Bhatia; Ian Magrath; Kurt W. Kohn

    We have assessed the role of the p5ĂŚ tumor suppressor gene in cell cycle arrest and cytotoxicity of ionizing radiation in 17 Burkitt's lymphoma and Ivmphoblastoid cell lines. Cell cycle arrest was assessed by flow cytometry of cells 16 h following irradiation. In addition to the usual G2 arrest, the cell lines exhibited three types of responses in I.,: Class

  5. Porcine Endogenous Retrovirus Infects but Does Not Replicate in Nonhuman Primate Primary Cells and Cell Lines

    PubMed Central

    Ritzhaupt, Armin; van der Laan, Luc J. W.; Salomon, Daniel R.; Wilson, Carolyn A.

    2002-01-01

    Porcine endogenous retroviruses (PERV) can infect human cell lines in vitro; hence, there is a presumed risk of viral exposure to a recipient when pig cells are transplanted into humans (xenotransplantation). Nonhuman primates (NHP) are considered a potential permissive animal model to study the risk of in vivo infection of PERV after xenotransplantation. We set out to determine whether PERV can infect and replicate in NHP primary cells or established cell lines from African green monkey, rhesus macaque, and baboon. We confirm that the NHP cell lines under investigation were infected with PERV as measured by detection of viral DNA and RNA by PCR and reverse transcription (RT)-PCR, respectively, indicating that a functional receptor must be present on the cell surface. However, the load of detectable viral DNA in infected NHP cells declined over time, and the cells never had detectable reverse transcriptase activity. Utilizing quantitative real-time TaqMan PCR we found detectable levels of unintegrated DNA intermediates, but the levels were approximately 100-fold lower compared to HEK 293 cells infected with PERV. Virions released from infected NHP cells could productively infect naďve human cell lines, HEK 293 and HeLa, as shown by RT-PCR and RT assay. However, naďve NHP cells remained negative in RT-PCR and RT assay after exposure to virions from infected NHP cells. Together our data demonstrate that NHP cells are not permissive to productive replication by PERV, presumably due to inefficient cell entry and replication. In light of these observations, the appropriateness of NHP as suitable animal models to study PERV infection in vivo needs to be reevaluated. PMID:12388691

  6. Radiosensitization of uterine cancer cell lines by cytotoxic agents.

    PubMed

    Nguyen, H N; Sevin, B U; Averette, H E; Gottlieb, C; Perras, J; Donato, D; Penalver, M

    1993-01-01

    Radiotherapy remains an integral part of uterine cancer therapy. Overcoming radioresistant tumors by sensitizers continues to be a prime objective in radiotherapy research. In this study, the effects of five cytotoxic agents on two radiosensitive and four radioresistant uterine cancer cell lines were investigated. The ATP bioluminescence was used to measure surviving fractions. Data analysis was done using the linear quadratic model and radiosensitivity index D. Both AN3 and SKUT1B were radiosensitive with Ds of 1.73 and 1.72 Gy, respectively. The resistant cell lines had the following D values: AE7, 3.50; ECC, 6.61; HEC1A, 4.59; and HEC1B, 13.49 Gy. The average radiosensitization effects for various drugs were measured by reduction of D: DXR 45 +/- 7, DDP 40 +/- 9, 5FU 55 +/- 10, MITO 59 +/- 14, and HU 1.7 +/- 7%. Except for HU, Wilcoxon analyses revealed that these sensitizing effects were significant with P < 0.02. In summary, Adriamycin, 5-fluorouracil, cisplatin, and mitomycin-C have the potential to be radiosensitizers in uterine cancer cell lines. PMID:8423017

  7. Diverse hematopoietic potentials of five human embryonic stem cell lines

    PubMed Central

    Chang, Kai-Hsin; Nelson, Angelique M.; Fields, Paul A.; Hesson, Jennifer L.; Ulyanova, Tatiana; Cao, Hua; Nakamoto, Betty; Ware, Carol B.; Papayannopoulou, Thalia

    2009-01-01

    Despite a growing body of literature concerning the hematopoietic differentiation of human embryonic stem cells (hESCs), the full hematopoietic potential of the majority of existing hESC lines remains unknown. In this study, the hematopoietic response of five NIH-approved hESC lines (H1, hSF6, BG01, BG02, and BG03) was compared. Our data show that despite expressing similar hESC markers under self-renewing conditions and initiating mesodermal differentiation under spontaneous differentiation conditions, marked differences in subsequent hematopoietic differentiation potential among these lines existed. A high degree of hematopoietic differentiation was attained only by H1 and BG02, whereas this process appeared to be abortive in nature for hSF6, BG01, and BG03. This difference in hematopoietic differentiation predisposition was readily apparent during spontaneous differentiation, and further augmented under hematopoietic-inducing conditions. This predisposition appeared to be intrinsic to the specific hESC line and independent of passage number or gender karyotype. Interestingly, H1 and BG02 displayed remarkable similarities in their kinetics of hematopoietic marker expression, hematopoietic colony formation, erythroid differentiation, and globin expression, suggesting that a similar, predetermined differentiation sequence is followed. The identification of intrinsic and extrinsic factors governing the hematopoietic differentiation potential of hESCs will be of great importance for the putative clinical utility of hESC lines. PMID:18692044

  8. Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes

    Microsoft Academic Search

    Joan M. Cook-Milis; Joan S. Gallagher; Thomas L. Feldbush

    1996-01-01

    Summary  Two long-term cultured cell lines were established from BALB\\/c mouse axillary and cervical lymph nodes that exhibited a combination\\u000a of functional, morphological, and phenotypic characteristics consistent only with high endothelial venule cells. Spleen lymphocytes\\u000a selectively bound and migrated across the cell lines. On Matrigel, these cell lines formed tubules with lumens, a characteristic\\u000a unique to endothelial cells. Morphologically the cells

  9. Secretome analysis of Glioblastoma cell line--HNGC-2.

    PubMed

    Gupta, Manoj Kumar; Polisetty, Ravindra Varma; Ramamoorthy, Kalidoss; Tiwary, Shivani; Kaur, Navjot; Uppin, Megha S; Shiras, Anjali; Sirdeshmukh, Ravi

    2013-06-01

    Glioblastoma multiforme (GBM) is the most common and aggressive type of primary malignant tumor of the central nervous system. We have carried out a deep analysis of the secretome of a rapidly proliferating and tumorigenic cell line HNGC-2, representing GBM, in an effort to identify proteins, which may be targeted in the plasma of GBM patients as markers for diagnosis and disease surveillance. Prefractionation of the proteins from the conditioned medium of HNGC-2 cells in SDS gels followed by LC-MS/MS analysis using an ESI-IT mass spectrometer (LTQ) led to a total of 996 protein identifications with ?2 peptides each. Of them, 664 proteins were observed in the transcriptome of HNGC-2 cells. The dataset of 996 proteins was mapped to important functional groups, such as cellular assembly and organisation, DNA recombination and repair, and other classes. Actin cytoskeleton signalling, phosphatidyl inositol 3 kinase (PI3K/AKT) and integrin linked kinase (ILK) signalling pathways were seen as enriched pathways. Comparisons with the published secretome of cell lines from 12 different cancers, including GBM, revealed that 348 proteins shared a commonality with a secretome of at least one other cell line, 321 of which were found to contain signal sequences or transmembrane domains and 335 could be linked to a plasma membrane or extracellular localization. Through intergration of this data we arrived at a non-redundant list of 597 protein identifications with the potential for secretion either by classical secretory pathways or by non-secretory processes; 233 of them have been detected in cerebrospinal fluid or plasma as per the published literature, and 172 have been implicated in GBM or other cancers. The HNGC-2 secretome dataset could serve as a useful resource for designing a targeted investigation of GBM biomarkers in plasma. PMID:23483059

  10. Cytogenetics of malignant epithelial cells and lymphoblastoid cell lines from nasopharyngeal carcinoma.

    PubMed Central

    Finerty, S.; Jarvis, J. E.; Epstein, M. A.; Trumper, P. A.; Ball, G.; Giovanella, B. C.

    1978-01-01

    The malignant epithelial cells of nasopharyngeal carcinoma (NPC) and cells of lines derived form the lymphoid cells which infiltrate this tumour have been investigated cytogenetically. Chromosome spreads of lymphoblastoid cells of lines established from 7 different NPC biopsy specimens were examined after banding staining. Banding was also applied to the epithelial tumour cells of 5 further biopsy specimens freed from non-malignant infiltrating cells by passage through nude mice; epithelial cell spreads were obtained by in vivo splindle arrest. Five of the lymphoblastoid lines were found to be diploid, and 2 tetraploid; the karyotypes were essentially normal. The squamous epithelial nature of the cells in the nude-mouse-grown NPC tumours was established by light and electronmicroscopy, and 3 tumours were found to be near-triploid, and 2 near-diploid. The cells of the near-triploid tumours contained grossly abnormal chromosomes but those of the near-diploid tumours showed only relatively minor changes. Although abnormalities were observed which were specific for cells from each individual tumour, no discernible change was common to cells from all the tumours. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:629860

  11. Arsenic trioxide-induced apoptosis through oxidative stress in cells of colon cancer cell lines.

    PubMed

    Nakagawa, Yoshihito; Akao, Yukihiro; Morikawa, Hiroshi; Hirata, Ichiro; Katsu, Kenichi; Naoe, Tomoki; Ohishi, Nobuko; Yagi, Kunio

    2002-03-29

    Exposure of three colon cancer cell lines, SW480, DLD-1, and COLO201, to arsenic trioxide in the medium induced a marked concentration-dependent suppression of cell growth. The intracellular contents of reduced glutathione (GSH) in these cell lines tended to be inversely correlated with the sensitivity of the cells to arsenic trioxide. Among the cell lines, SW480 cells underwent apoptosis at the low arsenic trioxide concentration of 2 microM, which was prevented by pretreatment of the cells with N-acetylcysteine and was enhanced by buthionine sulfoximine. The production of reactive oxygen intermediates which were examined by 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA), increased with time after treatment with arsenic trioxide. The apoptosis was executed by the activation of caspase 3, which was shown by Western blot, enzymatic activity, and apoptosis inhibition assay. The mitochondrial membrane potential of adherent apoptotic SW480 cells and the cells from intermediate layer separated by density gradient centrifugation, both of which showed the active form of caspase 3 by Western blot analysis, was not lost. The overexpression of Bcl-2 protein in SW480 cells could not prevent the apoptosis induced by the treatment with arsenic trioxide. All these findings indicate that arsenic trioxide-induced apoptosis in SW480 cells is executed by the activation of caspase 3 without mediating by mitochondria under the overproduction of reactive oxygen species. PMID:12005185

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

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)] [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States); Frey, Teryl K., E-mail: tfrey@gsu.edu [Department of Biology, Georgia State University, P.O. Box 4010, Atlanta GA 30302-4010 (United States)

    2012-07-20

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

  13. Lycopene Treatment of Prostate Cancer Cell Lines Inhibits Adhesion and Migration Properties of the Cells

    PubMed Central

    Elgass, Simone; Cooper, Alan; Chopra, Mridula

    2014-01-01

    Background: Consumption of lycopene through tomato products has been suggested to reduce the risk of prostate cancer. Cellular adhesion and migration are important features of cancer progression and therefore a potential target for cancer interception. In the present study we have examined the in vitro effect of lycopene on these processes. Methods: Prostate cancer cell lines PC3, DU145 and immortalised normal prostate cell line PNT-2 were used. The adhesion assay consisted of seeding pre-treated cells onto Matrigel™, gently removing non-adherent cells and quantitating the adherent fraction using WST-1. Migratory potential was assessed using ibidi™ migration chamber inserts, in which a cell-free zone between two confluent areas was allowed to populate over time and the migration measured. Results: 24 hour incubation of prostate cell lines with 1.15µmol/l lycopene showed a 40% reduction of cellular motility in case of PC3 cells, 58% in DU145 cells and no effect was observed for PNT2 cells. A dose related inhibition of cell adhesion to a basement membrane in the form of Matrigel™ was observed in all three cell lines and it reached statistical significance for PC3 and PNT2 cells at lycopene concentrations ?1.15µmol/l. However, in case of DU145, only a concentration of 2.3µmol/l showed a significant reduction. Conclusion: This in vitro investigation indicates that lycopene can influence the cell adhesion and migration properties of cancer cells at a dose which is arguably achievable in patients. The results of our study expand our understanding of a chemo preventive role of lycopene in prostate cancer. PMID:25076850

  14. A cell line derived from non-neoplastic human neuroretinal cells.

    PubMed

    Mancini, M A; Kennedy, A; Frank, R N; Trese, M T; Hartzer, M; Hukku, B; Lin, L R

    1989-03-01

    We have derived a cell line from an epiretinal membrane excised surgically from a premature female infant born at a gestational age of 25 weeks, and who developed stage 5 retinopathy of prematurity. The cell line, which in early passages appeared immunocytochemically to contain cells with both neuronal and glial characteristics, has been maintained in culture for 14 months at the time this manuscript was submitted, and has survived 20 passages. The cells have a diploid, human karyotype, with most cells possessing 46 normal appearing chromosomes, including 44 autosomes and two X-chromosomes. Morphologically, the cell line at early passages consisted of polygonal cells and also of cells possessing long, spindly branching processes. These two cell types were cloned. Nearly 100% of the cells of both morphologic types in mixed cultures stained immunocytochemically for neuron-specific enolase (NSE), a neuronal marker, and approximately 5-10% of the cells in mixed cultures (including about 50% of the cells with the spindly morphology, that were less prevalent in mixed cultures) stained for glial fibrillary acid protein (GFAP), a glial marker. We have not performed "double-label" immunocytochemistry, but it was evident from the proportion of cells that stained with each marker that many cells must contain both GFAP and NSE. At least 50% of the cells in most of the early cultures were positive for keratin, while all were (and remain) negative for muscle actin. No cells are found that are immunocytochemically positive for factor VIII, a vascular endothelial cell marker. These cultured cells have also been studied immunocytochemically for their production of extracellular matrix substances. The cultures are immunocytochemically positive for type IV (but not type I) collagen, laminin and fibronectin. In later passages, cells of both clones lost their immunocytochemical positivity for GFAP and NSE, and all became positive for keratin. Cells of both clones also developed a similar, polygonal morphology, lacking long processes. By electron microscopy, many of the cells were seen to possess nonmotile cilia, with a 9 + 0 pattern of microtubule doublets. This cell line may be useful for studies of human retinal cell development and metabolism, and responses to pathological processes. PMID:2925321

  15. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro

    Microsoft Academic Search

    Benjamin E. Reubinoff; Chui-Yee Fong; Alan Trounson; Ariff Bongso; Martin F. Pera

    2000-01-01

    We describe the derivation of pluripotent embryonic stem (ES) cells from human blastocysts. Two diploid ES cell lines have been cultivated in vitro for extended periods while maintaining expression of markers characteristic of pluripotent primate cells. Human ES cells express the transcription factor Oct-4, essential for development of pluripotential cells in the mouse. When grafted into SCID mice, both lines

  16. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan) [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan)] [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan)] [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)] [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

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

  17. Responses of human normal osteoblast cells and osteoblast-like cell line, MG63 cells, to pulse electromagnetic field (PEMF)

    Microsoft Academic Search

    Suttatip Kamolmatyakul; Uraiwan Jinorose; Peerapong Prinyaroj; Yiping Li

    The objective of this in vitro study is to investigate the effect of pulsed electromagnetic field (PEMF) on cellular proliferation and osteocalcin production of osteoblast-like cell line, MG-63 cells, and human normal osteoblast cells (NHOC) obtained from surgical bone specimens. The cells were placed in 24-well culture plates in the amount of 3x104 cell\\/wells with 2 ml ?MEM media supplemented

  18. Expression of junctional proteins in choroid plexus epithelial cell lines: a comparative study

    Microsoft Academic Search

    Joanna Szmydynger-Chodobska; Crissey L Pascale; Andrew N Pfeffer; Cassaundra Coulter; Adam Chodobski

    2007-01-01

    BACKGROUND: There is an increasing interest in using choroid plexus (CP) epithelial cell lines to study the properties of the blood-cerebrospinal fluid barrier (BCSFB). Currently, there are three major CP-derived cell lines available. Z310 and TR-CSFB3, two immortalized cell lines carrying the simian virus 40 large T-antigen gene, were derived from rat CP epithelium, whereas the CPC-2 cell line was

  19. Equilibrium and kinetic analysis of Autographa californica nuclear polyhedrosis virus attachment to different insect cell lines

    Microsoft Academic Search

    T. J. Wickham; M. L. Shuler; D. A. Hammer; R. R. Granados; H. A. Wood

    1992-01-01

    The kinetic and equilibrium attachment of Autographa californica nuclear polyhedrosis virus (AcMNPV) to seven insect cell lines was evaluated. Kinetic experi- ments revealed differences of up to 10-fold in the infection rates among cell lines. Equilibrium binding also varied between cell lines and was saturable. The Tn 5B1-4 and Tn F cell lines had the highest virus binding affinities and

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

    SciTech Connect

    Kirchheimer, J.C.; Wojta, J.; Christ, G.; Binder, B.R. (Univ. of Vienna (Austria))

    1989-07-01

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

  1. Functional estrogen receptors in a human preosteoclastic cell line.

    PubMed Central

    Fiorelli, G; Gori, F; Petilli, M; Tanini, A; Benvenuti, S; Serio, M; Bernabei, P; Brandi, M L

    1995-01-01

    The primary biological effect of the estrogen estradiol-17 beta (17 beta E2) on bone is to decrease bone resorption. However, whether 17 beta E2 affects osteoclast differentiation or function directly or through its action on osteoblasts is unclear. To investigate this question we examined the human preosteoclastic cell line FLG 29.1 for evidence of functional estrogen receptors (ERs). Southern blotting of reverse transcription-PCR amplification products with a 32P-labeled cDNA probe for the human ER mRNA demonstrated that FLG 29.1 cells express ER mRNA. Binding of [3H]17 beta E2 to nuclear ERs was steroid specific with approximately 400 saturable, high affinity (Kd approximately 1 nM) binding sites per cell nucleus. Nuclear ERs covalently labeled with [3H]tamoxifen aziridine showed an apparent molecular weight of 65,000 by SDS/PAGE and Western blotting with the D75 monoclonal antibody to human ER. Pretreatment of cells with 0.1, 1.0, or 10 nM 17 beta E2 induced a dose- and time-dependent specific binding of progesterone to FGL 29.1 cells, and stimulation of the cells with 10 nM and 100 nM 17 beta E2 significantly (P < 0.05) reduced cell proliferation. Transcriptional activity of the ER gene was detected by transient transfection of cells with the pERE-BLCAT plasmid containing the estrogen response element for the vitellogenin A2 gene and the bacterial chloramphenicol acetyltransferase reporter gene. Treatment of FLG 29.1 cells with 10 nM 17 beta E2 increased chloroamphenicol acetyltransferase expression from 5- to 29-fold compared to controls. These observations suggest a potential role for estrogen in osteoclastogenesis. Images Fig. 1 Fig. 4 PMID:7708703

  2. Molecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture

    E-print Network

    Kenny, Paraic

    together (e.g., ERBB2 amplified cell lines sharing the same morphometric properties as the grapeMolecular Predictors of 3D Morphogenesis by Breast Cancer Cell Lines in 3D Culture Ju Han1 , Hang generation. In this paper, a panel of 24 breast cell lines was grown in 3D culture, their morphology

  3. Cytotoxicity of cashew flavonoids towards malignant cell lines.

    PubMed

    Konan, Nzi André; Lincopan, Nilton; Díaz, Ingrit Elida Collantes; de Fátima Jacysyn, Jacqueline; Tiba, Mirtes Midori Tanae; Amarante Mendes, Joăo Gustavo Pessini; Bacchi, Elfriede Marianne; Spira, Beny

    2012-07-01

    The leaves of the Cashew plant (Anacardium occidentale L.) are used by the folk medicine in South America and West Africa. This plant is rich in flavonoids, which are polyphenolic compounds widespread in plants, and that have diverse physiological effects. In a sub-acute toxicity assay it was found that an ethanolic extract of Cashew leaves elicited lymphopenia in rats. The extract was also found to be cytotoxic and to induce apoptosis in Jurkat (acute lymphoblastic leukemia) cells. The crude ethanolic extract was fractionated and resolved by HPLC. One of the four fractions obtained led to the isolation of the biflavonoid agasthisflavone. [(3)H]-thymidine incorporation assays and flow cytometry analysis showed that the isolated compound displayed a high anti-proliferative effect in Jurkat cells with an IC(50) of 2.4 ?g/ml (4.45 ?M). The effect of agathisflavone on the acute promyelocytic leukemia cell line HL60, Burkitt lymphoma Raji cells and Hep-2 laryngeal carcinoma cells was also tested. The two latter ones were only mildly affected by agathisflavone. It is also shown that agathisflavone induces apoptosis in Jurkat cells and it this proposed that this is the likely mechanism of agathisflavone specific cytotoxicity. PMID:21106357

  4. Sensitization assays: monocyte-derived dendritic cells versus a monocytic cell line (THP-1).

    PubMed

    Tietze, Corinna; Blomeke, Brunhilde

    2008-01-01

    Dendritic cells (DCs) play a critical role in the skin sensitization process of contact allergens. Many efforts have been made to develop in vitro sensitization tests that employ DCs, but more recently protocols were introduced that use cell lines other than DCs. The potential of the cell line THP-1 compared to monocyte-derived DCs (MoDCs) was evaluated using a known potent sensitizer 2,4-dinitrochlorobenzene (DNCB) and the terpenoid ascaridol (1,4-epodioxy-p-menth-2-ene), an ingredient present in oxidized tea tree oil. Activation of these cells was studied by estimation of the CD86 and CD54 cell surface expression. Overall, comparable results were found. The expression of CD86 was augmented by ascaridol in THP-1 and MoDCs, while the expression of CD54 was not reproducibly increased. These results encourage the further development of THP-1 cells as a short-term model for sensitization testing. PMID:18569603

  5. Digital cell image analysis of verapamil-induced effects in chemosensitive and chemoresistant neoplastic cell lines

    Microsoft Academic Search

    Chantal Etiévant; Olivier Pauwels; Robert Kiss

    1993-01-01

    We used chemosensitive and chemoresistant variants of the neoplastic mouse MXT mammary and human J82 and T24 bladder cell lines to characterize verapamil-induced cell proliferation and morphonuclear modifications in drug-treated and untreated cells. Chemoresistance to vinorelbine (Navelbine, aVinca alkaloid derivative), to DIAM3 (an investigational alkylating compound) and to Adriamycin (an intercalating agent) in the presence or absence of verapamil was

  6. Curcumin downregulates cell survival mechanisms in human prostate cancer cell lines

    Microsoft Academic Search

    Asok Mukhopadhyay; Carlos Bueso-Ramos; Devasis Chatterjee; Panayotis Pantazis; Bharat B Aggarwal

    2001-01-01

    While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-?B (NF-?B) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-?B and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our

  7. 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 14-3-3?. 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 14-3-3? is key to determine if the cell proliferates or dies. In addition, our bioinformatics analysis suggests that the neoplastic phenotype is governed by a non-canonical regulatory pathway. PMID:21696634

  8. Raman spectra and discrimination of NPC cell line CNE1 and normal nasopharyngeal cell line NP69

    NASA Astrophysics Data System (ADS)

    Chen, Yang; Li, Yong-zeng; Su, Ying; Lin, Ju-qiang; Pan, Jian-ji; Chen, Rong; Zou, Chang-yan; Lin, Shaojun; Li, Chao

    2009-08-01

    As a non-destructive and non-invasive technique, Raman spectroscopy (RS) plays an important role in the field of biomedical research. Great progress has been made in the research of biological samples from cellular level to macro-tissues. In this letter, advances of RS in tumor cells and some statistic algorithm developed in recent years for cancer differentiation and diagnosis are introduced. Also, Raman spectra of Nasopharyngeal Carcinoma (NPC) cell line CNE1 and normal nasopharyngeal cell line NP69 are acquired by confocal Raman micro-spectroscopy system. Raman bands are analyzed and compared to investigate the differences and relationship between CNE1 and NP69, Principle Components Analysis (PCA) is used to classify CNE1 and NP69 accurately with an accuracy of 100%. Comparing with CNE1, a blue-shift is observed in NP69 cells at band 936cm-1 and 2935cm-1 which are assigned to C-C stretch and CH3 stretching, respectively. Meanwhile, a red-shift is observed at 1338cm-1 assigned to A, G and C-H deformation vibration of protein. The results show that Raman spectroscopy has its potential and reliability to be one of the diagnostic methods for NPC and at the same time can provide valuable information for cancer early diagnosis.

  9. 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

  10. Small cell lung cancer cell lines secrete predominantly ACTH precursor peptides not ACTH.

    PubMed Central

    Stewart, M. F.; Crosby, S. R.; Gibson, S.; Twentyman, P. R.; White, A.

    1989-01-01

    A panel of 18 well characterised human small cell lung cancer (SCLC) cell lines was assessed for the production of adrenocorticotrophin (ACTH) and its precursor peptides, pro-opiomelanocortin (POMC) and pro-ACTH. These precursor peptides were measured directly using a novel two-site immunoradiometric assay (IRMA) based on monoclonal antibodies, in conjunction with a similar IRMA for ACTH 1-39. Significant concentrations of ACTH precursors were secreted by 10 of the 18 cell lines (56%). The low levels of ACTH immunoreactivity detected in seven cell lines could be accounted for by the known cross-reactivity of precursors in the ACTH IRMA. This suggests there is little, if any, processing of ACTH precursors to ACTH. Cell pellet extracts contained undetectable or low levels of ACTH precursors and ACTH, indicating that these peptides are not stored intracellularly. During the growth of the SCLC cells in vitro ACTH precursors accumulated progressively in the culture medium. Thus the combination of a direct assay for the ACTH precursors and the panel of SCLC cell lines provides a valuable in vitro model for the expression of POMC in human tumours. PMID:2553086

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

    PubMed Central

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

    2010-01-01

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

  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.

  13. Establishment and characterization of a SV40T-transformed human bronchial epithelial cell line

    Microsoft Academic Search

    Yong-Jie Lu; Shu-Ping Guo; Tong Tong; Li-Hua Xu; Xiang-Yang Dong; Nai-Jun Hana; Shu-Jun Cheng

    1998-01-01

    The majority of human lung cancers originate from the carcinogenesis of bronchial epithelial cells. To study the malignant progression of human bronchial epithelial cells, we established a SV40T-transformed human bronchial epithelial cell line, and observed some biological and genetic changes of the cell line at different passages. In a 2-year culture, this cell line was approaching malignancy without obvious senescence.

  14. Retinal ganglion cell line apoptosis induced by hydrostatic pressure.

    PubMed

    Agar, Ashish; Li, Shaojuan; Agarwal, Neeraj; Coroneo, Minas T; Hill, Mark A

    2006-05-01

    Cellular responses to changes in pressure are implicated in numerous disease processes. In glaucoma apoptosis of retinal ganglion cells (RGCs) is associated with elevated intra-ocular pressure, however, the exact cellular mechanisms remain unclear. We have previously shown that pressure can induce apoptosis in B35 and PC12 neuronal cell lines, using an in vitro model for pressure elevation. A novel RGC line allows us to study the effects of pressure on retinal neurons. 'RGC-5' cultures were subjected to elevated ambient hydrostatic pressure conditions in our model. Experimental pressure conditions were 100 mm Hg and 30 mm Hg, representing acute (high) and chronic (lower-pressure) glaucoma, and 15 mm Hg for normal intra-ocular pressure, set above atmospheric pressure for 2 h. Negative controls were treated identically except for the application of pressure, while positive controls were generated by treatment with a known apoptotic stimulus. Apoptosis was determined by a combination of cell morphology and specific TUNEL and Annexin V fluorescent markers. These were assessed simultaneously by laser scanning cytometry (LSC), which also enabled quantitative marker analysis. RGC-5 neurons showed a significantly increased proportion of apoptotic cells compared with controls; maximal at 100 mm Hg, moderate at 30 mm Hg and not statistically significant at 15 mm Hg. This graded response, proportionate to the level of pressure elevation, is representative of the severity of analogous clinical settings (acute, chronic glaucoma and normal). These results complement earlier findings of pressure-induced apoptosis in other neuronal cultures. They suggest the possibility of novel mechanisms of pressure-related mechanotransduction and cell death, relevant to the pathogenesis of diseases such as glaucoma. PMID:16638612

  15. 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

  16. Detection of luxol-fast-blue positive cells in human promyelocytic leukemia cell line HL-60.

    PubMed

    Lu, L; Broxmeyer, H E; Pelus, L M; Andreeff, M; Moore, M A

    1981-10-01

    Differentiation of HL-60 cells toward the eosinophilic series has not been reported previously. Eosinophil granule specific staining with Luxol-fast-blue was used to determine if HL-60 cells could differentiate into the eosinophilic lineage. The specificity of the Luxol-fast-blue stain for cells of the eosinophilic series was substantiated by comparison of the staining of cells from a patient with an eosinophilic syndrome by Wright-Giemsa and Luxol-fast-blue. Luxol-fast-blue positivity was most notable in cells found in colonies formed from HL-60 clonogenic cells in semisolid agar medium. Colony and cluster formation was spontaneous but in the presence of medium conditioned by either human placental cells or the human monocyte-like cell line, GCT, Luxol-fast-blue positive colonies and clusters were detected at a higher frequency. PMID:6175532

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

    PubMed

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

    2015-08-01

    Resistance to chemotherapy leading to poor outcome and survival remains a challenge for developing strategies for therapeutic interventions in all types of cancer, including head and neck cancer. In vitro chemoresistant cell line models are an indispensable resource towards delineating the mechanisms involved in drug resistance/response and for the development of novel drugs. Current treatment for head and neck cancer includes chemotherapy with cisplatin, docetaxel and 5?fluorouracil (5?FU) and the response rates to these drugs in patients is 60?80%. The present study aimed to generate head and neck cancer triple drug?resistant cell lines in an effort towards elucidating the mechanisms underlying chemoresistance and providing a resourceful tool for drug design. Using two head and neck squamous cell carcinoma cell lines, Hep?2 (larynx) and CAL?27 (oral cavity), the present study sequentially exposed these cells to increasing concentrations of the combination of docetaxel, cisplatin and 5?FU (TPF) to generate triple drug?resistant cells, termed Hep?2 TPF resistant (TPFR) and CAL?27 TPFR. The effect of the drug treatments on the cell viability, apoptosis, cell cycle and the expression of genes associated with multidrug resistance were analyzed in the parental cells and drug?resistant counterparts. The Hep?2 TPFR and CAL?27 TPFR cells exhibited a higher resistance index (RI?2) compared with that of the parental cells. Cell cycle analysis revealed a decreased number of TPFR cells in G0/G1 phase (P<0.05) and a corresponding accumulation of cells in G2/M phase. A reduced degree of apoptosis in these cells (Hep?2, 33 vs 20%, P=0.003; and CAL?27, 18 vs 9.7%) was complemented by an increased expression of genes involved in drug resistance, including MDR1, MRP2, ERCC1, CTR, survivin and thymidylate synthase. The present study, therefore, established two multi drug?resistant head and neck squamous cell carcinoma cell lines and characterized these cells on a cellular and molecular level. Development of these tools accentuates their requirement in the field of drug discovery and in mechanistic studies elucidating the underlying mechanisms of drug resistance. PMID:25962396

  18. Role of Alpha-Synuclein Protein Levels in Mitochondrial Morphology and Cell Survival in Cell Lines

    PubMed Central

    Zhu, Min; Li, Wenwei; Lu, Chuanzhen

    2012-01-01

    ?-Synuclein is highly associated with some neurodegeneration and malignancies. Overexpressing wild-type or mutant ?-synuclein promotes neuronal death by mitochondrial dysfunction, the underlying mechanisms of which remain poorly defined. It was recently reported that ?-synuclein expression could directly lead to mitochondrial fragmentation in vitro and in vivo, which may be due to ?-synuclein localization on mitochondria. Here, we applied a double staining method to demonstrate mitochondrial morphogenetic changes in cells overexpressed with ?-synuclein. We show that mitochondrial localization of ?-synuclein was increased following its overexpression in three distinct cell lines, including HeLa, SH-SY5Y, and PC12 cells, but no alteration in mitochondrial morphology was detected. However, ?-synuclein knockdown prevents MPP+-induced mitochondrial fragmentation in SH-SY5Y and PC12 cells. These data suggest that ?-synuclein protein levels hardly affect mitochondrial morphology in normal cell lines, but may have some influence on that under certain environmental conditions. PMID:22558453

  19. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues

    PubMed Central

    Barallon, Rita; Bauer, Steven R.; Butler, John; Capes-Davis, Amanda; Dirks, Wilhelm G.; Furtado, Manohar; Kline, Margaret C.; Kohara, Arihiro; Los, Georgyi V.; MacLeod, Roderick A. F.; Masters, John R. W.; Nardone, Mark; Nardone, Roland M.; Nims, Raymond W.; Price, Paul J.; Reid, Yvonne A.; Shewale, Jaiprakash; Sykes, Gregory; Steuer, Anton F.; Storts, Douglas R.; Thomson, Jim; Taraporewala, Zenobia; Alston-Roberts, Christine; Kerrigan, Liz

    2010-01-01

    Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues. PMID:20614197

  20. Recommendation of short tandem repeat profiling for authenticating human cell lines, stem cells, and tissues.

    PubMed

    Barallon, Rita; Bauer, Steven R; Butler, John; Capes-Davis, Amanda; Dirks, Wilhelm G; Elmore, Eugene; Furtado, Manohar; Kline, Margaret C; Kohara, Arihiro; Los, Georgyi V; MacLeod, Roderick A F; Masters, John R W; Nardone, Mark; Nardone, Roland M; Nims, Raymond W; Price, Paul J; Reid, Yvonne A; Shewale, Jaiprakash; Sykes, Gregory; Steuer, Anton F; Storts, Douglas R; Thomson, Jim; Taraporewala, Zenobia; Alston-Roberts, Christine; Kerrigan, Liz

    2010-10-01

    Cell misidentification and cross-contamination have plagued biomedical research for as long as cells have been employed as research tools. Examples of misidentified cell lines continue to surface to this day. Efforts to eradicate the problem by raising awareness of the issue and by asking scientists voluntarily to take appropriate actions have not been successful. Unambiguous cell authentication is an essential step in the scientific process and should be an inherent consideration during peer review of papers submitted for publication or during review of grants submitted for funding. In order to facilitate proper identity testing, accurate, reliable, inexpensive, and standardized methods for authentication of cells and cell lines must be made available. To this end, an international team of scientists is, at this time, preparing a consensus standard on the authentication of human cells using short tandem repeat (STR) profiling. This standard, which will be submitted for review and approval as an American National Standard by the American National Standards Institute, will provide investigators guidance on the use of STR profiling for authenticating human cell lines. Such guidance will include methodological detail on the preparation of the DNA sample, the appropriate numbers and types of loci to be evaluated, and the interpretation and quality control of the results. Associated with the standard itself will be the establishment and maintenance of a public STR profile database under the auspices of the National Center for Biotechnology Information. The consensus standard is anticipated to be adopted by granting agencies and scientific journals as appropriate methodology for authenticating human cell lines, stem cells, and tissues. PMID:20614197

  1. Duck lymphocytes. VIII. T-lymphoblastoid cell lines from reticuloendotheliosis virus-induced tumours

    Microsoft Academic Search

    Sarah W. S. Chan; Yuki Bando; G. W. Warr; Darlene L. Middleton; D. A. Higgins

    1999-01-01

    The T strain of reticuloendotheliosis virus (REV-T) obtained, along with the helper chicken syncytia virus (CSV), from the CSO4 cell line was highly oncogenic and rapidly fatal in ducks. Tumours were mainly seen in spleen, but neoplastic cells were observed microscopically in many organs. In vitro REV transformation of duck lymphocytes failed to yield stable cell lines, so cells from

  2. Human Hepatocellular Carcinoma Cell Lines Secrete the Major Plasma Proteins and Hepatitis B Surface Antigen

    Microsoft Academic Search

    Barbara B. Knowles; Chin C. Howe; David P. Aden

    1980-01-01

    Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B is injected into athymic mice, metastatic hepatocellular carcinomas

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

    PubMed Central

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

    2013-01-01

    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

  4. 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

  5. A novel 2,6-diisopropylphenyl–docosahexaenoamide conjugate induces apoptosis in T cell acute lymphoblastic leukemia cell lines

    Microsoft Academic Search

    Jeffrey D. Altenburg; Kevin A. Harvey; Sharon McCray; Zhidong Xu; Rafat A. Siddiqui

    2011-01-01

    We have previously characterized the effects of 2,6-diisopropylphenyl–docosahexaenoamide (DIP–DHA) conjugates and their analogs on the proliferation and progression of breast cancer cell lines. For this study, we investigated the effects of the DIP–DHA conjugate on 2 representative T cell acute lymphoblastic leukemia (T-ALL) cell lines: CEM and Jurkat. Treatment of both cell lines with DIP–DHA resulted in significantly greater inhibition

  6. Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line

    Microsoft Academic Search

    Toru Kondo; Takao Setoguchi; Tetsuya Taga

    2004-01-01

    Both stem cells and cancer cells are thought to be capable of unlimited proliferation. Paradoxically, however, some cancers seem to contain stem-like cells (cancer stem cells). To help resolve this paradox, we investigated whether established malignant cell lines, which have been maintained for years in culture, contain a subpopulation of stem cells. In this article, we show that many cancer

  7. 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

  8. A forensic path to RGC-5 cell line identification: lessons learned.

    PubMed

    Krishnamoorthy, Raghu R; Clark, Abbot F; Daudt, Donald; Vishwanatha, Jamboor K; Yorio, Thomas

    2013-08-01

    In 2001, a transformed cell line RGC-5 was developed from the rat retina that was thought to be of retinal ganglion cell origin. Since that time many investigators have used this line in a wide variety of studies to understand better retinal ganglion cell activity, cell signaling, and neuroprotection. Recently, a publication emerged that claimed that this RGC-5 cell line was derived from mouse and not rat, and other studies also indicated the expression of certain proteins that typically were not associated with retinal ganglion cells. This certainly came as a shock not only to the originators of this cell line, but also to others who have been using this as an in vitro model of rat retinal ganglion cells. As a result, we undertook experiments to determine if the RGC-5 cell line currently in use may have been mischaracterized. We, indeed, found that the RGC-5 cell line was of mouse and not rat origin, as was claimed originally in the original research report. We further determined whether these cells were of retinal ganglion origin. Our findings showed conclusively that RGC-5 cells were, indeed, of mouse origin and, using additional cytogenetic profile testing, karyotyping, and genetic and protein profiling, we concluded that these cells were not of retinal ganglion cell origin, but were the cell line 661W, a mouse SV-40 T antigen transformed photoreceptor cell line. The 661W cell line also was present in the laboratory of the originating laboratory and probably resulted in cross-contamination. The present study reviews some of the errors that were made in misidentifying the RGC-5 cell line and offers some insight as to how this may have happened, and ways one can avoid mischaracterization of a potentially important cell line. PMID:23975727

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

    PubMed Central

    2015-01-01

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

  10. How Reliable Are Sino-Nasal Cell Lines for Studying the Pathophysiology of Chronic Rhinosinusitis?

    PubMed Central

    Suwara, Monika I.; Borthwick, Lee A.; Wilson, Janet A.; Mann, Derek A.; Fisher, Andrew J.

    2015-01-01

    Background: Well-characterized cell lines represent useful scientific tools to study the pathophysiology of human disease. Chronic rhinosinusitis (CRS) is a very common condition, though the number of CRS cell lines is limited, as are data showing how closely they resemble primary cells. Methodology: Searches for available human cell lines were performed using the American Type Culture Collection (ATCC) and European Collection of Cell Cultures (ECACC). Identified cells were cultured and characterized with tinctorial and immunohistochemical staining and ELISA to assess their response to common, disease-relevant inflammatory stimuli. Carefully phenotyped CRS patients were recruited with informed consent. Primary nasal epithelial cell (PNEC) brushings were harvested, cultured, and compared to the available cell lines. Results: Searches identified 1 relevant CRS sino-nasal cell line, RPMI 2650. Cultured PNECs showed strong expression of epithelial markers while being negative for mesenchymal markers. However, RPMI 2650 cells show an atypical mixed epithelial/mesenchymal phenotype. When stimulated by pro-inflammatory ligands, PNECs responded in a dose-dependent manner, whereas RPMI 2650 cells showed limited response. Conclusions: The number and availability of cell lines to study the pathophysiology of CRS greatly underrepresent the disease burden. Additionally, the sole commercially available cell line appears to have a different phenotype and behavior to primary patient-derived cells. The development of further reproducible cell lines would be beneficial in our understanding of CRS. PMID:25539661

  11. The role of NEFL in cell growth and invasion in head and neck squamous cell carcinoma cell lines.

    PubMed

    Huang, Zhiquan; Zhuo, Ying; Shen, Zhuojian; Wang, Youyuan; Wang, Lili; Li, Haigang; Chen, Ju; Chen, Weiliang

    2014-03-01

    The neurofilament light polypeptide (NEFL) gene located on chromosome 8q21 is associated with the cancer of several organs and is regarded as a potential tumor suppressor gene. However, the role of the NEFL protein has not yet been studied in cancer cells. Although evidence suggests that there is a correlation between NEFL expression and cancer, studies regarding the role of the NEFL protein have been mostly limited to neurological diseases, such as Charcot-Marie-Tooth's disease (CMT). Most of these studies have not explored the role of NEFL in cancer cell apoptosis and/or invasion. In this study, NEFL expression was manipulated, and apoptosis and invasion were compared in head and neck squamous cell carcinoma cell lines. The results show that the expression of NEFL induces cancer cell apoptosis and inhibits invasion in these cell lines, suggesting that NEFL may play a role in cancer cell apoptosis and invasion. PMID:23992471

  12. Cytotoxic effects of fascaplysin against small cell lung cancer cell lines.

    PubMed

    Hamilton, Gerhard

    2014-03-01

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

  13. Interaction of the hemolytic lectin, CEL-III, with cultured human leukemic cell lines.

    PubMed

    Sallay, I; Moriwaki, S; Nakamura, O; Yasuda, S; Kimura, M; Yamasaki, N; Itoh, K; Ohba, H

    2000-12-01

    We studied interaction of CEL-III with cultured human leukemic cell lines and lymphocytes from normal adults by evaluating the extent of cytotoxicity and cytoagglutination. Among acute T lymphoblastic leukemia (T-ALL) cell lines, CEL-III displayed increased toxicity against different acute lymphoblastic leukemia (ALL) cell lines as a function of increasing differentiation stage. In the case of acute B lymphoblastic leukemia (B-ALL) cell lines, CEL-III showed strong cytotoxicity against relatively immature cell lines. We found that CEL-III was more toxic for ALL cell lines than leukocytes obtained from peripheral blood of healthy adults. Strong influence of the additional amount of calcium ion on the extent of cytotoxicity was observed. In addition, we describe a new way to evaluate the extent of cytoagglutination in "% of agglutinated cells". These findings make CEL-III a promising candidate in research for lectins which bind to and destroy only the targeted leukemic cells. PMID:11177600

  14. 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.

  15. A rare, human prostate oncocyte cell originates from the prostatic carcinoma (DU145) cell line.

    PubMed

    Gilloteaux, Jacques; Eze, Nkechinyere; Jamison, James M; McGuire, Karen; Summers, Jack L

    2013-12-01

    DU145 human prostate carcinoma cells are typically poorly differentiated and contain only scantily distributed organelles. However, among numerous tumor cells randomly examined by electron microscopy out of in vitro cultivation, a peculiar, rare oncocyte-like cell type has been observed whose nucleus appears to be of small dimension and with a cytoplasm almost entirely filled with often distorted mitochondria. A few small, dispersed lysosomal bodies, small cisterns of the endoplasmic reticulum and a few glycogen patches can be found among highly osmiophilic contrasted, cytosolic spaces filled by innumerable ribonucleoproteins. The excessive population of mitochondria may have arisen from a more populated tumor cell type wherein the altered mitochondria are found to appear burgeoning into a spherical-like size progeny crowding the tumor cells. Literature cited between 1950 and the present suggests that this rare, oncocytic, benign prostatic tumor cell type is likely appear epigenetically, stemming from an original secretory cell, which is confirmed by the origin of the cell line originally maintained as cell line out of a brain metastatic, adenocarcinoma niche. PMID:23957452

  16. Selective Killing of Human Malignant Cell Lines Deficient in Methylthioadenosine Phosphorylase, a Purine Metabolic Enzyme

    Microsoft Academic Search

    Naoyuki Kamatani; Walter A. Nelson-Rees; Dennis A. Carson

    1981-01-01

    Seven out of 31 (23%) human malignant tumor cell lines had no detectable methylthioadenosine phosphorylase activity (<0.001 nmol\\/min per mg of protein), assayed with 5'-chloroadenosine as substrate. The enzyme-deficient cell lines were derived from five leukemias, one melanoma, and one breast cancer. None of 16 cell lines of nonmalignant origin, derived from lymphocytes, fibroblasts, and epithelial cells, lacked the enzyme

  17. Signaling network of paclitaxel-induced apoptosis in the LNCaP prostate cancer cell line

    Microsoft Academic Search

    R Panvichian; K Orth; M. J Pilat; M. L Day; K. C Day; C Yee; J. M Kamradt; K. J Pienta

    1999-01-01

    Objectives. To attempt to identify the relationship of the key regulator molecules in paclitaxel-induced apoptosis using two metastatic cell lines: the human prostate carcinoma LNCaP line and the cervical carcinoma HeLa cell line.Methods. Both LNCaP and HeLa cells were continuously exposed to clinically achievable concentrations of paclitaxel and observed for activation of programmed cell death as measured by cytotoxic dose-response

  18. Characterization of a Human Squamous Carcinoma Cell Line Resistant to m-Diamminedichloroplatinum(II)1

    Microsoft Academic Search

    Beverly A. Teicher; Sylvia A. Holden; Michael J. Kelley; Thomas C. Shea; Carol A. Cucchi; Andre Rosowsky; W. David Henner; Emil Frei

    1987-01-01

    We have developed a human head and neck squamous cell carcinoma cell line (SCC-25\\/CP) which is relatively stably resistant to m-diam- minedichloroplatinum(II) (('1)1)1') after repeated exposure to escalating doses of the drug. The studies reported elucidate the mechanism(s) by which the SCC-25\\/CP cell line is resistant to CDDP. The SCC-25\\/CP cell line is approximately 30-fold resistant to CDDP, approximately 10-

  19. Tick cell lines: tools for tick and tick-borne disease research

    Microsoft Academic Search

    Lesley Bell-Sakyi; Erich Zweygarth; Edmour F. Blouin; Ernest A. Gould; Frans Jongejan

    2007-01-01

    Over 40 cell lines are currently available from 13 ixodid\\u000aand one argasid tick species. The successful isolation\\u000aand propagation of several economically important tickborne\\u000apathogens in tick cell lines has created a useful\\u000amodel to study interactions between tick cells and\\u000athese viral and bacterial disease agents. Tick cell lines\\u000ahave already proved to be a useful tool in

  20. Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats

    PubMed Central

    Men, Hongsheng; Bauer, Beth A.

    2012-01-01

    Germline-competent rat embryonic stem (ES) cell lines are important resources for the creation of mutant rat models using ES-cell-based gene targeting technology. The ability to isolate germline-competent ES cell lines from any rat strain, including genetically modified strains, would allow for more sophisticated genetic manipulations without extensive breeding. Sprague Dawley (SD) males carrying an enhanced green fluorescent protein (EGFP) transgene were used as the founder animals for the derivation of ES cell lines. A number of ES cell lines were established and subjected to rigorous quality control testing that included assessment of pluripotency factor expression, karyotype analysis, and pathogen/sterility testing. Two male ES cell lines, SD-Tg.EC1/Rrrc and SD-Tg.EC8/Rrrc, were injected into blastocysts recovered from a cross of Dark Agouti (DA) males with SD females. Resulting chimeric animals were bred with wild-type SD mates to verify the germline transmissibility of the ES cell lines by identifying pups carrying the ES cell line–derived EGFP transgene. While both ES cell lines gave rise to chimeric animals, only SD-Tg.EC1 was germline competent. This confirms the feasibility of deriving germline-competent ES cell lines from transgenic rat strains and provides a novel ES cell line with a stable green fluorescent protein (GFP) reporter for future genetic manipulations to create new rat models. PMID:22455749

  1. Adult T-Cell Leukemia: Antigen in an ATL Cell Line and Detection of Antibodies to the Antigen in Human Sera

    Microsoft Academic Search

    Yorio Hinuma; Kinya Nagata; Masao Hanaoka; Masuyo Nakai; Tadashi Matsumoto; Ken-Ichiro Kinoshita; Shigeru Shirakawa; Isao Miyoshi

    1981-01-01

    Indirect immunofluorescence of certain human sera demonstrated an antigen(s) in the cytoplasm of 1-5% of the cells of a T-cell line, MT-1, from a patient with adult T-cell leukemia (ATL), which is endemic in southwestern Japan. The antigen was not detected in other human lymphoid cell lines, including six T-cell lines, seven B-cell lines, and four non-T non-B cell lines.

  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. Gene expression profiling analysis of osteosarcoma cell lines.

    PubMed

    Sun, Lu; Li, Jie; Yan, Bing

    2015-09-01

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

  4. The cytotoxic effects of bendamustine in combination with cytarabine in mantle cell lymphoma cell lines

    Microsoft Academic Search

    Carlo Visco; Silvia Castegnaro; Katia Chieregato; Martina Bernardi; Elena Albiero; Cristina Zanon; Domenico Madeo; Francesco Rodeghiero

    Bendamustine is clinically useful in mantle-cell lymphoma (MCL). Its favorable toxicity profile in-vivo favors its combination with other cytotoxic drugs. Cytarabine is a key drug in the treatment of younger patients with MCL. The current study investigated the in-vitro cytotoxic effect of bendamustine and cytarabine, alone or combined, on two MCL cell lines representing the classic and blastoid variant of

  5. Host cell protein dynamics in the supernatant of a mAb producing CHO cell line.

    PubMed

    Tait, A S; Hogwood, C E M; Smales, C M; Bracewell, D G

    2012-04-01

    The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile. PMID:22124969

  6. Bromodeoxyuridine Induces p53Dependent and Independent Cell Cycle Arrests in Human Gastric Carcinoma Cell Lines

    Microsoft Academic Search

    Dun-Fa Peng; Hiroyuki Sugihara; Takanori Hattori

    2001-01-01

    Objectives: This study was designed to clarify the effects of bromodeoxyuridine (BrdU) on cell cycle progression and induction of apoptosis, and to demonstrate the role of P53 in these processes. Methods: We continuously exposed four human gastric carcinoma cell lines with different P53 status (P53 wild-type AGS and MKN-45, P53-mutated MKN-28 and P53-deleted KATO-III) to BrdU in asynchronous and synchronous

  7. Isolation and characterization of cell lines of Nicotiana tabacum lacking nitrate reductase

    Microsoft Academic Search

    Andreas J. Mtiller; Reinhard Grafe

    1978-01-01

    Chlorate-resistant cell lines were established from survivors after plating allodihaploid cells of Nicotiana tabacum into solid medium containing 20 mM chlorate and amino acids as sole nitrogen source. Data characterizing 9 of the most resistant lines are presented. The mutational origin of these lines was inferred on the basis of the enhancement of the variant frequency by mutagen treatment, and

  8. A one-mesh method for the cell-centered discretization of slide lines , B. Despresb

    E-print Network

    Paris-Sud XI, Université de

    A one-mesh method for the cell-centered discretization of slide lines G. Claira, , B. Despresb , E on several basic problems. Keywords: Slide lines, Constraints, Minimization method, Lagrangian Hydrodynamic of slide lines were described for staggered schemes. Recently, new methods adapted to cell

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

    Hasibeder, Astrid; Venkataramani, Vivek; Thelen, Paul; Radzun, Heinz-Joachim; Schweyer, Stefan

    2013-11-01

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

  11. ACTION OF CYTOCHALASIN D ON CELLS OF ESTABLISHED LINES

    PubMed Central

    Miranda, Armand F.; Godman, Gabriel C.; Deitch, Arline D.; Tanenbaum, Stuart W.

    1974-01-01

    HeLa, Vero, L, HEp2, and MDBK cells respond immediately to 0.2–0.5 µg/ml cytochalasin D (CD) with sustained contraction (contracture), loss of microvilli, expression of endoplasmic contents (zeiosis), nuclear protrusion, and extension of cytoplasmic processes. The development of these changes is depicted, and the dose-response patterns in these cell lines are described. MDBK is generally most resistant and HeLa most sensitive to these effects of CD. Cells in G1 are most sensitive to CD; responsiveness decreases progressively during early S and is least in mid S through G2. CD inhibits transport of [14C]deoxyglucose in HeLa by about 45% but has no significant effect on hexose uptake in Vero and MDBK; sugar transport is thus apparently unrelated to any morphologic effect of CD. Although spreading and attachment are impeded, CD does not decrease and may even enhance the adhesiveness of established monolayers. Contraction appears to be a primary early effect of CD, upon which other visible changes follow. It is prevented by some inhibitors of energy metabolism (deoxyglucose and dinitrophenol) and does not occur in glycerinated models without ATP. The possible bases of the contractile response to CD are discussed. Although direct or indirect action of CD on some microfilaments may occur, a generalized structural disruption of contractile filaments by CD is considered unlikely. PMID:4208074

  12. The conformational alteration of the mutated extracellular domain of Fas in an adult T cell leukemia cell line

    Microsoft Academic Search

    Takahiro Maeda; Susumu Nakayama; Yasuaki Yamada; Kazuyuki Sugahara; Hajime Isomoto; Masayuki Tawara; Reishi Yamasaki; Yasuyuki Onimaru; Tetsuro Matsushita; Yoshiyuki Ohzono; Shimeru Kamihira

    2002-01-01

    Fas (APO-1\\/CD95) is a cell surface receptor involved in apoptosis. Almost all adult T cell leukemia (ATL) cells express abundant Fas antigen and show apoptosis induced by IgM anti-Fas monoclonal antibody (mAb). We established the ATL cell line, RSO4, which was obtained from Fas-sensitive ATL cell line SO4 and showed resistance to apoptosis induced by the mAb. By sequencing analysis

  13. Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

    SciTech Connect

    Kawata, Shigehisa [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan); Suzuki, Jun [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan); Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 (Japan); Maruoka, Masahiro [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan); Mizutamari, Megumi [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan); Ishida-Kitagawa, Norihiro [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan); Yogo, Keiichiro [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan); Jat, Parmjit S. [Department of Neurodegenerative Disease, University College London, Square, London, WC1N 3BG (United Kingdom); Shishido, Tomoyuki [Laboratory of Molecular Oncology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 (Japan)]. 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.

  14. Experimental exposure of arsenic in cultured rat intestinal epithelial cells and cell line: Toxicological consequences

    Microsoft Academic Search

    Raj K. Upreti; A. Kannan; A. B. Pant

    2007-01-01

    Arsenic is a naturally occurring metalloid and the drinking water contamination by inorganic arsenic remains a major public health problem. The trivalent arsenic (arsenite) is more toxic than the pentavalent form (arsenate), and is known to cause gastrointestinal toxicity. Specific immortal cell lines are considered to be suitable for toxicity screening and testing of chemicals as they are easy to

  15. Cell division promotes efficient retrotransposition in a stable L1 reporter cell line

    PubMed Central

    2013-01-01

    Background Long interspersed element type one (L1) actively modifies the human genome by inserting new copies of itself. This process, termed retrotransposition, requires the formation of an L1 ribonucleoprotein (RNP) complex, which must enter the nucleus before retrotransposition can proceed. Thus, the nuclear import of L1 RNP presents an opportunity for cells to regulate L1 retrotransposition post-translationally. The effect of cell division on L1 retrotransposition has been investigated by two previous studies, which observed varied degrees of inhibition in retrotransposition when primary cell strains or cancer cell lines were experimentally arrested in different stages of the cell cycle. However, seemingly divergent conclusions were reached. The role of cell division on retrotransposition remains highly debated. Findings To monitor both L1 expression and retrotransposition quantitatively, we developed a stable dual-luciferase L1 reporter cell line, in which a bi-directional tetracycline-inducible promoter drives the expression of both a firefly luciferase-tagged L1 element and a Renilla luciferase, the latter indicative of the level of promoter induction. We observed an additional 10-fold reduction in retrotransposition in cell-cycle arrested cells even after retrotransposition had been normalized to Renilla luciferase or L1 ORF1 protein levels. In synchronized cells, cells undergoing two mitoses showed 2.6-fold higher retrotransposition than those undergoing one mitosis although L1 expression was induced for the same amount of time. Conclusions Our data provide additional support for an important role of cell division in retrotransposition and argue that restricting the accessibility of L1 RNP to nuclear DNA could be a post-translational regulatory mechanism for retrotransposition. PMID:23497436

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

    PubMed

    Genzel, Yvonne

    2015-05-01

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

  17. Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line.

    PubMed

    Hughes, Elizabeth D; Qu, Yun Yan; Genik, Suzanne J; Lyons, Robert H; Pacheco, Christopher D; Lieberman, Andrew P; Samuelson, Linda C; Nasonkin, Igor O; Camper, Sally A; Van Keuren, Margaret L; Saunders, Thomas L

    2007-08-01

    Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background. PMID:17828574

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

    PubMed Central

    Yan, Bin; Peng, Zhi-Yong

    2015-01-01

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

  19. Establishment and characterization of a new cell line of Chilo suppressalis Walker (Lepidoptera: Pyralididae).

    PubMed

    Liu, Guangfu; Xu, Yipeng; Yu, Xiaoping

    2015-03-01

    A new cell line, designated as ZJBIQ-Chsu-I, was initiated from the fat body of larval Chilo suppressalis (Walker) (Lepidoptera: Pyralididae) in TNM-FH insect medium containing 15% fetal bovine serum. The polygonal cells (65.6%) were predominant among various cell types, and the diameter range was from 12.63 to 22.50 ?m. The cell line showed a typical lepidopteran chromosome pattern ranging from 108 to 136 chromosomes in the majority of the cells. The population doubling time (PDT) of the cell line at the 15th passage was 62 h. This cell line was found to be susceptible to Spodoptera exigua nuclear polyhedrosis virus (SeNPV). By the DNA amplification fingerprinting polymerase chain reaction (DAF-PCR) technique, it was confirmed that cell line ZJBIQ-Chsu-I really originated from C. suppressalis. PMID:25381037

  20. DNA Fingerprint Comparison of Rainbow Trout and RTG-2 Cell Line Using Random Amplified Polymorphic DNA

    Microsoft Academic Search

    Concepcion Becerril; Helda Acevedo; Mar Ferrero; Felix Sanz; Argelia Castańo

    2001-01-01

    The detection of genotoxic effects using in vitro cell systems can be extremely useful in risk assessment procedures. However, care should be taken in the extrapolation of in vitro results since, amongst other factors, established cell lines may deviate from the genetic characteristics of their species. In this work, the genetic similarities between the RTG-2 cell line and rainbow trout

  1. Transplantation of Germ Line Stem Cells for the Study and Manipulation of Spermatogenesis

    Microsoft Academic Search

    I. Dobrinski

    Transplantation of male germ line stem cells from a fertile donor to the testis of an infertile recipient restores donor-derived spermatogenesis in the recipient testis and the resulting sperm pass the donor genotype to the offspring of the recipient. Germ cell transplantation has been an invaluable tool to elucidate the biology of male germ line stem cells and their niche

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Effect of radiation combined with hyperthermia on human prostatic carcinoma cell lines in culture

    Microsoft Academic Search

    I. Kaver; J. L. Ware; J. D. Wilson; W. W. Jr. Koontz

    1991-01-01

    The effect of radiation combined with heat on three human prostatic carcinoma cell lines growing in vitro was investigated. Cells were exposed to different radiation doses followed by heat treatment at 43 degrees C for one hour. Heat treatment, given ten minutes after radiation, significantly enhanced the radiation response of all the cell lines studied. The combined effect of radiation

  4. Quantitative gene expression assessment identifies appropriate cell line models for individual cervical cancer pathways

    Microsoft Academic Search

    Mark W Carlson; Vishwanath R Iyer; Edward M Marcotte

    2007-01-01

    BACKGROUND: Cell lines have been used to study cancer for decades, but truly quantitative assessment of their performance as models is often lacking. We used gene expression profiling to quantitatively assess the gene expression of nine cell line models of cervical cancer. RESULTS: We find a wide variation in the extent to which different cell culture models mimic late-stage invasive

  5. Cytotoxic activity of pierisin, from the cabbage butterfly, Pieris rapae, in various human cancer cell lines

    Microsoft Academic Search

    Takuo Kono; Masahiko Watanabe; Kotaro Koyama; Taketoshi Kishimoto; Shoji Fukushima; Takashi Sugimura; Keiji Wakabayashi

    1999-01-01

    Pierisin, a protein purified from pupae of the cabbage butterfly, Pieris rapae, exhibits cytotoxic effects against the human gastric cancer TMK-1 cell line, inducing apoptosis. The present study was performed to determine whether pierisin might exert a similar influence on nine other human cancer cell lines and human umbilical vein endothelial cells (HUVECs). Pierisin showed cytotoxic effects in all the

  6. Bit line coupling memory tests for single-cell fails in SRAMs

    Microsoft Academic Search

    Sandra Irobi; Zaid Al-Ars; Said Hamdioui

    2010-01-01

    Due to the decreasing dimensions of manufactured devices, the effect of bit line capacitive coupling on the behavior of faulty memory cells cannot be ignored. Neighboring cells influence the faulty behavior of defective cells through coupling. This paper analyzes and validates this behavior theoretically and through electrical simulations. The paper evaluates the impact of bit line coupling in SRAMs on

  7. Genetic Inheritance of Gene Expression in Human Cell Lines

    PubMed Central

    Monks, S. A.; Leonardson, A.; Zhu, H.; Cundiff, P.; Pietrusiak, P.; Edwards, S.; Phillips, J. W.; Sachs, A.; Schadt, E. E.

    2004-01-01

    Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value <.000005. Of these, 13 genes possessed a QTL within 5 Mb of their physical location. Hierarchical clustering was performed on the basis of both Pearson correlation of gene expression and genetic correlation. Both reflected biologically relevant activity taking place in the lymphoblastoid cell lines, with greater coherency represented in Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways than in Gene Ontology database pathways. However, more pathway coherence was observed in KEGG pathways when clustering was based on genetic correlation than when clustering was based on Pearson correlation. As more expression data in segregating populations are generated, viewing clusters or networks based on genetic correlation measures and shared QTLs will offer potentially novel insights into the relationship among genes that may underlie complex traits. PMID:15514893

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

    SciTech Connect

    Youakim, A.; Herscovics, A.

    1985-11-01

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

  9. Morphological changes in amphibian and fish cell lines infected with Andrias davidianus ranavirus.

    PubMed

    Gao, X C; Chen, Z Y; Yuan, J D; Zhang, Q Y

    2015-01-01

    Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe disease in Chinese giant salamanders, the largest extant amphibian in the world. A fish cell line, Epithelioma papulosum cyprinid (EPC), and a new amphibian cell line, Chinese giant salamander spleen cell (GSSC), were infected with ADRV and observed by light and electron microscopy. The morphological changes in these two cell lines infected with ADRV were compared. Cytopathic effect (CPE) began with rounding of the cells, progressing to cell detachment in the cell monolayer, followed by cell lysis. Significant CPE was visualized as early as 24 h post infection (hpi) in EPC cells and at 36 hpi in GSSC cells. Microscopical examination showed clear and significant CPE in EPC cells, while less extensive and irregular CPE with some adherent cells remaining was observed in GSSC cells. Following ADRV infection, CPE became more extensive. Transmission electron micrographs showed many virus particles around cytoplasmic vacuoles, formed as crystalline arrays or scattered in the cytoplasm of infected cells. Infected cells showed alteration in nuclear morphology, with condensed and marginalized nuclear chromatin on the inner aspect of the nuclear membrane and formation of a cytoplasmic viromatrix adjacent to the nucleus in both cell lines. Some virus particles were also detected in the nucleus of infected GSSC cells. Both cell lines are able to support replication of ADRV and can therefore be used to investigate amphibian ranaviruses. PMID:25728809

  10. Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

    SciTech Connect

    Ageberg, Malin, E-mail: Malin.Ageberg@med.lu.se [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden)] [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden); Rydstroem, Karin, E-mail: Karin.Rydstom@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden)] [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linden, Ola, E-mail: Ola.Linden@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden)] [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Linderoth, Johan, E-mail: Johan.Linderoth@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden)] [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Jerkeman, Mats, E-mail: Mats.Jerkeman@skane.se [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden)] [Department of Oncology, Skanes University Hospital, Allmaenmott, Onkologiska kliniken i Lund, 221 85 Lund (Sweden); Drott, Kristina, E-mail: Kristina.Drott@med.lu.se [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden)] [Division of Hematology and Transfusion Medicine, Lund University, BMC C14, 221 84 Lund (Sweden)

    2011-05-01

    Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.

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

    PubMed

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

    2012-11-01

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

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

    PubMed

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

    2015-01-01

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

  13. Downstream targets of HOXB4 in a cell line model of primitive hematopoietic progenitor cells

    PubMed Central

    Lee, Han M.; Zhang, Hui; Schulz, Vincent; Tuck, David P.

    2010-01-01

    Enforced expression of the homeobox transcription factor HOXB4 has been shown to enhance hematopoietic stem cell self-renewal and expansion ex vivo and in vivo. To investigate the downstream targets of HOXB4 in hematopoietic progenitor cells, HOXB4 was constitutively overexpressed in the primitive hematopoietic progenitor cell line EML. Two genome-wide analytical techniques were used: RNA expression profiling using microarrays and chromatin immunoprecipitation (ChIP)–chip. RNA expression profiling revealed that 465 gene transcripts were differentially expressed in KLS (c-Kit+, Lin?, Sca-1+)-EML cells that overexpressed HOXB4 (KLS-EML-HOXB4) compared with control KLS-EML cells that were transduced with vector alone. In particular, erythroid-specific gene transcripts were observed to be highly down-regulated in KLS-EML-HOXB4 cells. ChIP-chip analysis revealed that the promoter region for 1910 genes, such as CD34, Sox4, and B220, were occupied by HOXB4 in KLS-EML-HOXB4 cells. Side-by-side comparison of the ChIP-chip and RNA expression profiling datasets provided correlative information and identified Gp49a and Laptm4b as candidate “stemness-related” genes. Both genes were highly ranked in both dataset lists and have been previously shown to be preferentially expressed in hematopoietic stem cells and down-regulated in mature hematopoietic cells, thus making them attractive candidates for future functional studies in hematopoietic cells. PMID:20404135

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

    NASA Astrophysics Data System (ADS)

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

    2014-10-01

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

  15. MC3 Mucoepidermoid carcinoma cell line enriched cancer stem-like cells following chemotherapy

    PubMed Central

    ZHANG, LOUQIANG; LI, LONGJIANG; WANG, YIN; LIU, YING; LI, CHUNJIE

    2014-01-01

    Mucoepidermoid carcinoma (MEC) is common in human salivary glands. Surgery is the preferred treatment method for MEC and chemotherapy is often administered following surgery as an adjuvant cancer treatment; however, chemotherapy does not completely prevent tumor recurrence. Emerging evidence has indicated the existence of cancer stem-like (CSL)-cells in tumors. CSL-cells are important in the development, invasion and drug resistance of carcinomas. The present study aimed to investigate whether chemotherapy enriched the CSL-cells in the MEC cell line of MC3 using 5-fluorouracil (5-Fu). The MC3 cells were treated with 5-Fu, which enhanced the spherogenesis and vitality of the cells and upregulated the pluripotency gene, octamer-binding transcription factor 4. Side population analysis demonstrated that the proportion of CSL-cells also increased. These findings showed that compared with other types of cancer cells, chemotherapy was unable to effectively kill the CSL-cells resulting in an enriched CSL-cell subpopulation with a higher resistance to chemotherapy, which may have been key the recurrence of MEC. PMID:24765178

  16. MC3 Mucoepidermoid carcinoma cell line enriched cancer stem-like cells following chemotherapy.

    PubMed

    Zhang, Louqiang; Li, Longjiang; Wang, Yin; Liu, Ying; Li, Chunjie

    2014-05-01

    Mucoepidermoid carcinoma (MEC) is common in human salivary glands. Surgery is the preferred treatment method for MEC and chemotherapy is often administered following surgery as an adjuvant cancer treatment; however, chemotherapy does not completely prevent tumor recurrence. Emerging evidence has indicated the existence of cancer stem-like (CSL)-cells in tumors. CSL-cells are important in the development, invasion and drug resistance of carcinomas. The present study aimed to investigate whether chemotherapy enriched the CSL-cells in the MEC cell line of MC3 using 5-fluorouracil (5-Fu). The MC3 cells were treated with 5-Fu, which enhanced the spherogenesis and vitality of the cells and upregulated the pluripotency gene, octamer-binding transcription factor 4. Side population analysis demonstrated that the proportion of CSL-cells also increased. These findings showed that compared with other types of cancer cells, chemotherapy was unable to effectively kill the CSL-cells resulting in an enriched CSL-cell subpopulation with a higher resistance to chemotherapy, which may have been key the recurrence of MEC. PMID:24765178

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

    PubMed Central

    2013-01-01

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

  18. Morphologic, molecular, and ultrastructural characterization of a feline synovial cell sarcoma and derived cell line.

    PubMed

    Cazzini, Paola; Frontera-Acevedo, Karelma; Garner, Bridget; Howerth, Elizabeth; Torres, Bryan; Northrup, Nicole; Sakamoto, Kaori

    2015-05-01

    A 2.5-year-old, male, neutered cat presented with a 5-month history of progressive right hind limb lameness and an enlarged right popliteal lymph node. Radiographs revealed significant bony lysis of the tarsus and distal tibia, and fine-needle aspirate of the bone lesion and lymph node revealed a neoplastic population of cells with uncertain origin. Amputation was elected, and the mass was submitted for histology and cellular culture for better characterization. Histologic examination revealed a mixture of spindle-shaped cells and larger, round to polygonal cells. All cells were immunoreactive for vimentin, and only the larger polygonal cells were also positive for cytokeratin. All cells were negative for desmin, smooth muscle actin, cluster of differentiation (CD)3, CD18, CD79a, macrophage antibody (MAC)387, and glial fibrillary acidic protein. Cultured neoplastic cells failed to express CD18, and were not able to secrete the pro-inflammatory cytokines tumor necrosis factor-?, interleukin-1 (IL-1)?, and IL-6 when stimulated by lipopolysaccharide, disproving that the cells originated from the macrophage or monocyte line. Ultrastructurally, neoplastic cells were characterized by abundant rough endoplasmic reticulum, interdigitating cellular processes, and membrane condensations. Based on location and cytologic, histologic, ultrastructural, and functional studies, this neoplasm was considered a synovial cell sarcoma. PMID:25901004

  19. A new cell line from larval fat bodies of the bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae).

    PubMed

    Zhang, Huan; Zhang, Yong-An; Qin, Qilian; Wang, Yuzhu; Li, Xuan; Miao, Lin; Yin, Zhenxian; Zhang, Aijun; Qu, Liangjian; Ding, Cui

    2006-01-01

    A new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells. It was confirmed to have originated from the H. armigera by the DNA amplification- fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera. PMID:17316061

  20. Proliferation of mitochondria during the cell cycle of the human cell line (HL-60)

    PubMed Central

    1981-01-01

    Using rhodamine 123 to stain mitochondria of the human cell line HL-60, we have followed their increase over the cell cycle by flow cytometry. A near-linear synthesis of mitochondrial mass was shown to occur over the cell cycle. A comparison with the cell's DNA synthesis pattern obtained by the same technique established a common time-base. The mitochondrial synthesis curve changes with culture age. As a control, thd dye was tested for its binding specificity and for its use to resolve mitochondria microscopically. Its stoichiometric range was established and, above 0.25 microgram/ml, it was shown to reduce growth rate and cell viability in culture. PMID:7195902

  1. A Niche Maintaining Germ Line Stem Cells in the Drosophila Ovary

    Microsoft Academic Search

    Ting Xie; Allan C. Spradling

    2000-01-01

    Stromal cells are thought to generate specific regulatory microenviroments or ``niches'' that control stem cell behavior. Characterizing stem cell niches in vivo remains an important goal that has been difficult to achieve. The individual ovarioles of the Drosophila ovary each contain about two germ line stem cells that maintain oocyte production. Here we show that anterior ovariolar somatic cells comprising

  2. Phenotypic and functional heterogeneity of human cloned natural killer cell lines

    Microsoft Academic Search

    Thierry Hercend; Ellis L. Reinherz; Stefan Meuer; Stuart F. Schlossman; Jerome Ritz

    1983-01-01

    Extensive efforts have recently been made to characterize cells capable of mediating natural killing activity (see ref. 1 for review) and increasing evidence has arisen that these cells were heterogeneous2,3. By using the methods we have recently developed for cloning natural killer (NK) cells derived from peripheral blood4, we have analysed the heterogeneity of human NK cells. Seven cell lines

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

    PubMed

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

    2015-06-01

    Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUP(r)GEMCI(20)), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer. PMID:26055179

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

    PubMed Central

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

    2015-01-01

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

  5. Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development

    PubMed Central

    Zhan, Yu; Su, Juanjuan; Du, Yarui; Xu, Guoliang; Shi, Yufang; Siebenlist, Ulrich; Zhang, Xiaoren

    2013-01-01

    Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs in vitro. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-?B subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells in vivo. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells in vitro and in vivo. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors. PMID:24086471

  6. Characterization of a porcine intestinal epithelial cell line for influenza virus production

    PubMed Central

    Sun, Zhi; Huber, Victor C.; McCormick, Kara; Kaushik, Radhey S.; Boon, Adrianus C. M.; Zhu, Longchao; Hause, Ben; Webby, Richard J.

    2012-01-01

    We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log10 TCID50 ml?1. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin–Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 ml?1, respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses. PMID:22739061

  7. Understanding pathogenetic aspects and clinical presentation of primary effusion lymphoma (PEL) through its derived cell lines

    PubMed Central

    Carbone, Antonino; Cesarman, Ethel; Gloghini, Annunziata; Drexler, Hans G.

    2013-01-01

    Primary effusion lymphoma (PEL) is a very rare subgroup of B-cell lymphomas presenting as pleural, peritoneal and pericardial neoplastic effusions in the absence of a solid tumor mass or recognizable nodal involvement. There is strong evidence that Kaposi’s sarcoma associated herpesvirus (KSHV) is a causal agent of PEL. PEL tumor cells are latently infected by KSHV with consistent expression of several viral proteins and microRNAs that can affect cellular proliferation, differentiation and survival. The most relevant data on pathogenesis and biology of KSHV have been provided by studies on PEL derived cell lines. Fourteen continuous cell lines have been established from the malignant effusions of patients with AIDS-and non-AIDS-associated PEL. These KSHV+ EBV+/? cell lines are wellcharacterized, authenticated and mostly available from public biological ressource centers. The PEL cell lines display unique features and are clearly distinct from other lymphoma cell lines. PEL cell lines represent an indispensable tool for the understanding of KSHV biology and its impact on the clinical manifestation of PEL. Studies on PEL cell lines have shown that a number of viral genes, expressed during latency or lytic life cycle, have effects on cell binding, proliferation, angiogenesis and inflammation. Also PEL cell lines are important model systems for the study of the pathology of PEL including the lack of invasive or destructive growth patterns and the peculiar propensity of PEL to involve body cavity surfaces. PMID:20051807

  8. Systematic variation in gene expression patterns in human cancer cell lines

    Microsoft Academic Search

    Douglas T. Ross; Uwe Scherf; Michael B. Eisen; Charles M. Perou; Christian Rees; Paul Spellman; Vishwanath Iyer; Stefanie S. Jeffrey; Matt Van de Rijn; Mark Waltham; Alexander Pergamenschikov; Jeffrey C. F. Lee; Deval Lashkari; Dari Shalon; Timothy G. Myers; David Botstein; John N. Weinstein; Patrick O. Brown

    2000-01-01

    We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were

  9. Immunohistochemical localization of glial cell line-derived neurotrophic factor in the human central nervous system

    Microsoft Academic Search

    Y Kawamoto; S Nakamura; A Matsuo; I Akiguchi; H Shibasaki

    2000-01-01

    Glial cell line-derived neurotrophic factor, initially purified from the rat glial cell line B49, has the ability to promote the survival and differentiation of various types of neurons in the central and peripheral nervous systems. In the present study, to evaluate the physiological role of glial cell line-derived neurotrophic factor in the central nervous system, we investigated the cellular and

  10. DNA fingerprinting of human cell lines using PCR amplification of fragment length polymorphisms

    Microsoft Academic Search

    Rui Yan; Mark Ottenbreit; Bharati Hukku; Michael Mally; Sharong Chou; Joseph Kaplan

    1996-01-01

    Summary  Methods for monitoring cell line identification and authentication include species-specific immunofluorescence, isoenzyme\\u000a phenotyping, chromosome analysis, and DNA fingerprinting. Most previous studies of DNA fingerprinting of cell lines have used\\u000a restriction fragment length polymorphism analysis. In this study, we examined the utility of an alternative and simpler method\\u000a of cell line DNA fingerprinting—polymerase chain reaction (PCR) amplification of fragment length polymorphisms.

  11. In vitro growth of microsporidia Anncaliia algerae in cell lines from warm water fish

    Microsoft Academic Search

    S. Richelle Monaghan; Rebecca L. Rumney; Nguyen T. K. Vo; Niels C. Bols; Lucy E. J. Lee

    2011-01-01

    Anncaliia algerae is an aquatic microsporidium that most commonly infects mosquitoes but can be grown on the rabbit kidney cell line, RK-13.\\u000a Spores were purified from RK-13 cultures and added to cell lines from warm water fish and from an insect. The cell lines were\\u000a GFSK-S1 and GFB3C-W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from

  12. Establishment and characterization of 13 cell lines from a green turtle ( Chelonia mydas ) with fibropapillomas

    Microsoft Academic Search

    Yuanan Lu; Vivek R. Nerurkar; Alonso A. Aguirre; Thierry M. Work; George H. Balazs; Richard Yanagihara

    1999-01-01

    Summary  Thirteen cell lines were established and characterized from brain, kidney, lung, spleen, heart, liver, gall bladder, urinary\\u000a bladder, pancreas, testis, skin, and periorbital and tumor tissues of an immature male green turtle (Chelonia mydas) with fibropapillomas. Cell lines were optimally maintained at 30° C in RPMI 1640 medium supplemented with 10% fetal bovine\\u000a serum. Propagation of the turtle cell lines

  13. Establishment and characterization of insect cell lines from 10 lepidopteran species

    Microsoft Academic Search

    Cynthia L. Goodman; Galal N. El Sayed; Arthur H. Mcintosh; James J. Grasela; Brad Stiles

    2001-01-01

    Summary  Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A\\u000a total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European\\u000a corn borer,Ostrinia nubilalis, a plutellid, the diamondback moth,Plutella xylostella, as well as eight noctuids: the black cutworm,Agrotis ipsilon, the celery looper,Anagrapha falcifera,

  14. Lymphokine-activated killer cells lyse human renal cancer cell lines and cultured normal kidney cells.

    PubMed Central

    Miltenburg, A M; Meijer-Paape, M E; Daha, M R; Paul, L C

    1988-01-01

    In this study, we investigated whether or not lymphokine-activated killer (LAK) cells can damage renal tissue and therefore whether they may contribute to graft destruction during kidney allograft rejection. Human peripheral blood mononuclear cells were activated with a lymphokine preparation and the resulting LAK cells were tested against kidney cells from various sources. Renal cancer cells as well as cultured normal kidney cells were efficiently lysed by LAK cells, as assessed with Cr-labelled target cells, showing that both cell types are sensitive to LAK cell-mediated cytolysis. PMID:3259208

  15. Characterization of Resistance to Rhabdovirus and Retrovirus Infection in a Human Myeloid Cell Line

    PubMed Central

    Boso, Guney; Somia, Nikunj V.

    2015-01-01

    Viruses interact with various permissive and restrictive factors in host cells throughout their replication cycle. Cell lines that are non-permissive to viral infection have been particularly useful in discovering host cell proteins involved in viral life cycles. Here we describe the characterization of a human myeloid leukemia cell line, KG-1, that is resistant to infection by retroviruses and a Rhabdovirus. We show that KG-1 cells are resistant to infection by Vesicular Stomatits Virus as well as VSV Glycoprotein (VSVG) pseudotyped retroviruses due to a defect in binding. Moreover our results indicate that entry by xenotropic retroviral envelope glycoprotein RD114 is impaired in KG-1 cells. Finally we characterize a post- entry block in the early phase of the retroviral life cycle in KG-1 cells that renders the cell line refractory to infection. This cell line will have utility in discovering proteins involved in infection by VSV and HIV-1. PMID:25811758

  16. Antitumor effects of saikosaponins, baicalin and baicalein on human hepatoma cell lines.

    PubMed

    Motoo, Y; Sawabu, N

    1994-10-28

    Antitumor effects of nine components of a herbal medicine, 'Sho-saiko-to', were investigated on human hepatoma cell lines (PLC/PRF/5, Hep-G2), human liver cells (Chang) and a human pancreatic cancer cell line (BxPC-3). The concentration of each component required for 50% inhibition of cell growth of PLC/PRF/5 cells was as follows: saikosaponin-d, baicalin, 20 micrograms/ml; saikosaponin-a, baicalein, 50 micrograms/ml; saikosaponin-b2, -c, ginsenoside-Rb1, -Rg1, glycyrrhizin, > 1000 micrograms/ml. Saikosaponin-a in 50-micrograms/ml quantities inhibited the cell growth and DNA synthesis of all the cell lines tested. These results indicate that 'Sho-saiko-to' includes potent antitumor components such as saikosaponin-a, -d, baicalin against human hepatoma cells as well as other human cell lines. PMID:7954360

  17. Time line of redox events in aging postmitotic cells

    PubMed Central

    Brandes, Nicolas; Tienson, Heather; Lindemann, Antje; Vitvitsky, Victor; Reichmann, Dana; Banerjee, Ruma; Jakob, Ursula

    2013-01-01

    The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death. DOI: http://dx.doi.org/10.7554/eLife.00306.001 PMID:23390587

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

    SciTech Connect

    Fiorica, E.; Spector, S.

    1988-01-01

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

  19. The importance of molecular cytogenetic analysis prior to using cell lines in research: The case of the KG-1a leukemia cell line

    PubMed Central

    PELLICCIA, FRANCA; UBERTINI, VALENTINA; BOSCO, NAZARIO

    2012-01-01

    KG-1 and its less differentiated subline KG-1a are leukemia cell lines used in research in a number of laboratories. The karyotypes of the two lines were initially identical. In the following years, further analysis revealed that the cell lines had acquired additional karyotypical abnormalities and differed in the presence of certain typical chromosomal rearrangements. To obtain cytogenetic authentication prior to the use of the two cell lines, we analyzed their karyotype by combining DAPI- and CMA-chromosome bandings and a fluorescence in situ hybridization (FISH)-based approach by using BAC clones useful for the identification of chromosome regions of interest. Sequences of the MYC, PLZF, RARA and BCR genes, that are known to play a critical role in leukemogenesis, and certain BAC clones mapped to five known common fragile sites (CFS) were used for the FISH analysis. A telomeric probe (TTAGGG)n and a set of BAC clones were used to characterize the marker chromosome der(1) that was observed in the cell line KG-1a. The existence of notable differences between the karyotype of the KG-1a cell line previously described, and that described in this study, demonstrate that the use of established cancer cell lines should be preceded by cytogenetic and/or molecular characterization. PMID:22844360

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

    PubMed Central

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

    2012-01-01

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

  1. [Isolation of classical swine pest virus from homologous and heterologic cell lines].

    PubMed

    Vergun, L Iu

    2005-01-01

    This study was devoted to the choice of cell line for isolation of CSPV with high sensitivity. For this purpose the homologous transplantable cell lines from the collection of European References Laboratory of CSF (National Veterinary Research Institute, Pulawy, Poland) and heterological primary cell culture from the collection "Biotest-Laboratory". Cell cultures were cultivated as a monolayer in 96-hole microtitration plates. Antigen of CSFV was detected in peroxides-linked assay (PLA). Cell culture SK-6 proved to be the best line for isolation of CSFV from organ samples and samples of infected cell culture. The cell line--SK-6 is recommended for cultivation of CSFV other lines (LT, PK-15) are not fit for this purpose. PMID:15765885

  2. Establishment and exploitation of hyperdiploid and non-hyperdiploid human myeloma cell lines

    PubMed Central

    Li, Xin; Pennisi, Angela; Zhan, Fenghuang; Sawyer, Jeffrey R.; Shaughnessy, John D.; Yaccoby, Shmuel

    2009-01-01

    Summary The establishment of clinically relevant human myeloma cell lines is central for our understanding of myeloma pathogenesis and development of novel therapies for the disease. Unfortunately, most available lines were generated from extramedullary sites, harbored multiple genetic abnormalities and categorized as non-hyperdiploid. In contrast, hyperdiploid myeloma cell lines, which represent more than 50% of patients, are rare. We established procedures for establishment of stroma-dependent myeloma lines by passaging primary myeloma cells, in severe combined immunodeficient-human (SCID-hu) or SCID-rab mice followed by maintenance in co-culture with stromal cells. We described the establishment and characterization of two hyperdiploid (LD and CF) and two non-hyperdiploid (JB and BN) cell lines. Using our animal models, we also established bortezomib-sensitive and -resistant BN lines. These cell lines were cellularly, phenotypically and molecularly characterized using flow cytometry immunophenotyping, DNA content, G-band and multicolor spectral karyotyping (SKY) and global gene expression profiling. All four cell lines were infected with lentiviral-expressing luciferase for detection of tumour cells at high sensitivity level and for monitoring myeloma growth in co-cultures and in vivo by live animal imaging. These myeloma cell lines and the procedures used for their establishment provide essential tools for studying myeloma biology and therapy. PMID:17760811

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

    NASA Astrophysics Data System (ADS)

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

    2012-06-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2014-10-01

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

  6. Bilirubin- and light induced cell death in a murine lymphoma cell line.

    PubMed

    Christensen, T; Roll, E B; Jaworska, A; Kinn, G

    2000-11-01

    Cells from the mouse lymphoma cell line L5178Y-R were exposed to blue light from phototherapy lamps in the presence of solutions of 160 microM bilirubin supplemented with serum albumin. HPLC analysis showed that the bilirubin solution was photooxidised as a function of increasing light dose. The cells were stained with trypan blue to score necrosis, and apoptosis was assayed by the terminal deoxynucleotide transferase assay (TdT) or by studying the nuclear structure in cells stained with propidium iodide. A rapidly developing apoptosis was observed after light doses killing 60-80% of the cells as judged from the trypan blue exclusion test. The fraction of apoptotic cells was smaller than the fraction of necrotic cells. Exposure of the cells to fractions of light at a high dose rate was compared to the effect of the same total dose at a lower dose rate given as a single fraction. No large differences were found, however, there was a tendency of a higher degree of necrosis as well as apoptosis in the cells receiving the light in fractions at a high dose rate. PMID:11233646

  7. Paclitaxel-induced apoptosis in non–small cell lung cancer cell lines is associated with increased caspase-3 activity

    Microsoft Academic Search

    Tracey L. Weigel; Michael T. Lotze; Peter K. Kim; Andrew A. Amoscato; James D. Luketich; Christine Odoux

    2000-01-01

    Objective: Our objective was to determine whether paclitaxel-induced apoptosis in human lung cancer cells is Fas dependent. Methods: Human lung cancer cell lines were evaluated for morphologic evidence of apoptosis, DNA fragmentation (TUNEL positivity), and caspase-3 activation after paclitaxel treatment. Human lung adenocarcinoma, squamous cell carcinoma, undifferentiated lung carcinoma, and bronchoalveolar carcinoma cell lines were each cultured in 10 ?mol\\/L

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  9. Whole-exome characterization of pancreatic neuroendocrine tumor cell lines BON-1 and QGP-1.

    PubMed

    Vandamme, Timon; Peeters, Marc; Dogan, Fadime; Pauwels, Patrick; Van Assche, Elvire; Beyens, Matthias; Mortier, Geert; Vandeweyer, Geert; de Herder, Wouter; Van Camp, Guy; Hofland, Leo J; Op de Beeck, Ken

    2015-04-01

    The human BON-1 and QGP-1 cell lines are two frequently used models in pancreatic neuroendocrine tumor (PNET) research. Data on the whole-exome genetic constitution of these cell lines is largely lacking. This study presents, to our knowledge, the first whole-exome profile of the BON-1 and QGP-1 cell lines. Cell line identity was confirmed by short tandem repeat profiling. Using GTG-banding and a CytoSNP-12v2 Beadchip array, cell line ploidy and chromosomal alterations were determined in BON-1 and QGP-1. The exomes of both cell lines were sequenced on Ilumina's HiSeq next-generation sequencing (NGS) platform. Single-nucleotide variants (SNVs) and insertions and deletions (indels) were detected using the Genome Analysis ToolKit. SNVs were validated by Sanger sequencing. Ploidy of BON-1 and QGP-1 was 3 and 4 respectively, with long stretches of loss of heterozygosity across multiple chromosomes, which is associated with aggressive tumor behavior. In BON-1, 57 frameshift indels and 1725 possible protein-altering SNVs were identified in the NGS data. In the QGP-1 cell line, 56 frameshift indels and 1095 SNVs were identified. ATRX, a PNET-associated gene, was mutated in both cell lines, while mutation of TSC2 was detected in BON-1. A mutation in NRAS was detected in BON-1, while KRAS was mutated in QGP-1, implicating aberrations in the RAS pathway in both cell lines. Homozygous mutations in TP53 with possible loss of function were identified in both cell lines. Various MUC genes, implicated in cell signaling, lubrication and chemical barriers, which are frequently expressed in PNET tissue samples, showed homozygous protein-altering SNVs in the BON-1 and QGP-1 cell lines. PMID:25612765

  10. Comparative properties of untreated and N-nitrosobenzylmethyl-amine-transformed rat esophageal epithelial cell lines.

    PubMed

    Stoner, G D; Babcock, M S; McCorquodale, M M; Gunning, W T; Jamasbi, R; Budd, N; Hukku, B

    1989-10-01

    A culture system utilizing rat esophageal epithelial cells has been developed. Four normal and eight N-nitrosobenzylmethylamine-treated lines were compared with respect to chromosome number, anchorage-independent growth in agarose, and tumorigenic potential in syngeneic rats. All cell lines were aneuploid with nine in the near-tetraploid range and three in the near-diploid range. No relation between tumorigenic potential and chromosome number or structure was apparent. Similarly, anchorage-independent growth in agarose did not correlate with tumorigenic potential. Three of the 12 immortalized lines (two carcinogen-treated and 1 untreated) induced well-differentiated squamous cell carcinomas in syngeneic rats. These tumors had weak metastatic potentials suggesting that tumorigenic potential and metastatic ability are separately controlled. These cell lines will be useful for the investigation of factors involved in the conversion of immortalized rat esophageal epithelial cell lines to lines of high metastatic potential. PMID:2808222

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

    E-print Network

    George, Carol Charlene

    2013-07-03

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

  12. New Helicoverpa armigera Hbn cell line from larval hemocyte for baculovirus studies.

    PubMed

    Sudeep, A B; Shouche, Y S; Mourya, D T; Pant, U

    2002-01-01

    A new cell line from the larval hemocytes of H. armigera was established in Grace's medium modified by adding lactalbumin hydrolysate and yeastolate (3.3g/l), and supplemented with fetal bovine serum (20%). The cell line was designated as NIV-HA-1195. The cell population at P-78 consisted mainly of epithelial-like cells (89.36%), fibroblast-like cells (8.31%) and giant cells (2.13%). The population doubling time was 96hr at P-8, 60hr at P-43. The chromosome number ranged from 45 to 200. The cell line is susceptible to the baculoviruses, Autographa californica nucleopolyhedrovirus (AcNPV), Spodoptera litura NPV and the homologous HaNPV. Isoenzyme profile and results of 16S rRNA heteroduplex analysis clearly indicated the species specificity of the new cell line. PMID:12561972

  13. Autophagy Protects Against Aminochrome-Induced Cell Death in Substantia Nigra-Derived Cell Line

    PubMed Central

    Paris, Irmgard; Muńoz, Patricia; Huenchuguala, Sandro; Couve, Eduardo; Sanders, Laurie H.; Greenamyre, John Timothy; Caviedes, Pablo; Segura-Aguilar, Juan

    2011-01-01

    Aminochrome, the precursor of neuromelanin, has been proposed to be involved in the neurodegeneration neuromelanin-containing dopaminergic neurons in Parkinson’s disease. We aimed to study the mechanism of aminochrome-dependent cell death in a cell line derived from rat substantia nigra. We found that aminochrome (50?M), in the presence of NAD(P)H-quinone oxidoreductase, EC 1.6.99.2 (DT)-diaphorase inhibitor dicoumarol (DIC) (100?M), induces significant cell death (62 ± 3%; p < 0.01), increase in caspase-3 activation (p < 0.001), release of cytochrome C, disruption of mitochondrial membrane potential (p < 0.01), damage of mitochondrial DNA, damage of mitochondria determined with transmission electron microscopy, a dramatic morphological change characterized as cell shrinkage, and significant increase in number of autophagic vacuoles. To determine the role of autophagy on aminochrome-induced cell death, we incubated the cells in the presence of vinblastine and rapamycin. Interestingly, 10?M vinblastine induces a 5.9-fold (p < 0.001) and twofold (p < 0.01) significant increase in cell death when the cells were incubated with 30?M aminochrome in the absence and presence of DIC, respectively, whereas 10?M rapamycin preincubated 24 h before addition of 50?M aminochrome in the absence and the presence of 100?M DIC induces a significant decrease (p < 0.001) in cell death. In conclusion, autophagy seems to be an important protective mechanism against two different aminochrome-induced cell deaths that initially showed apoptotic features. The cell death induced by aminochrome when DT-diaphorase is inhibited requires activation of mitochondrial pathway, whereas the cell death induced by aminochrome alone requires inhibition of autophagy-dependent degrading of damaged organelles and recycling through lysosomes. PMID:21427056

  14. Development and characterization of a cell line WAF from freshwater shark Wallago attu.

    PubMed

    Dubey, Akhilesh; Goswami, Mukunda; Yadav, Kamalendra; Sharma, Bhagwati S

    2014-02-01

    A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants. PMID:24381102

  15. Lymphocyte Migration Through Monolayers of Endothelial Cell Lines Involves VCAM-1 Signaling Via Endothelial Cell NADPH Oxidase1

    Microsoft Academic Search

    Heather E. Matheny; Tracy L. Deem; Joan M. Cook-Mills

    Lymphocytes migrate from the blood across endothelial cells to reach foreign substances sequestered in peripheral lymphoid organs and inflammatory sites. To study intracellular signaling in endothelial cells during lymphocyte migration, we used murine endothelial cell lines that promote lymphocyte migration and constitutively express VCAM-1. The maximum rate of resting splenic lymphocyte migration across monolayers of the endothelial cells occurred at

  16. Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines.

    PubMed

    Lazar, Ikrame; Clement, Emily; Ducoux-Petit, Manuelle; Denat, Laurence; Soldan, Vanessa; Dauvillier, Stéphanie; Balor, Stéphanie; Burlet-Schiltz, Odile; Larue, Lionel; Muller, Catherine; Nieto, Laurence

    2015-07-01

    Exosomes are important mediators in cell-to-cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma-specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro-migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells' aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment. PMID:25950383

  17. CD133 positive cells isolated from A549 cell line exhibited high liver metastatic potential.

    PubMed

    Zhang, H; Yang, N; Sun, B; Jiang, Y; Hou, C; Ji, C; Zhang, Y; Liu, Y; Zuo, P

    2014-01-01

    Lung cancer remains a major cause of cancer-related lethality because of high incidence and recurrence in spite of significant advances in staging and therapy. In present study, we identified a subpopulation of cells isolated from the A549 cell line with marker CD133. In vivo results showed that A549 CD133+ cells displayed high liver metastatic potential. Severe liver cell damage with tumor cell invasion revealed by pathological examination and these changes were consistent with the results of serological tests where the plasma GPT and GOT level are significantly higher than that of the control group. Compared with A549 cells, A549 CD133+ cells expressed high levels of VEGF and exhibited high migration and invasion capability. In conclusion, we first reported that A549 CD133+ cells exhibited characteristic of high liver metastatic potential which makes it be a suitable model for further study of liver metastasis of lung adenocarcinoma and provide a potential platform for anti-metastatic drug discovery or evaluation. PMID:24299311

  18. Sceptrin, a marine natural compound, inhibits cell motility in a variety of cancer cell lines.

    PubMed

    Cipres, Angel; O'Malley, Daniel P; Li, Ke; Finlay, Darren; Baran, Phil S; Vuori, Kristiina

    2010-02-19

    Sceptrin, a natural compound produced by various marine sponges, was tested for its effect on cell motility. We report for the first time that sceptrin inhibits cell motility in several cancer cell lines. The compound shows no toxicity at concentrations that are double the amount of sceptrin required for maximal inhibitory effect. Both random and factor-induced migration were impaired, suggesting that sceptrin targets a central process of cell motility machinery. Activity of de novo synthesized sceptrin was indistinguishable from sceptrin purified from Agelas nakamurai, and the inhibitory activity was found to be, at least partially, due to sceptrin's capability to inhibit cell contractility. Additionally, sceptrin was found to bind to monomeric actin, further suggesting a mechanism involving the actin cytoskeleton. Close analogues of sceptrin were synthesized, tested for their effect on cell motility, and found to be either equimolar or less potent compared to the parental compound. Inadvertent cell motility is a key contributing factor in various human diseases, including cancer and chronic inflammation. Marine compounds isolated from sponges have been proven to be an excellent source of metabolites that show biological activities. Given the recently achieved total synthesis of sceptrin in multigram quantities, sceptrin could prove to be an attractive lead molecule for further preclinical testing and development for therapeutic purposes, as well as a useful research tool to elucidate the mechanisms involved in cell motility. PMID:20030414

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

    SciTech Connect

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

    2010-05-04

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

  20. Human B cell activating factor (BCAF): production by a human T cell tumor line.

    PubMed

    Fevrier, M; Diu, A; Mollier, P; Abadie, A; Olive, D; Mawas, C; Theze, J

    1989-01-01

    In a previous study, we demonstrated that supernatants from human T cell clones stimulated by a pair of anti-CD2 monoclonal antibodies cause resting human B cells to become activated and to proliferate in the absence of any other signals. The activity responsible for these effects was shown to be different from already characterized lymphokines and in particular from IL-2 and IL-4, and was named B Cell Activating Factor or BCAF. In this paper, we describe the production of BCAF by a human T cell tumor line T687 after phorbol myristate acetate (PMA) stimulation; this production can be potentiated by phytohemagglutinin (PHA). We further show that the stimulatory phase can be separated from the secretory phase thereby avoiding contamination of BCAF-containing supernatant by PMA and PHA. Supernatants produced under these conditions do not contain either IL-4 or IFN but contain traces of lymphotoxin and 2 to 10 ng/ml of IL-2. The T687 cell line will allow us to obtain a large volume of supernatant for biochemical study and purification of the molecule(s) responsible for BCAF activity. PMID:2497279

  1. The Ah Receptor Regulates Growth Factor Expression in Head and Neck Squamous Cell Carcinoma Cell Lines

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-10-01

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

  4. Okadaic Acid Toxin at Sublethal Dose Produced Cell Proliferation in Gastric and Colon Epithelial Cell Lines

    PubMed Central

    del Campo, Miguel; Toledo, Héctor; Lagos, Néstor

    2013-01-01

    The aim of this study was to analyze the effect of Okadaic Acid (OA) on the proliferation of gastric and colon epithelial cells, the main target tissues of the toxin. We hypothesized that OA, at sublethal doses, activates multiple signaling pathways, such as Erk and Akt, through the inhibition of PP2A. To demonstrate this, we carried out curves of doses and time response against OA in AGS, MKN-45 and Caco 2 cell lines, and found an increase in the cell proliferation at sublethal doses, at 24 h or 48 h exposure. Indeed, cells can withstand high concentrations of the toxin at 4 h exposure, the time chosen considering the maximum time before total gastric emptying. We have proved that this increased proliferation is due to an overexpression of Cyclin B, a cyclin that promotes the passage from G2 to mitosis. In addition, we have demonstrated that OA induces activation of Akt and Erk in the three cells lines, showing that OA can activate pathways involved in oncogenesis. In conclusion, this study contributes to the knowledge about the possible effects of chronic OA consumption. PMID:24317467

  5. Okadaic acid toxin at sublethal dose produced cell proliferation in gastric and colon epithelial cell lines.

    PubMed

    del Campo, Miguel; Toledo, Héctor; Lagos, Néstor

    2013-12-01

    The aim of this study was to analyze the effect of Okadaic Acid (OA) on the proliferation of gastric and colon epithelial cells, the main target tissues of the toxin. We hypothesized that OA, at sublethal doses, activates multiple signaling pathways, such as Erk and Akt, through the inhibition of PP2A. To demonstrate this, we carried out curves of doses and time response against OA in AGS, MKN-45 and Caco 2 cell lines, and found an increase in the cell proliferation at sublethal doses, at 24 h or 48 h exposure. Indeed, cells can withstand high concentrations of the toxin at 4 h exposure, the time chosen considering the maximum time before total gastric emptying. We have proved that this increased proliferation is due to an overexpression of Cyclin B, a cyclin that promotes the passage from G2 to mitosis. In addition, we have demonstrated that OA induces activation of Akt and Erk in the three cells lines, showing that OA can activate pathways involved in oncogenesis. In conclusion, this study contributes to the knowledge about the possible effects of chronic OA consumption. PMID:24317467

  6. Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line

    Microsoft Academic Search

    WOLFGANG LENZ; MONIKA HERTEN; RUPERT GERZER; CHRISTIAN DRUMMER

    1999-01-01

    Regulation of natriuretic peptide (urodilatin) release in a human kidney cell line.BackgroundTo identify the molecular mechanisms underlying the release of a renal natriuretic peptide (NP) we selected a human kidney cell line (HEK 293) that displays several characteristics of distal tubular cells.MethodsCells were exposed to different extracellular and intracellular stimuli, and the effect on NP release was measured with a

  7. Trichostatin A induces cell death at the concentration recommended to differentiate the RGC-5 cell line.

    PubMed

    Schnichels, Sven; Schultheiss, Maximilian; Hofmann, Johanna; Szurman, Peter; Bartz-Schmidt, Karl Ulrich; Spitzer, Martin S

    2012-05-01

    Supplementation with Trichostatin A (TSA) has been described as the method of choice for differentiating the RGC-5 cell line into cells with neuronal properties. However, TSA is known to induce apoptosis. We therefore investigated whether TSA at the recommended concentration for differentiation (500 nM) and at three additional concentrations (40, 150 and 2000 nM) induces apoptosis or cell death in the RGC-5 cell line. Morphological changes of the RGC-5 cells occurred after 24 and 48 hours (h) of treatment with 500 and 2000 nM TSA. Differentiation of RGC-5 cell began at 150 nM. A decrease in the cell count was observed from 150 nM TSA onwards compared to controls. Five hundred nanomolar of TSA reduced the amount of cells to 51% (p<0.005) after 24h and to 24% (p<0.005) after 48 h compared to controls on crystal violet staining. At 500 nM TSA a massive induction of apoptosis after 24 and 48 h was noted. Supplementation of 500 nM TSA increased caspase 3/7 activity 5.0-fold (p<0.005). Furthermore, 27× more TUNEL-positive cells were found and the cleaved caspase 3/caspase 3 ratio was 1.8-fold (p<0.1) higher 24h after the addition of 500 nM TSA. The Bax/Bcl-2 ratio was 3.4-fold (p<0.05) higher after 48 h. Cell viability decreased to 70% (p<0.005) and to 35% (p<0.005) after 24 and 48 h, respectively. Moreover, 103× (p<0.05) more dead cells (via propidium iodide staining) were found after 48 h of treatment with 500 nM TSA. In conclusion, TSA induces cell death and apoptosis at the concentration recommended for differentiation. The induction of apoptosis occurred dose and time dependently and already at even lower concentrations of TSA which did not lead to differentiation induced apoptosis. Thus, studies with RGC-5 cells should not be performed within the first 48 h after supplementation with TSA. PMID:22391323

  8. Isolation of Cancer Stem Cells from Three Human Glioblastoma Cell Lines: Characterization of Two Selected Clones

    PubMed Central

    Piacentini, Roberto; Biamonte, Filippo; Mangiola, Annunziato; Maira, Giulio; Grassi, Claudio; Sica, Gigliola

    2014-01-01

    Cancer stem cells (CSC) were isolated via a non-adherent neurosphere assay from three glioma cell lines: LI, U87, and U373. Using a clonal assay, two clones (D2 and F11) were selected from spheres derived from LI cells and were characterized for the: expression of stem cell markers (CD133, Nestin, Musashi-1 and Sox2); proliferation; differentiation capability (determined by the expression of GalC, ?III-Tubulin and GFAP); Ca2+ signaling and tumorigenicity in nude mice. Both D2 and F11 clones expressed higher levels of all stem cell markers with respect to the parental cell line. Clones grew more slowly than LI cells with a two-fold increase in duplication time. Markers of differentiation (?III-Tubulin and GFAP) were expressed at high levels in both LI cells and in neurospheres. The expression of Nestin, Sox2, and ?III-Tubulin was down-regulated in D2 and F11 when cultured in serum-containing medium, whereas Musashi-1 was increased. In this condition, duplication time of D2 and F11 increased without reaching that of LI cells. D2, F11 and parental cells did not express voltage-dependent Ca2+-channels but they exhibited increased intracellular Ca2+ levels in response to ATP. These Ca2+ signals were larger in LI cells and in spheres cultured in serum-containing medium, while they were smaller in serum-free medium. The ATP treatment did not affect cell proliferation. Both D2 and F11 induced the appearance of tumors when ortotopically injected in athymic nude mice at a density 50-fold lower than that of LI cells. All these data indicate that both clones have characteristics of CSC and share the same stemness properties. The findings regarding the expression of differentiation markers and Ca2+-channels show that both clones are unable to reach the terminal differentiation. Both D2 and F11 might represent a good model to improve the knowledge on CSC in glioblastoma and to identify new therapeutic approaches. PMID:25121761

  9. Identification and verification of rodent cell lines by polymerase chain reaction

    PubMed Central

    Koelz, Anne-Leena; Drexler, Hans G.

    2007-01-01

    Cell lines represent valuable tools for basic research and diagnostic applications as well as for the production of biological products such as antibodies or vaccines. For all cell culturists, a well-identified origin of their cell lines as well as the periodic re-examination of their identity should be a basic requirement. We established a simple polymerase chain reaction (PCR) to verify or identify rodent and human cell lines. Since mouse-, rat-, Chinese hamster- and Syrian hamster-derived cell lines represent the most frequently used rodent cell lines, our investigations were focused on these species. Our assay used oligonucleotide primers annealing to sequences within the ?-actin and the ?-globin gene and to repetitive DNA. Primers were designed mostly from intron sequences of the genes aiming to amplify only one specific DNA segment and thus enabling to exclude easily false DNA. More than 130 cells lines originating from the five species were analyzed in that study. Our PCR revealed specific profiles for all species investigated. No further methods like DNA sequencing or fragment length polymorphism analysis were needed to differentiate these species. The results introduce our PCR-assay as a rapid, specific and routinely feasible tool in order to identify or distinguish rodent cell lines from each other and from human cell lines. PMID:19002841

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

    PubMed Central

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

    2015-01-01

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

  11. Experimental impact of aspirin exposure on rat intestinal bacteria, epithelial cells and cell line

    Microsoft Academic Search

    Raj K Upreti; A. Kannan; AB Pant

    2010-01-01

    Aspirin, a commonly used therapeutic non-steroidal anti-inflammatory drug (NSAID) is known to cause gastric mucosal damage. Intestinal bacteria having a regulatory effect on intestinal homeostasis play significant role in NSAID-induced intestinal injury. Bacteria and specific cell lines are considered to be suitable for toxicity screening and testing of chemicals. Therefore, to evaluate and compare in vitro toxicity, cultures of rat

  12. A tumorigenic murine Sertoli cell line that is temperature-sensitive for differentiation.

    PubMed

    Boekelheide, K; Lee, J W; Hall, S J; Rhind, N R; Zaret, K S

    1993-10-01

    The Sertoli cell is the epithelial cell within the seminiferous tubule responsible for supporting germ cells. Most current in vitro studies of Sertoli cell function use primary cultures because of the limited number of available Sertoli cell lines. In addition, few in vivo models of Sertoli cell malignancy have been described. In this study, a tumorigenic Sertoli cell line was developed by infection of isolated murine Sertoli cells by simian virus 40 tsA255; the ts mutation causes the inactivation of the large T antigen at elevated temperatures. A cloned Sertoli cell line, called S14-1, demonstrated temperature-dependent growth in soft agar and formed tumors in nude mice. Electron microscopy of the S14-1-derived tumor revealed extensive basal intercellular junctions and tubulobulbarlike processes supporting its Sertoli cell origin. Cytogenetic analysis showed that S14-1 cells were aneuploid with an average of 70 chromosomes per cell. At the nonpermissive (40 C) temperature, S14-1 cells in vitro demonstrated a reduced growth rate, enhanced secretion of transferrin, and increased expression of sulfated glycoprotein-2 messenger RNA, indicating the cells manifested increased differentiation following large T antigen inactivation. The murine S14-1 Sertoli cell line should be useful for both in vitro studies of Sertoli cell function and in vivo studies of Sertoli cell malignancy. PMID:8214009

  13. ECC-1 cells: a well-differentiated steroid-responsive endometrial cell line with characteristics of luminal epithelium.

    PubMed

    Mo, Bilan; Vendrov, Aleksandr E; Palomino, Wilder A; DuPont, Barbara R; Apparao, K B C; Lessey, Bruce A

    2006-09-01

    Endometrial cancer cell lines have provided a valuable model to study endometrial epithelial cells in vitro. Since the first development of HEC1B over 35 yr ago, many different cell lines have been isolated and described. One valuable cell line that maintains hormone responsiveness and unique stability over time is the ECC-1 cell line, developed originally by the late P.G. Satyaswaroop. In this study, we investigated some of the properties of these cells and present their salient characteristics. Like Ishikawa cells, ECC-1 cells maintain both estrogen receptors (ESR1 [ER alpha] and ESR2 [ER beta]), progesterone receptors (PR A and B; PGRs), and androgen receptors (ARs), along with the p160 steroid receptor coactivators NCOA1 (formerly SRC1), NCOA2 (formerly TIF2), and NCOA3 (formerly AIB1). The karyotype of these cells is abnormal, with multiple structural rearrangements in all cells analyzed. Unlike Ishikawa cells that express glandular epithelial antigens, ECC-1 cells maintain a luminal phenotype, with expression of KRT13 (cytokeratin 13) and KRT18 (cytokeratin 18). Apparent differences in the regulation of ESR2 also were evident in ECC-1 cells compared to Ishikawa cells. Like other endometrial cell lines, ECC-1 cells express the steroid receptor coactivators and exhibit epidermal growth factor-stimulated expression of known luminal proteins thought to be involved in implantation, including the hyaluronate receptor CD44 and SPP1 (formerly osteopontin) and CD55 (decay-accelerating factor). These characteristics appear to be stable and persistent over multiple cell passages, making this well-differentiated cell line an excellent choice to study endocrine and paracrine regulation of endometrial epithelium in vitro. PMID:16707768

  14. Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line.

    PubMed

    Solimene, A C; Carneiro, C R; Melati, I; Lopes, J D

    2001-05-01

    The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, Săo Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified. PMID:11323753

  15. The avian cell line AGE1.CR.pIX characterized by metabolic flux analysis

    PubMed Central

    2014-01-01

    Background In human vaccine manufacturing some pathogens such as Modified Vaccinia Virus Ankara, measles, mumps virus as well as influenza viruses are still produced on primary material derived from embryonated chicken eggs. Processes depending on primary cell culture, however, are difficult to adapt to modern vaccine production. Therefore, we derived previously a continuous suspension cell line, AGE1.CR.pIX, from muscovy duck and established chemically-defined media for virus propagation. Results To better understand vaccine production processes, we developed a stoichiometric model of the central metabolism of AGE1.CR.pIX cells and applied flux variability and metabolic flux analysis. Results were compared to literature dealing with mammalian and insect cell culture metabolism focusing on the question whether cultured avian cells differ in metabolism. Qualitatively, the observed flux distribution of this avian cell line was similar to distributions found for mammalian cell lines (e.g. CHO, MDCK cells). In particular, glucose was catabolized inefficiently and glycolysis and TCA cycle seem to be only weakly connected. Conclusions A distinguishing feature of the avian cell line is that glutaminolysis plays only a minor role in energy generation and production of precursors, resulting in low extracellular ammonia concentrations. This metabolic flux study is the first for a continuous avian cell line. It provides a basis for further metabolic analyses to exploit the biotechnological potential of avian and vertebrate cell lines and to develop specific optimized cell culture processes, e.g. vaccine production processes. PMID:25077436

  16. Classification of potassium and chlorine ionic currents in retinal ganglion cell line (RGC-5) by whole-cell patch clamp.

    PubMed

    Wang, Shu-Jie; Xie, Lai-Hua; Heng, Bin; Liu, Yan-Qiang

    2012-11-01

    Retinal ganglion cell line (RGC-5) has been widely used as a valuable model for studying pathophysiology and physiology of retinal ganglion cells in vitro. However, the electrophysiological characteristics, especially a thorough classification of ionic currents in the cell line, remain to be elucidated in details. In the present study, we determined the resting membrane potential (RMP) in RGC-5 cell line and then identified different types of ionic currents by using the whole-cell patch-clamp technique. The RMP recorded in the cell line was between -30 and -6 mV (-17.6 ± 2.6 mV, n = 10). We observed the following voltage-gated ion channel currents: (1) inwardly rectifying Cl- current (I Cl,ir), which could be blocked by Zn2+; (2) Ca2+-activated Cl- current (I Cl,Ca), which was sensitive to extracellular Ca2+ and could be inhibited by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate; (3) inwardly rectifying K+ currents (I K1), which could be blocked by Ba2+; (4) a small amount of delayed rectifier K+ current (I K). On the other hand, the voltage-gated sodium channels current (I Na) and transient outward potassium channels current (I A) were not observed in this cell line. These results further characterize the ionic currents in the RGC-5 cell line and are beneficial for future studies especially on ion channel (patho)physiology and pharmacology in the RGC-5 cell line. PMID:23110755

  17. A genetically engineered human pancreatic ? cell line exhibiting glucose-inducible insulin secretion

    PubMed Central

    Ravassard, Philippe; Hazhouz, Yasmine; Pechberty, Séverine; Bricout-Neveu, Emilie; Armanet, Mathieu; Czernichow, Paul; Scharfmann, Raphael

    2011-01-01

    Despite intense efforts over the past 30 years, human pancreatic ? cell lines have not been available. Here, we describe a robust technology for producing a functional human ? cell line using targeted oncogenesis in human fetal tissue. Human fetal pancreatic buds were transduced with a lentiviral vector that expressed SV40LT under the control of the insulin promoter. The transduced buds were then grafted into SCID mice so that they could develop into mature pancreatic tissue. Upon differentiation, the newly formed SV40LT-expressing ? cells proliferated and formed insulinomas. The resulting ? cells were then transduced with human telomerase reverse transcriptase (hTERT), grafted into other SCID mice, and finally expanded in vitro to generate cell lines. One of these cell lines, EndoC-?H1, expressed many ? cell–specific markers without any substantial expression of markers of other pancreatic cell types. The cells secreted insulin when stimulated by glucose or other insulin secretagogues, and cell transplantation reversed chemically induced diabetes in mice. These cells represent a unique tool for large-scale drug discovery and provide a preclinical model for cell replacement therapy in diabetes. This technology could be generalized to generate other human cell lines when the cell type–specific promoter is available. PMID:21865645

  18. Identification of Cancer Stem Cell-Like Side Population Cells in Human Nasopharyngeal Carcinoma Cell Line

    Microsoft Academic Search

    Jing Wang; Li-Ping Guo; Li-Zhen Chen; Yi-Xin Zeng

    2007-01-01

    Side population (SP) cells have been isolated from several solid tumors. They lack distinct molecular markers for cancer stem cells (CSC) and increasing evidence suggests that they may play an important role in tumorigenesis and cancer therapy. However, there are no reports about the existence and function of SP cells in nasopharyngeal carcinoma (NPC) cells thus far. In this study,

  19. Establishment of an immortal chicken embryo liver-derived cell line.

    PubMed

    Lee, Jeongyoon; Foster, Douglas N; Bottje, Walter G; Jang, Hyeon-Min; Chandra, Yohanna G; Gentles, Lauren E; Kong, Byung-Whi

    2013-06-01

    A continuously growing immortal cell substrate can be used for virus propagation, diagnostic purposes, and vaccine production. The aim of this study was to develop an immortal chicken cell line for efficient propagation of avian infectious viruses. From the various chicken embryo cells that were tested for life span extension, an immortalized chicken embryo liver (CEL) cell line, named CEL-im, was derived spontaneously without either oncogenic viruses or carcinogenic chemical treatment. Currently, CEL-im cells are growing 0.8 to 1.1 population doublings per day and have reached 120 passages. The CEL-im cell line is permissive for poultry infectious viruses, including avian metapneumovirus (AMPV), Marek's disease virus serotype 1 (MDV-1), and infectious laryngotracheitis virus. The CEL-im cells produced high AMPV titer (>10(5) pfu/mL), whereas very low titers (~10 pfu/mL) for MDV-1 and infectious laryngotracheitis virus were produced. To identify genetic alterations in the immortal CEL-im cell line, telomerase activity and mRNA expression for major cell cycle regulatory genes were determined during the immortalizing process. The CEL-im cell line has negative telomerase activity, and when compared with the primary passage 2 CEL cell counterpart, mRNA expression of tumor suppressor protein p53, mouse double minute 2 (Mdm2), cyclin dependent kinase (CDK) inhibitor p21 (p21(WAF)), and CDK inhibitor p16 (p16(INK4)) were downregulated in the CEL-im cell line, whereas retinoblastoma (Rb), transcription factor E2F, member 1 (E2F-1), and alternative reading frame of p16(INK4) (ARF) were upregulated. These results are similar to genetic alterations found previously in immortal chicken embryo fibroblast (CEF) cell lines that showed efficient propagation of MDV-1. Therefore, this newly established CEL-im cell line can serve as an alternative cell substrate for the propagation of poultry viruses, such as AMPV. PMID:23687157

  20. Similar biological characteristics of human embryonic stem cell lines with normal and abnormal karyotypes

    PubMed Central

    Sun, Xiaofang; Long, Xiaolin; Yin, Yifei; Jiang, Yonghua; Chen, Xinjie; Liu, Weiqiang; Zhang, Wenhong; Du, Hongzi; Li, Shaoying; Zheng, Yuhong; Kong, Shu; Pang, Qianying; Shi, Yu; Huang, Yulin; Huang, Shengchan; Liao, Baoping; Xiao, Guohong; Wang, Weihua

    2008-01-01

    BACKGROUND Human embryonic stem cell (hESC) lines derived from poor quality embryos usually have either normal or abnormal karyotypes. However, it is still unclear whether their biological characteristics are similar. METHODS Seven new hESC lines were established using discarded embryos. Five cell lines had normal karyotype, one was with an unbalanced Robertsonian translocation and one had a triploid karyotype. Their biological characteristics, short tandem repeat loci, HLA typing, differentiation capability and imprinted gene, DNA methylation and X chromosome inactivation status were compared between different cell lines. RESULTS All seven hESC lines had similar biological characteristics regardless of karyotype (five normal and two abnormal), such as expression of stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-81 and TRA-1-60 proteins, transcription factor octamer binding protein 4 mRNA, no detectable expression of SSEA-1 protein and high levels of alkaline phosphatase activity. All cell lines were able to undergo differentiation. Imprinted gene expression and DNA methylation were also similar among these cell lines. Non-random X chromosome inactivation patterns were found in XX cell lines. CONCLUSIONS The present results suggest that hESC lines with abnormal karyotype are also useful experimental materials for cell therapy, developmental biology and genetic research. PMID:18611919

  1. Robust regeneration of adult zebrafish lateral line hair cells reflects continued precursor pool maintenance.

    PubMed

    Cruz, Ivan A; Kappedal, Ryan; Mackenzie, Scott M; Hailey, Dale W; Hoffman, Trevor L; Schilling, Thomas F; Raible, David W

    2015-06-15

    We have examined lateral line hair cell and support cell maintenance in adult zebrafish when growth is largely complete. We demonstrate that adult zebrafish not only replenish hair cells after a single instance of hair cell damage, but also maintain hair cells and support cells after multiple rounds of damage and regeneration. We find that hair cells undergo continuous turnover in adult zebrafish in the absence of damage. We identify mitotically-distinct support cell populations and show that hair cells regenerate from underlying support cells in a region-specific manner. Our results demonstrate that there are two distinct support cell populations in the lateral line, which may help explain why zebrafish hair cell regeneration is extremely robust, retained throughout life, and potentially unlimited in regenerative capacity. PMID:25869855

  2. Good manufacturing practice and clinical-grade human embryonic stem cell lines

    Microsoft Academic Search

    Christian Unger; Heli Skottman; Pontus Blomberg; M. Sirac Dilber; Outi Hovatta

    2008-01-01

    Human embryonic stem cell (hESC) lines, after directed differentiation, hold the greatest potential for cell transplantation treatment in many severe diseases. Good manufacturing practice (GMP) quality, defined by both the European Medicines Agency and the Food and Drug Administration, is a requirement for clinical- grade cells, offering optimal defined quality and safety in cell transplantation. Using animal substance-free culture media,

  3. Establishment of Immortalized Human Glomerular Endothelial Cell Lines and Their Application

    Microsoft Academic Search

    Takashi Harada; Stephen Batsford; Tetsuo Morioka; Jian Yao; Masaaki Arakawa; Fumitake Gejyo; Takashi Oite

    2005-01-01

    Background: The experimental use of cultured endothelial cells derived from the microvasculature such as glomerular endothelial cells possesses many problems, including limited growth rates, heterogeneity and loss of specific cell properties dependent on culture passage. In this study, we attempted to establish immortalized, human glomerular endothelial cell (HGEC) lines. Methods: HGECs of up to 5 passages were transformed by infection

  4. An Ixodes scapularis cell line with a predominantly neuron-like phenotype.

    PubMed

    Oliver, Jonathan D; Chávez, Adela S Oliva; Felsheim, Roderick F; Kurtti, Timothy J; Munderloh, Ulrike G

    2015-07-01

    The Ixodes scapularis embryo-derived cell line ISE6 is the most widely utilized tick-derived cell line due to its susceptibility to a wide variety of tick- and non-tick-vectored pathogens. Little is known about its tissue origin or biological background. Protein expression of ISE6 cells was compared with that of another I. scapularis-derived cell line, IDE12, and dissected tick synganglia. Results demonstrated the presence of a neuronal marker protein, type 3 ?-tubulin, in all three samples, as well as other shared and unique neuronal and immune response-associated proteins. Of neuronal proteins shared between the two cell lines, ISE6 expressed several in significantly greater quantities than IDE12. Stimulation of ISE6 cells by in vivo exposure to the hemocoel environment in unfed larval and molting nymphal ticks, but not unfed nymphal ticks, resulted in the development of neuron-like morphologic characteristics in the implanted cells. PMID:25894426

  5. Establishment of an agamid cell line and isolation of adenoviruses from central bearded dragons (Pogona vitticeps).

    PubMed

    Ball, Inna; Hoferer, Marc; Marschang, Rachel E

    2014-03-01

    A cell line was established from whole 6-8-week-old central bearded dragon (Pogona vitticeps) embryos. Cells were mid-sized and showed an elongated and polymorphic form. The cell line grew in a monolayer and has been serially passaged for 17 passages at time of publication. This cell line has been used with samples from adenovirus polymerase chain reaction (PCR)-positive bearded dragons, and 2 virus isolates have been obtained so far. The isolates show a clear cytopathic effect in inoculated cells. Both virus isolates have been serially passaged on this cell line, and have been identified by PCR amplification and sequencing of a portion of the DNA-dependent DNA polymerase gene and show 100% nucleotide identity to the corresponding region of an agamid adenovirus. Electron microscopic examination of supernatant from infected cells demonstrated the presence of nonenveloped particles, with a diameter of approximately 80 nm in both virus isolates. PMID:24569225

  6. Comparing the level of bystander effect in a couple of tumor and normal cell lines.

    PubMed

    Soleymanifard, Shokouhozaman; Bahreyni, Mohammad T Toossi

    2012-04-01

    Radiation-induced bystander effect refers to radiation responses which occur in non-irradiated cells. The purpose of this study was to compare the level of bystander effect in a couple of tumor and normal cell lines (QU-DB and MRC5). To induce bystander effect, cells were irradiated with 0.5, 2, and 4 Gy of (60)Co gamma rays and their media were transferred to non-irradiated (bystander) cells of the same type. Cells containing micronuclei were counted in bystander subgroups, non-irradiated, and 0.5 Gy irradiated cells. Frequencies of cells containing micronuclei in QU-DB bystander subgroups were higher than in bystander subgroups of MRC5 cells (P < 0.001). The number of micronucleated cells counted in non-irradiated and 0.5 Gy irradiated QU-DB cells was also higher than the corresponding values for MRC5 cells (P < 0.001). Another difference between the two cell lines was that in QU-DB bystander cells, a dose-dependent increase in the number of micronucleated cells was observed as the dose increased, but at all doses the number of micronucleated cells in MRC5 bystander cells was constant. It is concluded that QU-DB cells are more susceptible than MRC5 cells to be affected by bystander effect, and in the two cell lines there is a positive correlation between DNA damages induced directly and those induced due to bystander effect. PMID:22557800

  7. Comparing the level of bystander effect in a couple of tumor and normal cell lines

    PubMed Central

    Soleymanifard, Shokouhozaman; Bahreyni, Mohammad T. Toossi

    2012-01-01

    Radiation-induced bystander effect refers to radiation responses which occur in non-irradiated cells. The purpose of this study was to compare the level of bystander effect in a couple of tumor and normal cell lines (QU-DB and MRC5). To induce bystander effect, cells were irradiated with 0.5, 2, and 4 Gy of 60Co gamma rays and their media were transferred to non-irradiated (bystander) cells of the same type. Cells containing micronuclei were counted in bystander subgroups, non-irradiated, and 0.5 Gy irradiated cells. Frequencies of cells containing micronuclei in QU-DB bystander subgroups were higher than in bystander subgroups of MRC5 cells (P < 0.001). The number of micronucleated cells counted in non-irradiated and 0.5 Gy irradiated QU-DB cells was also higher than the corresponding values for MRC5 cells (P < 0.001). Another difference between the two cell lines was that in QU-DB bystander cells, a dose-dependent increase in the number of micronucleated cells was observed as the dose increased, but at all doses the number of micronucleated cells in MRC5 bystander cells was constant. It is concluded that QU-DB cells are more susceptible than MRC5 cells to be affected by bystander effect, and in the two cell lines there is a positive correlation between DNA damages induced directly and those induced due to bystander effect. PMID:22557800

  8. Short communication Antiproliferative activity of fish protein hydrolysates on human breast cancer cell lines

    Microsoft Academic Search

    L. Picot; S. Bordenave; S. Didelot; I. Fruitier-Arnaudin; F. Sannier; G. Thorkelsson; F. Guerard; A. Chabeaud; J. M. Piot

    Antiproliferative activity of 18 fish protein hydrolysates was measured on 2 human breast cancer cell lines grown in vitro. Three blue whiting, three cod, three plaice and one salmon hydrolysates were identified as significant growth inhibitors on the two cancer cell lines. Preliminary analysis of hydrolysates composition evidenced they contained a complex mixture of free amino acids, peptides with various

  9. Identification of a germ line transcript from the unrearranged kappa gene in human B cells.

    PubMed Central

    Martin, D J; Van Ness, B G

    1989-01-01

    A novel kappa immunoglobulin-hybridizing mRNA in cell lines derived from human B cells arrested at several stages of development has been identified. Hybridization studies demonstrate that this 1.5-kilobase mRNA species is the spliced product of a precursor germ line transcript initiating upstream of the unrearranged JKappa locus. Images PMID:2573834

  10. Expression Profiles of Cloned Channel Catfish (Ictalurus punctatus) Lymphoid Cell Lines and Mixed Lymphocyte Cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clonal channel catfish lymphoid cell lines and mixed lymphocyte cultures (MLC) have proven extremely useful in examining immune responses at the cellular and molecular levels. To date clonal catfish cell lines and MLC have been biologically and phenotypically characterized using a variety of techniq...

  11. Stable Alphavirus Packaging Cell Lines for Sindbis Virus-and Semliki Forest Virus-Derived Vectors

    Microsoft Academic Search

    John M. Polo; Barbara A. Belli; David A. Driver; Ilya Frolov; Scott Sherrill; Mangala J. Hariharan; Kay Townsend; Silvia Perri; Steven J. Mento; Douglas J. Jolly; Stephen M. W. Chang; Sondra Schlesinger; Thomas W. Dubensky Jr.

    1999-01-01

    Alphavirus vectors are being developed for possible human vaccine and gene therapy applications. We have sought to advance this field by devising DNA-based vectors and approaches for the production of recombinant vector particles. In this work, we generated a panel of alpha-virus vector packaging cell lines (PCLs). These cell lines were stably transformed with expression cassettes that constitutively produced RNA

  12. An ecosystem of cancer cell line factories to support a cancer dependency map.

    PubMed

    Boehm, Jesse S; Golub, Todd R

    2015-07-01

    Jesse Boehm and Todd Golub call for an international effort to establish >10,000 cancer cell line models as a community resource. Cancer cell line factories will facilitate the creation of a cancer dependency map, connecting cancer genomics to therapeutic dependencies. PMID:26077369

  13. Development of Fibroblast Cell Lines From the Cow Used to Sequence the Bovine Genome

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.BGCF.2 and MARC.BGCF.1-3, were initiated from skin biopsies obtained from the Hereford cow whose DNA was used in sequencing the bovine genome. These cell lines were submitted to American Type Culture Collection (ATCC, Manassas, VA, USA) and will be made publicly avai...

  14. RESEARCH Open Access Proteomic analysis of an Aedes albopictus cell line

    E-print Network

    Paris-Sud XI, Université de

    RESEARCH Open Access Proteomic analysis of an Aedes albopictus cell line infected with Dengue in an Aedes albopictus cell line. The potential of these viruses to cause severe disease at primary infection worldwide [1]. Aedes albopictus (Diptera, Culicidae) is a less effi- cient vector for this virus, although

  15. Integrated classification of lung tumors and cell lines by expression profiling

    Microsoft Academic Search

    Carl Virtanen; Yuichi Ishikawa; Daisuke Honjoh; Mami Kimura; Miyuki Shimane; Tatsu Miyoshi; Hitoshi Nomura; Michael H. Jones

    2002-01-01

    The utility of cancer cell lines depends largely on their accurate classification, commonly based on histopathological diagnosis of the cancers from which they were derived. However, because cancer is often heterogeneous, the cell line, which also has the opportunity to alter in vitro, may not be representative. Yet without the overall architecture used in histopathological diagnosis of fresh samples, reclassification

  16. Development of a new human breast cancer cell line Ia270

    Microsoft Academic Search

    Pao-Min Loh; Gerald Clamon; John MacIndoe; Mark White; Luis Urdaneta; Bharati Hukku; Ward D. Peterson

    1985-01-01

    Summary A new human breast cancer cell line (Ia-270) has been isolated from a malignant pleural effusion from a woman with metastatic infiltrating ductal carcinoma of the breast. This cell line contains cytoplasmic estrogen (ER) and progesterone (PR) receptors. Following estradiol (E2) administration, PR synthesis is augmented and a higher level of saturation density is reached. In an athymic mouse,

  17. Establishment and characterization of insect cell lines from 10 lepidopteran species.

    PubMed

    Goodman, C L; El Sayed, G N; McIntosh, A H; Grasela, J J; Stiles, B

    2001-06-01

    Cell lines from selected lepidopteran species were established for the overall purpose of use in baculovirus production. A total of 36 new cell lines from 10 lepidopteran species were generated, including cell lines from a pyralid, the European corn borer, Ostrinia nubilalis, a plutellid, the diamondback moth, Plutella xylostella, as well as eight noctuids: the black cutworm, Agrotis ipsilon, the celery looper, Anagrapha falcifera, the velvetbean caterpillar, Anticarsia gemmatalis, the corn earworm, Helicoverpa zea, the tobacco budworm, Heliothis virescens, the beet armyworm, Spodoptera exigua, the fall armyworm, Spodoptera frugiperda, and the cabbage looper, Trichoplusia ni. Tissues used for cell line establishment included fat bodies, ovaries, testes, or whole embryos/larvae/pupae. All the cell lines were subcultured numerous times, characterized by isoenzyme analysis and/or deoxyribonucleic acid amplification fingerprinting using polymerase chain reaction, and stored in liquid nitrogen. Many of the cell lines were adapted to grow in serum-free medium, with cell lines from A. ipsilon and H. virescens being adapted to suspension culture using shaker flasks. The potential use for these cell lines in baculovirus production is discussed. PMID:11515970

  18. ARPE-19, A Human Retinal Pigment Epithelial Cell Line with Differentiated Properties

    Microsoft Academic Search

    K. C. DUNN; A. E. AOTAKI-KEEN; F. R. PUTKEY; L. M. HJELMELAND

    1996-01-01

    The retinal pigment epithelium (RPE) plays a critical role in the development and maintenance of adjacent photoreceptors in the vertebrate retina. This study describes the development and characterization of ARPE-19, a spontaneously arising human RPE cell line with normal karyology which forms polarized epithelial monolayers on porous filter supports. The cell line was established by selective trypsinization of a primary

  19. Pediatric pancreatoblastoma: histopathologic and cytogenetic characterization of tumor and derived cell line

    Microsoft Academic Search

    Linda Barenboim-Stapleton; Xuezhong Yang; Maria Tsokos; Jon M. Wigginton; Hesed Padilla-Nash; Thomas Rieda; Carol J. Thiele

    Little is known of the molecular events underlying the genesis of pancreatoblastoma tumors in the pediatric population. Such studies have been limited by the rare nature of the disease, infrequent reports detailing cytogenetic alterations, and the lack of availability of cell lines for biologic studies. We present the isolation of a cell line from a 14-year-old boy with malignant pancreatoblastoma,

  20. Karyotype stability of the DT40 chicken B cell line: Macrochromosome variation and cytogenetic mosaicism

    E-print Network

    Delany, Mary E.

    Karyotype stability of the DT40 chicken B cell line: Macrochromosome variation and cytogenetic for publication by Herbert Macgregor 23 January 2004 Key words: aneuploidy, chicken, chromosomes, cytogenetics, DT40, karyotype, mosaicism Abstract The DT40 transformed chicken B-cell line is an important and widely

  1. Finding splitting lines for touching cell nuclei with a shortest path algorithm.

    PubMed

    Bai, Xiangzhi; Wang, Peng; Sun, Changming; Zhang, Yu; Zhou, Fugen; Meng, Cai

    2014-10-22

    A shortest path-based algorithm is proposed in this paper to find splitting lines for touching cell nuclei. First, an initial splitting line is obtained through the distance transform of a marker image and the watershed algorithm. The initial splitting line is then separated into different line segments as necessary, and the endpoint positions of these line segments are adjusted to the concave points on the contour. Finally, a shortest path algorithm is used to find the accurate splitting line between the starting-point and the end-point, and the final split can be achieved by the contour of the touching cell nuclei and the splitting lines. Comparisons of experimental results show that the proposed algorithm is effective for segmentation of different types of touching cell nuclei. PMID:25458811

  2. Highly passage of Spodoptera litura cell line causes its permissiveness to baculovirus infection.

    PubMed

    Zhang, Xuping; Lan, Wenjie; Deng, Yujie; Ma, Yuan; Liu, Kaiyu; Peng, Jianxin; Li, Yi; Hong, Huazhu

    2008-07-01

    It is well known that the characteristics of cell lines possibly alter when cell lines are at high-passage number because of the environmental selection. We do not know whether non-permissive or low-permissive cell lines could become permissive or more permissive to virus infection after over-high passage. In the present studies, the alteration of the permissiveness of Spodoptera litura cell line Sl-zsu-1 to three baculovirus infection was investigated after over-high passage, and the possible mechanisms are also investigated. Vigorous apoptosis in Sl-zsu-1 cells was induced by both the recombinant Autographa californica multiple nucleopolyhedrovirus AcMNPV-GFP-actin and the celery looper Anagrapha falcifera multiple nucleopolyhedrovirus AfMNPV, suggesting the replication of the two viruses was blocked by apoptosis. However, the cells infected by S. litura multicapsid nucleopolyhedrovirus SpltMNPV did not undergo apoptosis, but the SpltMNPV titre of the supernatant was not detectable, suggesting this cell line was low-permissive for this virus infection and other factor(s) involved in blockage of the virus replication except apoptosis. However, when Sl-zsu-1 cells had been subcultured continuously for more than 4 years (high-passage cell), which was named as Sl-HP cell line afterwards, no significant apoptosis was induced by the three baculovirus in Sl-HP cells, and many replicated virions or nucleocapsids were observed in the cells. But the permissiveness of Sl-HP cells to the three viruses was very different according to the titre of viruses in the cell cultures. Interestingly, the DNA extracted from SpltMNPV could induce vigorous apoptosis of Sl-HP cells. Altogether, Sl-zsu-1 cell line becomes more permissive to baculovirus infection after over-high passage and multiple paths can block the baculovirus infectivity. PMID:19003180

  3. Highly passage of Spodoptera litura cell line causes its permissiveness to baculovirus infection

    PubMed Central

    Zhang, Xuping; Lan, Wenjie; Deng, Yujie; Ma, Yuan; Peng, Jianxin; Li, Yi; Hong, Huazhu

    2008-01-01

    It is well known that the characteristics of cell lines possibly alter when cell lines are at high-passage number because of the environmental selection. We do not know whether non-permissive or low-permissive cell lines could become permissive or more permissive to virus infection after over-high passage. In the present studies, the alteration of the permissiveness of Spodoptera litura cell line Sl-zsu-1 to three baculovirus infection was investigated after over-high passage, and the possible mechanisms are also investigated. Vigorous apoptosis in Sl-zsu-1 cells was induced by both the recombinant Autographa californica multiple nucleopolyhedrovirus AcMNPV-GFP-actin and the celery looper Anagrapha falcifera multiple nucleopolyhedrovirus AfMNPV, suggesting the replication of the two viruses was blocked by apoptosis. However, the cells infected by S. litura multicapsid nucleopolyhedrovirus SpltMNPV did not undergo apoptosis, but the SpltMNPV titre of the supernatant was not detectable, suggesting this cell line was low-permissive for this virus infection and other factor(s) involved in blockage of the virus replication except apoptosis. However, when Sl-zsu-1 cells had been subcultured continuously for more than 4 years (high-passage cell), which was named as Sl-HP cell line afterwards, no significant apoptosis was induced by the three baculovirus in Sl-HP cells, and many replicated virions or nucleocapsids were observed in the cells. But the permissiveness of Sl-HP cells to the three viruses was very different according to the titre of viruses in the cell cultures. Interestingly, the DNA extracted from SpltMNPV could induce vigorous apoptosis of Sl-HP cells. Altogether, Sl-zsu-1 cell line becomes more permissive to baculovirus infection after over-high passage and multiple paths can block the baculovirus infectivity. PMID:19003180

  4. Casticin inhibits self-renewal of liver cancer stem cells from the MHCC97 cell line

    PubMed Central

    HE, GUICHENG; CAO, XIAOCHENG; HE, MENG; SHENG, XIFENG; WU, YOUHUA; AI, XIAOHONG

    2014-01-01

    Casticin exerts anticarcinogenic activity in several types of cancers, including human hepatocellular carcinoma (HCC). The aim of the present study was to investigate the effects of casticin, which is derived from Fructus Viticis Simplicifoliae, on the self-renewal capacity of liver cancer stem cells (LCSCs) derived from the HCC MHCC97 cell line. The present study demonstrated that casticin significantly inhibited the proliferation of LCSCs from the MHCC97 cell line in a dose-dependent manner (P<0.05), the half maximal inhibitory concentration of the parental cells and LCSCs was 17.9 and 0.5 ?mol/l, respectively. Furthermore, casticin reduced the sphere-forming capacity of LCSCs and downregulated ?-catenin protein expression in a concentration-dependent manner. Lithium chloride, an agonist known to activate the Wnt/?-catenin signaling pathway, attenuated the casticin-induced downregulation of ?-catenin protein expression and inhibited the self-renewal capacity. To the best of our knowledge, the present study is the first to demonstrate that casticin effectively eradicates LCSCs and ?-catenin was identified as the potential target. Thus, casticin may offer a novel therapeutic approach for the treatment of HCC. PMID:24932283

  5. Mipu1 overexpression protects macrophages from oxLDL-induced foam cell formation and cell apoptosis.

    PubMed

    Qu, Shun-Lin; Fan, Wen-Jing; Zhang, Chi; Guo, Fang; Han, Dan; Pan, Wen-Jun; Li, Wei; Feng, Da-Ming; Jiang, Zhi-Sheng

    2014-12-01

    Mipu1 (myocardial ischemic preconditioning upregulated protein 1) is a novel N-terminal Kruppel-associated box (KRAB)/C2H2 zinc finger superfamily protein, that displays a powerful effect in protecting H9c2 cells from oxidative stress-induced cell apoptosis. The present study aims to investigate the effect of Mipu1 overexpression on oxidized low-density lipoprotein (oxLDL)-induced foam cell formation, cell apoptosis, and its possible mechanisms. New Zealand healthy rabbits were used to establish atherosclerosis model, and serum levels of triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol were detected by an automatic biochemical analyzer. Sudan IV staining was used to detect atherosclerotic lesions. The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining, high-performance liquid chromatography, and Dil-labeled lipoprotein were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to determine cell apoptosis. Real-time quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as ABCA1, ABCG1, SR-BI, and CD36. Western blot analysis was used to detect the protein expression of Mipu1. There were atherosclerotic lesions in the high-fat diet group with Sudan IV staining. High-fat diet decreased Mipu1 expression and increased CD36 expression significantly at the 10th week compared with standard-diet rabbits. Mipu1 overexpression decreased oxLDL-induced cholesterol accumulation, oxLDL uptake, cell apoptosis, and cleaved caspase-3. Mipu1 overexpression inhibited the oxLDL-induced CD36 mRNA and protein expression, but it did not significantly inhibit the mRNA expression of ABCA1, ABCG1, and SR-BI. Mipu1 overexpression inhibits oxLDL-induced foam cell formation and cell apoptosis. Mipu1 overexpression reduces the lipid intake of macrophages and might be associated with the downregulation of CD36 expression in the presence of oxLDL. PMID:25141035

  6. In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum

    Microsoft Academic Search

    Řystein Bruserud; Karl Johan Tronstad; Rolf Berge

    2005-01-01

    Purpose: Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum compo- nent then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we

  7. Animal model of naturally occurring bladder cancer: Characterization of four new canine transitional cell carcinoma cell lines

    PubMed Central

    2014-01-01

    Background Development and further characterization of animal models for human cancers is important for the improvement of cancer detection and therapy. Canine bladder cancer closely resembles human bladder cancer in many aspects. In this study, we isolated and characterized four primary transitional cell carcinoma (K9TCC) cell lines to be used for future in vitro validation of novel therapeutic agents for bladder cancer. Methods Four K9TCC cell lines were established from naturally-occurring canine bladder cancers obtained from four dogs. Cell proliferation rates of K9TCC cells in vitro were characterized by doubling time. The expression profile of cell-cycle proteins, cytokeratin, E-cadherin, COX-2, PDGFR, VEGFR, and EGFR were evaluated by immunocytochemistry (ICC) and Western blotting (WB) analysis and compared with established human bladder TCC cell lines, T24 and UMUC-3. All tested K9TCC cell lines were assessed for tumorigenic behavior using athymic mice in vivo. Results Four established K9TCC cell lines: K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly were confirmed to have an epithelial-cell origin by morphology analysis, cytokeratin, and E-cadherin expressions. The tested K9TCC cells expressed UPIa (a specific marker of the urothelial cells), COX-2, PDGFR, and EGFR; however they lacked the expression of VEGFR. All tested K9TCC cell lines confirmed a tumorigenic behavior in athymic mice with 100% tumor incidence. Conclusions The established K9TCC cell lines (K9TCC#1Lillie, K9TCC#2Dakota, K9TCC#4Molly, and K9TCC#5Lilly) can be further utilized to assist in development of new target-specific imaging and therapeutic agents for canine and human bladder cancer. PMID:24964787

  8. Morphologic, immunologic, biochemical, and cytogenetic characteristics of the human glioblastoma-derived cell line, SNB-19

    Microsoft Academic Search

    William C. Welch; Richard S. Morrison; Janet L. Gross; Susanne M. Gollin; Richard B. Kitson; Ronald H. Goldfarb; Kenneth A. Giuliano; Margaret K. Bradley; Paul L. Kornblith

    1995-01-01

    Summary  Human glioma-derived cell cultures and lines have proven to be of significant value in the study of the basic properties that\\u000a contribute to the highly malignant, invasive and angiogenic phenotype of glioblastoma multiforme tumors. It is frequently\\u000a difficult to establish lines that retain glial tumor properties in long term culture. The SNB-19 cell line has maintained\\u000a and exhibited properties of

  9. Propagation of arthropod-borne Rickettsia spp. in two mosquito cell lines.

    PubMed

    Sakamoto, Joyce M; Azad, Abdu F

    2007-10-01

    Rickettsiae are obligate intracellular alphaproteobacteria that include pathogenic species in the spotted fever, typhus, and transitional groups. The development of a standardized cell line in which diverse rickettsiae can be grown and compared would be highly advantageous to investigate the differences among and between pathogenic and nonpathogenic species of rickettsiae. Although several rickettsial species have been grown in tick cells, tick cells are more difficult to maintain and they grow more slowly than insect cells. Rickettsia-permissive arthropod cell lines that can be passaged rapidly are highly desirable for studies on arthropod-Rickettsia interactions. We used two cell lines (Aedes albopictus cell line Aa23 and Anopheles gambiae cell line Sua5B) that have not been used previously for the purpose of rickettsial propagation. We optimized the culture conditions to propagate one transitional-group rickettsial species (Rickettsia felis) and two spotted-fever-group rickettsial species (R. montanensis and R. peacockii) in each cell line. Both cell lines allowed the stable propagation of rickettsiae by weekly passaging regimens. Stable infections were confirmed by PCR, restriction digestion of rompA, sequencing, and the direct observation of bacteria by fluorescence in situ hybridization. These cell lines not only supported rickettsial growth but were also permissive toward the most fastidious species of the three, R. peacockii. The permissive nature of these cell lines suggests that they may potentially be used to isolate novel rickettsiae or other intracellular bacteria. Our results have important implications for the in vitro maintenance of uncultured rickettsiae, as well as providing insights into Rickettsia-arthropod interactions. PMID:17766452

  10. CD133 positive progenitor endothelial cell lines from human cord blood.

    PubMed

    Paprocka, Maria; Krawczenko, Agnieszka; Dus, Danuta; Kantor, Aneta; Carreau, Aude; Grillon, Catherine; Kieda, Claudine

    2011-08-01

    Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB.1 and HEPC-CB.2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers: CD133, CD13, CD271, CD90 and also endothelial cell markers: CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells: CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells. PMID:21710642

  11. A Comparative Study of the FcεRI Molecule on Human Mast Cell and Basophil Cell Lines

    Microsoft Academic Search

    B. M. Jensen; S. Dissing; P. S. Skov; L. K. Poulsen

    2005-01-01

    Background: Mast cells and basophils express the high-affinity IgE receptor FcεRI. We have analysed the human mast cell line LAD2 and four subclones of the basophil cell line KU812 in order to reveal possible differences concerning the FcεRI surface regulation, anti-IgE-triggered activation, FcεRI? protein stability and the mRNA level of FcεRI?-, ?- and the truncated ?-chain (?T), and thereby determine

  12. Derivation, characterization, and gene expression profile of two new human ES cell lines from India.

    PubMed

    Mandal, Arundhati; Bhowmik, Subhanjan; Patki, Ameet; Viswanathan, Chandra; Majumdar, Anish Sen

    2010-11-01

    Human embryonic stem cells (hESCs) offer new avenues for studying human development and disease progression in addition to their tremendous potential toward development of cell-replacement therapies for various cellular disorders. We have earlier reported the derivation and characterization of Relicell(®) hES1, the first fully characterized hESC line generated from the Indian subcontinent. Recent studies have demonstrated discrete differences among hESC lines, in terms of both their growth properties and their differentiation propensity. To address some of these issues in the context of hESC research in India, we have recently generated two new hESC lines: Relicell(®) hES2 and Relicell(®)hES3. Both these cell lines were derived using a combinatorial approach of immunosurgery followed by mechanical surgery for inner cell mass isolation. The cell lines exhibit the usual hESC characteristics including their ability to differentiate both in vitro and in vivo to yield the three germinal layers. Whole genome microarray analysis of these cell lines was compared with Relicell(®)hES1 and it showed that approximately 9000 genes were expressed by these lines. As expected the expression pattern of these new cell lines bore close resemblance to that of Relicell(®)hES1. A majority of the pluripotency genes and the genes known to inhibit various differentiation pathways were also expressed by these cell lines. We also observed that each of these cell lines expressed a unique set of genes that are mutually exclusive from each other. These results represent the first detailed characterization of a set of hESC lines originating from India. PMID:20826120

  13. An eIF4E-interacting peptide induces cell death in cancer cell lines

    PubMed Central

    Masse, M; Glippa, V; Saad, H; Le Bloas, R; Gauffeny, I; Berthou, C; Czjzek, M; Cormier, P; Cosson, B

    2014-01-01

    The eukaryotic initiation factor eIF4E is essential for cap-dependent initiation of translation in eukaryotes. Abnormal regulation of eIF4E has been implicated in oncogenic transformation. We developed an eIF4E-binding peptide derived from Angel1, a partner of eIF4E that we recently identified. We show here that this peptide fused to a penetratin motif causes drastic and rapid cell death in several epithelial cancer cell lines. This necrotic cell death was characterized by a drop in ATP levels with F-actin network injury being a key step in extensive plasma membrane blebbing and membrane permeabilization. This synthetic eIF4E-binding peptide provides a candidate pharmacophore for a promising new cancer therapy strategy. PMID:25356869

  14. Development, characterization and use of a porcine epiblast-derived liver stem cell line: ARS-PICM-19

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Totipotent embryonic stem cell lines have not been established from ungulates, however, we have developed several somatic cell lines from the in vitro culture of pig epiblast cells. One such cell line, PICM-19, was isolated via colony-cloning and was found to spontaneously differentiate into hepati...

  15. Behavior of Disclination Lines Induced by a Nonuniform Electric Field in a Nematic Liquid Crystal Cell

    Microsoft Academic Search

    T. Nose; T. Sato; S. Sato

    1996-01-01

    The disclination lines appearing in the liquid crystal cell with a slit-patterned electrode structure are investigated in detail. Behaviors of disclination lines depend directly upon the motion of the reverse tilted molecular orientation domain, and there seem to be multi-stable molecular orientation states related to the disclination lines. Moreover, it is interesting that the disclination line has a zig-zag structure

  16. Characterization of the Murine Myeloid Precursor Cell Line MuMac-E8

    PubMed Central

    Fricke, Stephan; Riemschneider, Sina; Kohlschmidt, Janine; Hilger, Nadja; Fueldner, Christiane; Knauer, Jens; Sack, Ulrich; Emmrich, Frank; Lehmann, Jörg

    2014-01-01

    Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies. PMID:25546418

  17. Prodigiosin activates endoplasmic reticulum stress cell death pathway in human breast carcinoma cell lines

    SciTech Connect

    Pan, Mu-Yun [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China)] [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Shen, Yuh-Chiang [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China) [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); National Research Institute of Chinese Medicine, Taipei, Taiwan (China); Lu, Chien-Hsing [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China) [Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Obstetrics and Gynecology, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yang, Shu-Yi [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China)] [Institute of Biomedical Sciences, National Chung Hsing University, Taichung, Taiwan (China); Ho, Tsing-Fen [Department of Medical Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan (China)] [Department of