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Sample records for h9c2 cell line

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

    PubMed

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

    2014-10-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Lee, Kang Pa; Kim, Jai-Eun

    2015-01-01

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

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

    SciTech Connect

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

    2010-03-19

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

  8. 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

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

    PubMed Central

    Nguyen, Huy; Romani, Andrea

    2015-01-01

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

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

    PubMed

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

    2015-02-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

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

    2010-10-01

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

  14. Zinc inhibits doxorubicin-activated calcineurin signal transduction pathway in H9c2 embryonic rat cardiac cells.

    PubMed

    Merten, Kevyn E; Jiang, Youchun; Kang, Y James

    2007-05-01

    Elevation of the zinc-binding protein metallothionein (MT) in the heart inhibits doxorubicin (DOX)-induced myocardial apoptosis and heart hypertrophy. Zinc release from MT in response to oxidative stress has been suggested as a mechanism of action of MT protection from DOX toxicity, and calcineurin is involved in the signaling pathways leading to myocardial apoptosis and heart hypertrophy. The present study was undertaken to determine if zinc can modulate the DOX-activated calcineurin signaling pathway. H9c2 cells were treated with 1 muM DOX, and zinc release was monitored by a zinc ion-specific fluorophore, zinquin ethyl ester. Additionally, DOX-activated calcineurin signaling was detected by a calcineurin-dependent nuclear factor of activated T-cell reporter. DOX treatment induced an increase in intracellular labile zinc and activated calcineurin signaling. Pretreatment of H9c2 cells with a zinc-specific, membrane-permeable chelating agent, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), inhibited the increase in intracellular labile zinc and increased the DOX-activated calcineurin signaling. Pretreatment of H9c2 cells with exogenously added zinc attenuated the DOX-activated calcineurin signaling in a dose-dependent manner. However, neither TPEN nor addition of exogenous zinc affected DOX-induced cellular hypertrophy or DOX-induced decrease in cell viability. Additionally, inhibition of DOX-induced calcineurin signaling with the calcineurin inhibitors cyclosporine A or tacrolimus (FK506) failed to restrict the DOX-induced decrease in cell viability. These results indicate that zinc suppresses DOX-induced calcineurin signaling in H9c2 cells; however, calcineurin signaling is not involved in the DOX-induced decrease in cell viability in H9c2 cells. (It had been shown previously that calcineurin is also not necessary for DOX-induced H9c2 cell hypertrophy.). PMID:17463165

  15. 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

  16. Silibinin protects H9c2 cardiac cells from oxidative stress and inhibits phenylephrine-induced hypertrophy: potential mechanisms.

    PubMed

    Anestopoulos, Ioannis; Kavo, Anthula; Tentes, Ioannis; Kortsaris, Alexandros; Panayiotidis, Mihalis; Lazou, Antigone; Pappa, Aglaia

    2013-03-01

    Cardiac hypertrophy is the main response of the heart to various extrinsic and intrinsic stimuli, and it is characterized by specific molecular and phenotypic changes. Recent in vitro and in vivo studies indicate the involvement of reactive oxygen species in the hypertrophic response. In this study, silibinin, a plant flavonolignan extracted from milk thistle with potent antioxidant activity, was evaluated for its effects in (a) preventing hydrogen peroxide (H2O2)-induced cellular damage and (b) blocking the phenylephrine-induced hypertrophic response. Using the in vitro model of embryonic rat heart-derived H9c2 cells, we showed that silibinin has a rather safe profile as concentrations up to 200?M did not affect cell viability. Pretreatment of H9c2 cells with silibinin resulted in better protection of H9c2 cells under conditions of H2O2-induced cellular stress compared to untreated cells as indicated by cell viability and DNA fragmentation assays. Furthermore, silibinin attenuated the phenylephrine-induced hypertrophic response as evidenced by the measurement of cell surface, up-regulation of atrial natriuretic peptide and increase of cellular protein levels. Moreover, silibinin repressed the phenylephrine-induced phosphorylation of ERK1/2 kinases, while it appeared to inhibit the weakly activated by phenylephrine phosphorylation of Akt. Based on our results, silibinin may attenuate the phenylephrine-induced hypertrophic response of H9c2 cells via antioxidant mechanisms involving mainly the inhibition of the intracellular signaling pathways mediated by ERK1/2 MAPKs and Akt. PMID:22818713

  17. Interleukin-27 Protects Cardiomyocyte-Like H9c2 Cells against Metabolic Syndrome: Role of STAT3 Signaling

    PubMed Central

    Phan, Wei-Lian; Huang, Yu-Tzu; Ma, Ming-Chieh

    2015-01-01

    The present results demonstrated that high glucose (G), salt (S), and cholesterol C (either alone or in combination), as mimicking extracellular changes in metabolic syndrome, damage cardiomyocyte-like H9c2 cells and reduce their viability in a time-dependent manner. However, the effects were greatest when cells were exposed to all three agents (GSC). The mRNA of glycoprotein (gp) 130 and WSX-1, both components of the interleukin (IL)-27 receptor, were present in H9c2 cells. Although mRNA expression was not affected by exogenous treatment with IL-27, the expression of gp130 mRNA (but not that of WSX-1 mRNA) was attenuated by GSC. Treatment of IL-27 to H9c2 cells increased activation of signal transducer and activator of transcription 3 (STAT3) and protected cells from GSC-induced cytochrome c release and cell damage. The protective effects of IL-27 were abrogated by the STAT3 inhibitor, stattic. The results of the present study clearly demonstrate that the STAT3 pathway triggered by anti-inflammatory IL-27 plays a role in protecting cardiomyocytes against GSC-mediated damage. PMID:26339633

  18. Effect of alteration of caveolin-1 expression on doxorubicin-induced apoptosis in H9c2 cardiac cells.

    PubMed

    Takaguri, Akira; Kamato, Maiko; Satoh, Yoshiaki; Ohtsuki, Kazuaki; Satoh, Kumi

    2015-09-01

    Doxorubicin is an anthracycline antibiotic widely used in cancer treatment. Although its antitumor efficacy appears to be dose dependent, its clinical use is greatly restricted by the development of cardiotoxicity associated with apoptosis. Although caveolin-1, the major structural protein in caveolae, can positively or negatively regulate apoptosis depending on the stimulus or cell types, the contribution of caveolin-1 to doxorubicin-induced apoptosis remains unknown. This study was performed to identify the regulatory role of caveolin-1 on doxorubicin-induced apoptosis in H9c2 cardiac cells using a genetic approach. Caveolin-1 knockdown with a short hairpin (sh) RNA adenovirus, but not overexpression of wild-type caveolin-1, resulted in a marked inhibition of doxorubicin-induced caspase-3 cleavage. However, caveolin-1 knockdown tended to protect against doxorubicin-induced decrease in cell viability, but it did not significantly reverse cell death induced by doxorubicin. Doxorubicin stimulated the phosphorylation of p38 and extracellular signal regulated kinase (ERK). Doxorubicin-induced caspase-3 cleavage was inhibited by U0126, a MEK inhibitor or SB203580, a p38 inhibitor. Caveolin-1 knockdown markedly inhibited doxorubicin-induced p-38 phosphorylation but not ERK-mediated p-53 phosphorylation in H9c2 cardiac cells. Our results suggest that reduced caveolin-1 expression plays an anti-apoptotic role in doxorubicin-induced apoptosis but that it is insufficient to prevent such an apoptosis in H9c2 cardiac cells. PMID:25858022

  19. 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

  20. Pharmacological discrimination of plasmalemmal and mitochondrial sodium-calcium exchanger in cardiomyocyte-derived H9c2 cells.

    PubMed

    Namekata, Iyuki; Hamaguchi, Shogo; Tanaka, Hikaru

    2015-01-01

    We examined the effects of SEA0400 and CGP-37157 on the plasmalemmal Na(+)-Ca(2+) exchanger (NCX) and mitochondrial NCX using H9c2 cardiomyocytes loaded with Ca(2+)-sensitive fluorescent probes. The plasmalemmal NCX activity, which was measured as the increase in cytoplasmic Ca(2+) concentration after application of low Na(+) extracellular solution, was inhibited by SEA0400 but not by CGP-37157. The mitochondrial NCX activity, which was measured in permeabilized H9c2 cells as the decrease in mitochondrial Ca(2+) concentration after application of Ca(2+)-free extramitochondrial solution, was inhibited by CGP-37157 but not by SEA0400. These results indicate that SEA0400 and CGP-37157 act as selective inhibitors towards plasmalemmal and mitochondrial NCX, respectively, and provide pharmacological evidence that the plasmalemmal and mitochondrial NCX are distinct molecular entities. PMID:25744471

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

    PubMed

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

    2015-06-01

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

  2. 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

  3. 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 M 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 M) 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

  4. The mechanisms of propofol-induced block on ion currents in differentiated H9c2 cardiac cells.

    PubMed

    Liu, Yen-Ching; Wang, Ya-Jean; Wu, Sheng-Nan

    2008-08-20

    General anesthetic propofol (2,6-bis(isopropyl)-phenol) possess a chemical structure unrelated to other anesthetic drugs. It has been known to block a variety of ion currents. This study is designed to determine the effect of this drug on ion currents in differentiated H9c2 cardiac cells. The effects of propofol, an intravenous anesthetic agent with a distinct chemical structure, on ion currents of differentiated clonal cardiac (H9c2) cells were investigated in this study. Propofol (10-300 microM) suppressed the amplitude of delayed rectifier K(+) current (I(K(DR))) in a concentration-dependent manner with an IC(50) value of 36 microM. This compound reduced activation time constant and increased current inactivation, although no voltage dependency of propofol-induced block of I(K(DR)) can demonstrated. Neither diazoxide, pinacidil, nor caffeic acid phenethyl ester had any effect on propofol-induced block of I(K(DR)). Propofol (30 microM) had no effect on erg-mediated K(+) current in these cells; however, it suppressed L-type Ca(2+) current (I(Ca,L)) of cardiac and skeletal types to a similar extent. Intracellular dialysis with propofol (100 microM) had no effects on I(K(DR)) or I(Ca,L). Numerical simulations of I(K(DR)) based on a Markovian model reproduce the experimental results and show that propofol-induced blockade of I(K(DR)) is associated with an decrease in forward rate of the activation process and an increase in transitional rate into the inactivated state. Propofol can suppress I(K(DR)) in differentiated H9c2 cardiac cells in a concentration- and state-dependent manner. These effects can significantly contribute its action on functional activity of heart cells. PMID:18582866

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

    SciTech Connect

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

    2006-09-22

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2015-10-30

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

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

    PubMed

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

    2006-01-01

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

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2014-03-01

    Evidences suggest that apple peel has a wide range of polyphenols having antioxidant activity and its consumption has been linked with improved health benefits. Arsenic trioxide (ATO) is a very effective drug for the treatment of acute promyelocytic leukemia (APL) but it leads to cardiotoxicity mediated through alterations in various cardiac ion channels and by increasing the intracellular calcium level and reactive oxygen species (ROS). The aim of the present investigation was to study the effect of methanolic extract of apple peel (APME) and aqueous extract of apple peel (APAE) on ATO (5 ?M) induced toxicity in the H9c2 cardiac myoblast cell line. We estimated the cellular status of innate antioxidant enzymes, level of ROS, mitochondrial superoxide, glutathione and intracellular calcium with ATO and apple peel extracts. Prior to the cell line based study, we had evaluated the antioxidant potential of apple peel extract by 1,1-diphenyl-2-picrylhydrazyl (DPPH), total reducing power (TRP), superoxide anion and hydroxyl radical scavenging activity, in addition to quantifying total phenolic and flavonoid content. Both the extracts showed considerable antioxidant activity in cell-free chemical assays. In addition, both APME and APAE prevented the alteration in antioxidant status induced by ATO in H9c2 cells. Significant differential alterations had been observed in the activity of lactate dehydrogenase, superoxide dismutase, catalase, glutathione, glutathione peroxidase, thioredoxin reductase, xanthine oxidase, calcium overload and caspase3 activity with ATO. The overall result revealed the protective property of polyphenol-rich apple peel extract against ATO induced cardiac toxicity via its antioxidant activity. PMID:24441683

  11. Resveratrol prevents doxorubicin-induced cardiotoxicity in H9c2 cells through the inhibition of endoplasmic reticulum stress and the activation of the Sirt1 pathway.

    PubMed

    Lou, Yu; Wang, Zhen; Xu, Yi; Zhou, Ping; Cao, Junxian; Li, Yuanshi; Chen, Yeping; Sun, Junfeng; Fu, Lu

    2015-09-01

    Treatment with doxorubicin (DOX) is one of the major causes of chemotherapy-induced cardiotoxicity and is therefore, the principal limiting factor in the effectiveness of chemotherapy for cancer patients. DOX?induced heart failure is thought to result from endoplasmic reticulum (ER) stress and cardiomyocyte apoptosis. Resveratrol (RV), a polyphenol antioxidant found in red wine, has been shown to play a cardioprotective role. The aim of the present study was to examine the effects of RV on DOX?induced cardiotoxicity in H9c2 cells. We hypothesized that RV would protect H9c2 cells against DOX?induced ER stress and subsequent cell death through the activation of the Sirt1 pathway. Our results demonstrated that the decrease observed in the viability of the H9c2 cells following exposure to DOX was accompanied by a significant increase in the expression of the ER stress?related proteins, glucose?regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP). However, we found that RV downregulated the expression of ER stress marker protein in the presence of DOX and restored the viability of the H9c2 cells. Exposure to RV or DOX alone only slightly increased the protein expression of Sirt1, whereas a significant increase in Sirt1 protein levels was observed in the cells treated with both RV and DOX. The Sirt1 inhibitor, nicotinamide (NIC), partially neutralized the effects of RV on the expression of Sirt1 in the DOX?treated cells and completely abolished the effects of RV on the expression of GRP78 and CHOP. The findings of our study suggest that RV protects H9c2 cells against DOX?induced ER stress through ER stabilization, and more specifically through the activation of the Sirt1 pathway, thereby leading to cardiac cell survival. PMID:26202177

  12. Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    PubMed Central

    Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

    2014-01-01

    BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (?-tubulin) and novel (?-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

  13. Calcineurin activation is not necessary for Doxorubicin-induced hypertrophy in H9c2 embryonic rat cardiac cells: involvement of the phosphoinositide 3-kinase-Akt pathway.

    PubMed

    Merten, Kevyn E; Jiang, Youchun; Feng, Wenke; Kang, Y James

    2006-11-01

    The calcium/calmodulin-dependent phosphatase calcineurin has been shown to be both necessary and sufficient to induce cardiac hypertrophy in vivo and in vitro. Treatment with the antineoplastic agent doxorubicin (DOX) was shown to activate calcineurin signaling in H9c2 rat cardiac muscle cells; however, the effect of this activation on hypertrophy was not investigated. Therefore, the present study was undertaken to examine the involvement of calcineurin activation in DOX-induced cardiac cell hypertrophy. H9c2 cells were treated with 1 microM DOX for 2 h following pretreatment with and in the presence of calcineurin inhibitors cyclosporine A (CsA) or FK506 (tacrolimus). Subsequent analysis of calcineurin signaling and cellular hypertrophy was performed 8 to 48 h after the treatment. DOX treatment activated calcineurin signaling and resulted in cellular hypertrophy as assessed by an increase in cell volume and protein content per cell. Inhibition of calcineurin with CsA or FK506 blocked DOX-induced calcineurin signaling. However, this inhibition did not prevent the DOX-induced hypertrophic response in H9c2 cells. Further evaluation of the possible signaling pathways involved in DOX-induced H9c2 cellular hypertrophy revealed that DOX treatment resulted in phosphorylation of the serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K). Moreover, the DOX-induced hypertrophic response was blunted by LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a specific inhibitor for PI3K. These results demonstrate that, although calcineurin is activated by DOX treatment, it is not necessary for DOX-induced hypertrophy in H9c2 cells. Rather, the PI3K-Akt signaling pathway seems to be more critically involved in DOX-induced hypertrophy. PMID:16926266

  14. Factors Released from Embryonic Stem Cells inhibit Apoptosis in H9c2 cells through P1-3kinase/Akt but not ERK pathway

    PubMed Central

    Singla, Dinender K.; Singla, Reetu D.; McDonald, Debbie E.

    2008-01-01

    We recently reported that embryonic stem cells-conditioned medium (ES-CM) contains antiapoptotic factors that inhibit apoptosis in the cardiac myoblast, H9c2 cells. However, the mechanisms of inhibited apoptosis remain elusive. In this report, we provide evidences for novel mechanisms involved in the inhibition of apoptosis provided by ES-CM. ES-CM from mouse ES cells was generated. Apoptosis was induced after exposure with H2O2 (400?m) in H9c2 cells followed by replacement with ES-CM or culture medium. H9c2 cells treated with H2O2 were exposed to ES-CM, and ES-CM+cell survival protein phosphatidyl-inositol 3-kinase (PI-3k/Akt) inhibitor, LY294002 or extracellular signal-regulated kinase (ERK1/2), PD98050. After 24 hours, H9c2 cells treated with ES-CM demonstrated significant increase in cell survival. ES-CM significantly inhibited (p<0.05) apoptosis determined by TUNEL staining, apoptotic ELISA and caspase-3 activity. Importantly, enhanced cell survival and inhibited apoptosis with ES-CM was abolished with LY294002. In contrast, PD98050 shows no effect on ES-CM increased cell survival. Furthermore, H2O2 induced apoptosis is associated with decreased levels of phosphorylated (p) Akt activity. Following treatment with ES-CM, we observed a decrease in apoptosis with an increase in pAkt, and increased activity was attenuated with Akt inhibitor, suggesting that the Akt pathway is involved in the decreased apoptosis and cell survival provided by ES-CM. In contrast, we observed no change in ES-CM decreased apoptosis or pERK with PD98050. In conclusion, we suggest that ES-CM inhibited apoposis and is mediatd by Akt but not ERK pathway. PMID:17545477

  15. Differential behaviour of epicardial adipose tissue-secretomes with high and low orosomucoid levels from patients with cardiovascular disease in H9C2 cells.

    PubMed

    Lage, Ricardo; Moscoso, Isabel; Fernndez-Trasancos, ngel; Cebro, Mara; Couselo, Marinela; Fandio-Vaquero, Rubn; Bravo, Susana B; Sierra, Juan; Gonzlez-Juanatey, Jos Ramn; Eiras, Sonia

    2015-11-15

    Epicardial adipose tissue releases orosomucoid (ORM), an acute phase protein with multiple modulatory and protective properties. We aimed to identify the effect of EAT-supernatants according to their ORM levels on H9C2 cells. H9C2 were cultured with EAT-secretomes or ORM protein itself on a Real-Time Cell Analyser. Secretome proteins identification was performed by LC-mass spectrometry according to their ORM levels. Two of them were validated by ELISA in EAT-supernatants from 42 patients. ORM effect on H9C2 and neonatal rat cardiomyocytes apoptosis under hypoxia with or without fatty acid treatment was determined by Annexin-V flow cytometry measurement. Caspase-3 expression levels were determined by western blot in H9C2. Our results showed a differential effect of EAT-secretomes according their ORM levels. Although additional secreted proteins can contribute to their beneficial effects, ORM reduced hypoxia-induced apoptosis through caspase-3 inhibition. Our data showed the cardioprotective role of ORM and suggest that its quantification on EAT secretomes might help us to find new secreted factors with a cardioprotective role. PMID:26343163

  16. PI3K/Akt/FoxO3a signaling mediates cardioprotection of FGF-2 against hydrogen peroxide-induced apoptosis in H9c2 cells.

    PubMed

    Liu, Mi-Hua; Li, Guo-Hua; Peng, Li-Jun; Qu, Shun-Lin; Zhang, Yuan; Peng, Juan; Luo, Xin-Yuan; Hu, Heng-Jing; Ren, Zhong; Liu, Yao; Tang, Hui; Liu, Lu-Shan; Tang, Zhi-Han; Jiang, Zhi-Sheng

    2016-03-01

    Cardiovascular disease is a growing major global public health problem. Oxidative stress is regarded as one of the key regulators of pathological physiology, which eventually leads to cardiovascular disease. However, mechanisms by which FGF-2 rescues cells from oxidative stress damage in cardiovascular disease is not fully elucidated. Herein this study was designed to investigate the protective effects of FGF-2 in H2O2-induced apoptosis of H9c2 cardiomyocytes, as well as the possible signaling pathway involved. Apoptosis of H9c2 cardiomyocytes was induced by H2O2 and assessed using methyl thiazolyl tetrazolium assay, Hoechst, and TUNEL staining. Cells were pretreated with PI3K/Akt inhibitor LY294002 to investigate the possible PI3K/Akt pathways involved in the protection of FGF-2. The levels of p-Akt, p-FoxO3a, and Bim were detected by immunoblotting. Stimulation with H2O2 decreased the phosphorylation of Akt and FoxO3a, and induced nuclear localization of FoxO3a and apoptosis of H9c2 cells. These effects of H2O2 were abrogated by pretreatment with FGF-2. Furthermore, the protective effects of FGF-2 were abolished by PI3K/Akt inhibitor LY294002. In conclusion, our data suggest that FGF-2 protects against H2O2-induced apoptosis of H9c2 cardiomyocytes via activation of the PI3K/Akt/FoxO3a pathway. PMID:26899709

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  19. Overproduction of reactive oxygen species and activation of MAPKs are involved in apoptosis induced by PM2.5 in rat cardiac H9c2 cells.

    PubMed

    Cao, Jing; Qin, Gang; Shi, Ruizan; Bai, Feng; Yang, Guangzhao; Zhang, Mingsheng; Lv, Jiyuan

    2016-04-01

    Epidemiological studies show a positive correlation between the air levels of fine particulate matter (PM2.5 ) and cardiovascular disorders, but how PM2.5 affects cardiomyocytes has not been studied in great deal. The aim of the present study was to obtain an insight into the links among intracellular levels of reactive oxygen species (ROS), apoptosis and mitogen-activated protein kinases (MAPKs) in rat cardiac H9c2 cells exposed to PM2.5 . H9c2 cells were incubated with PM2.5 at 100-800 µg ml(-1) to evaluate the effects of PM2.5 on cell viability, cell apoptosis, intracellular levels of ROS and expression of apoptosis-related proteins as well as activation of MAPKs. PM2.5 decreased cell viability, increased the cell apoptosis rate and intracellular ROS production in a concentration-dependent manner. PM2.5 decreased the Bcl-2/Bax ratio and increased cleaved caspase-3 levels. A Western blots study showed up-regulation of phosphorylated MAPKs including extracellular signal-regulated protein kinases (ERKs), c-Jun NH2 -terminal kinases (JNKs) and p38 MAPK in the PM2.5 -treated cells. The p38 MAPK inhibitor SB239063 attenuated whereas the ERKs inhibitor PD98059 augmented the effects of PM2.5 on apoptosis and the expression of related proteins. In conclusion, PM2.5 decreases cell viability and increases apoptosis by enhancing intracellular ROS production and activating the MAPKs signaling pathway in H9c2 cells. The MAPKs signaling pathway could be a new promising target for clinical therapeutic strategies against PM2.5 -induced cardiac injury. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26472149

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

    A recent clinical study demonstrated that a testosterone supplementation improves functional capacity in elderly female patients suffering from heart failure. These findings prompted us to consider possible mechanisms of testosterone-induced cardioprotection in females. To address this question we have used a pure female population of rat heart embryonic H9c2 cells. Pre-treatment of cells with testosterone for 24h significantly increased survival of H9c2 cells exposed to 2,4-dinitrophenol (DNP), an inhibitor of oxidative phosphorylation. These cells expressed low level of androgen receptors and the effect of testosterone was not modified by hydroxyflutamide, an antagonist of androgen receptor. In contrast, cyclohexamide, an inhibitor of protein biosynthesis, and tamoxifene, a partial agonist of estrogen receptors, abolished cardioprotection afforded by testosterone. In addition, finasteride, an inhibitor of 5?-reductase, and anastrazole, an inhibitor of ?-aromatase, also blocked testosterone-induced cytoprotection. Real time RT-PCR revealed that testosterone did not regulate the expression of nine subunits and accessory proteins of sarcolemmal ATP-sensitive K+ (KATP) channels. On the other hand, testosterone, as well as 17?-estradiol, up-regulated a putative mitochondrial KATP channel subunit, mitochondrial sulfonylurea receptor 2B intraexonics splice variant (IES SUR2B), without affecting expression of IES SUR2A. Tamoxifene inhibited testosterone-induced up-regulation of IES SUR2B without affecting IES SUR2A. In conclusion, this study has shown that testosterone protect female embryonic heart H9c2 cells against severe metabolic stress by its conversion into metabolites that activate estrogen receptors and up-regulate IES SUR2B. PMID:23085378

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

    PubMed

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

    2013-02-01

    A recent clinical study demonstrated that a testosterone supplementation improves functional capacity in elderly female patients suffering from heart failure. These findings prompted us to consider possible mechanisms of testosterone-induced cardioprotection in females. To address this question we have used a pure female population of rat heart embryonic H9c2 cells. Pre-treatment of cells with testosterone for 24h significantly increased survival of H9c2 cells exposed to 2,4-dinitrophenol (DNP), an inhibitor of oxidative phosphorylation. These cells expressed low level of androgen receptors and the effect of testosterone was not modified by hydroxyflutamide, an antagonist of androgen receptor. In contrast, cyclohexamide, an inhibitor of protein biosynthesis, and tamoxifene, a partial agonist of estrogen receptors, abolished cardioprotection afforded by testosterone. In addition, finasteride, an inhibitor of 5?-reductase, and anastrazole, an inhibitor of ?-aromatase, also blocked testosterone-induced cytoprotection. Real time RT-PCR revealed that testosterone did not regulate the expression of nine subunits and accessory proteins of sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. On the other hand, testosterone, as well as 17?-estradiol, up-regulated a putative mitochondrial K(ATP) channel subunit, mitochondrial sulfonylurea receptor 2B intraexonics splice variant (IES SUR2B), without affecting expression of IES SUR2A. Tamoxifene inhibited testosterone-induced up-regulation of IES SUR2B without affecting IES SUR2A. In conclusion, this study has shown that testosterone protect female embryonic heart H9c2 cells against severe metabolic stress by its conversion into metabolites that activate estrogen receptors and up-regulate IES SUR2B. PMID:23085378

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

    Cell apoptosis induced by Angiotensin II (Ang II) has a critical role in the development of cardiovascular diseases. The aim of the present study was to investigate whether sanguinarine (SAN), a drug which was proved to have anti-oxidant, anti-proliferative and immune enhancing effects, can abolish cell apoptosis induced by Ang II. In the present study, H9c2 cardiac cells were stimulated with 10 M 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

  6. Protective effects of ginsenoside Rg2 against H2O2-induced injury and apoptosis in H9c2 cells

    PubMed Central

    Fu, Wenwen; Sui, Dayun; Yu, Xiaofeng; Gou, Dongxia; Zhou, Yifa; Xu, Huali

    2015-01-01

    Ginsenoside Rg2 is one of the major active components of ginseng and has many biological activities. This study aimed to investigate the protective effects of ginsenoside Rg2 against H2O2-induced injury and apoptosis in H9c2 cells. The results showed that pretreatment with ginsenoside Rg2 not only increased cell viability, but also decreased lactate dehydrogenase (LDH) release. Ginsenoside Rg2 inhibited the decrease of SOD, GSH-PX activities and the increase of MDA content induced by H2O2. Meanwhile, the levels of ROS generation and cardiomyocyte apoptosis in ginsenoside Rg2 group significantly reduced when compared with the model group. Western blot analyses demonstrated that ginsenoside Rg2 up-regulate level of Bcl-2 expression and down-regulate levels of Bax, Caspase-3, -9 expression. These findings indicated that ginsenoside Rg2 could protect H9c2 cells against H2O2-induced injury through its actions of anti-oxidant and anti-apoptosis. PMID:26884906

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

    PubMed Central

    Jia, Xiongfei; Zhang, Li; Mao, Xiaoqin

    2015-01-01

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

  8. Protection of Luteolin-7-O-Glucoside Against Doxorubicin-Induced Injury Through PTEN/Akt and ERK Pathway in H9c2 Cells.

    PubMed

    Yao, Hong; Shang, Zhimei; Wang, Penghong; Li, Shuixian; Zhang, Qianyun; Tian, Huiqin; Ren, Dongmei; Han, Xiuzhen

    2016-04-01

    Luteolin-7-O-glucoside (LUTG) was isolated from the plants of Dracocephalum tanguticum Maxim. Previous research has showed that LUTG pretreatment had a significant protective effect against doxorubicin (DOX)-induced cardiotoxicity by reducing intracellular calcium overload and leakage of creatine kinase and lactate dehydrogenase. But the underlying mechanisms have not been completely elucidated. In the present study, we investigated the effects of LUTG on H9c2 cell morphology, viability, apoptosis, reactive oxygen species generation, and the mitochondrial transmembrane potentials. The expression of p-PTEN, p-Akt, p-ERK, p-mTOR, and p-GSK-3β were detected by Western blotting. Compared with DOX alone treatment group, the morphological injury and apoptosis of the cells in groups treated by DOX plus LUTG were alleviated, cell viability was increased, ROS generation was lowered remarkably, and mitochondrial depolarization was mitigated. In DOX group, the expression of p-PTEN was lower than normal group and the expression of p-Akt and p-ERK was higher than normal group. In the groups treated with LUTG (20 μM), the expression of p-PTEN was upregulated and the expression of p-Akt, p-ERK, p-mTOR, and p-GSK-3β was downregulated. These results indicated that the protective effects of LUTG against DOX-induced cardiotoxicity may be related to anti-apoptosis through PTEN/Akt and ERK pathway. PMID:25724325

  9. Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation

    PubMed Central

    Cheng, Yuanyuan; Xia, Zhengyuan; Han, Yifan; Rong, Jianhui

    2016-01-01

    The opening of mitochondrial permeability transition pore (mPTP) is a major cause of cell death in ischemia reperfusion injury. Based on our pilot experiments, plant natural product formononetin enhanced the survival of rat cardiomyocyte H9c2 cells during oxygen glucose deprivation (OGD) and reoxygenation. For mechanistic studies, we focused on two major cellular factors, namely, reactive oxygen species (ROS) and glycogen synthase kinase 3β (GSK-3β), in the regulation of mPTP opening. We found that formononetin suppressed the formation of ROS and superoxide in a concentration-dependent manner. Formononetin also rescued OGD/reoxygenation-induced loss of mitochondrial membrane integrity. Further studies suggested that formononetin induced Akt activation and GSK-3β (Ser9) phosphorylation, thereby reducing GSK-3β activity towards mPTP opening. PI3K and PKC inhibitors abolished the effects of formononetin on mPTP opening and GSK-3β phosphorylation. Immunoprecipitation experiments further revealed that formononetin increased the binding of phosphor-GSK-3β to adenine nucleotide translocase (ANT) while it disrupted the complex of ANT with cyclophilin D. Moreover, immunofluorescence revealed that phospho-GSK-3β (Ser9) was mainly deposited in the space between mitochondria and cell nucleus. Collectively, these results indicated that formononetin protected cardiomyocytes from OGD/reoxygenation injury via inhibiting ROS formation and promoting GSK-3β phosphorylation.

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

    PubMed Central

    Alyane, Mohamed; Barratt, Gillian; Lahouel, Mesbah

    2015-01-01

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

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

    PubMed

    Alyane, Mohamed; Barratt, Gillian; Lahouel, Mesbah

    2016-03-01

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

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

    PubMed Central

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

    2014-01-01

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

  13. Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

    PubMed

    Sun, Lei; Zhao, Mei; Yang, Yang; Xue, Run-Qing; Yu, Xiao-Jiang; Liu, Jian-Kang; Zang, Wei-Jin

    2016-05-01

    Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh-elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN-induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were detected with fluorescence staining. We found that H/R-treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3-II/LC3-I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green-labeled mitochondria further confirmed ACh-induced mitochondrial translocation of Parkin, which was reversed by M2 receptor antagonist methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor 3-methaladenine abolished ACh-induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial-dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targets in the preservation of cardiac homeostasis against H/R injury. J. Cell. Physiol. 231: 1171-1181, 2016. © 2015 Wiley Periodicals, Inc. PMID:26465230

  14. 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

  15. 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

  16. The Protective Effect of INH2BP, a Novel PARP Inhibitor 5-Iodo-6-Amino-1,2-Benzopyrone, Against Hydrogen Peroxide-Induced Apoptosis Through ERK and p38 MAPK in H9c2 Cells.

    PubMed

    Back, Oun-Cheol; Jang, Hyung-Seok; Lee, Ji-Sook; Kim, Dae-Eun; Lee, Young; Park, Eun-Seok; Kim, In Sik

    2015-11-01

    INH2BP (5-iodo-6-amino-1,2-benzopyrone), a poly-ADP ribose polymerase inhibitor, has been shown to possess anti-cancer, anti-viral, and anti-inflammation properties. The aim of this study was to investigate the protective effect of INH2BP against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells. While the treatment of H9c2 cardiomyoblasts cells with hydrogen peroxide (H2O2) caused a loss of cell viability and an increase in the number of apoptotic cells, INH2BP significantly protected the cells against H2O2-induced cell death without any cytotoxicity. Our data also shows that INH2BP significantly scavenged intracellular reactive oxygen species (ROS), and markedly enhanced the expression of antioxidant enzymes such as Mn-SOD (superoxide) and Cu/Zn-SOD, and heme oxygenase-1, which was accompanied by the concomitant activation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation in H9c2 cells. The effects of INH2BP on ERK1/2 and p38 MAPK phosphorylation were abrogated by PD98059, an ERK1/2 inhibitor, and SB203580, a p38 inhibitor. In addition, inhibition of ERK1/2 and p38 MAPK by these inhibitors significantly attenuated INH2BP-mediated H9c2 viability as well as cleaved caspases-3, Bax, and Bcl-2 activation. Taken together, these results demonstrate that INH2BP prevents H2O2-induced apoptosis in H9c2 cells by reducing the production of intracellular ROS, regulating apoptotic-related proteins, and the activation of the ERK1/2 and p38 MAPK. PMID:26550832

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

    PubMed

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

    2016-01-01

    Ischemia-reperfusion(I/R) plays an important role in myocardial injury. In the present study, we aimed to examine the protective effects of Danshensu(DSS) against I/R injury and to elucidate the underlying mechanisms. For this purpose, H9c2 cells were cultured in hypoxic solution in a hypoxic incubator for 2h, and then cultured in a high oxygen incubator for various periods of time and pre-treated with or without DSS, ammonium pyrrolidine dithiocarbamate(PDTC) or SP600125[a c-Jun N-terminal kinase(JNK) inhibitor]. Cell apoptosis and cytosolic free Ca2+([Ca2+]i) levels were analyzed by flow cytometry. The protein expression levels of JNK, phosphorylated(p-)JNK, nuclear factor-?B(NF-?B) and transient receptor potential cation channel, subfamilyC, member6(TRPC6) were measured by western blot analysis. The mRNA expression levels of JNK were measured by RT-qPCR. The results revealed that TRPC6 protein expression, the cell apoptotic rate and the [Ca2+]i levels increased in a time-dependent manner in the H9c2 cells following the induction of I/R injury. The apoptotic rate and TRPC6 protein expression decreased when the cells were treated with DSS prior to the induction of I/R injury. The knockdown of JNK expression by siRNA decreased the p-JNK and TRPC6 protein expression levels in the H9c2 cells subjected to I/R injury. The protein expression levels of p-JNK and NF-?B in the nucleus increased significantly when the H9c2 cells were subjected to I/R injury, whereas NF-?B expression in the cytoplasm decreased in a time?dependent manner. However, p-JNK, NF-?B and TRPC6 protein expression, the [Ca2+]i level and cell apoptosis decreased when the H9c2 cells were pre-treated with DSS or SP600125. Therefore, our data suggest that DSS prevents myocardial I/R injury by inhibiting p-JNK activation and NF-?B translocation, which potentially upregulate TRPC6 expression, increase the [Ca2+]i level, and result in the apoptosis of H9c2 cells. PMID:26718129

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

    PubMed Central

    HAN, HUI; HU, JINQUAN; YAN, QIANG; ZHU, JINZHOU; ZHU, ZHENGBIN; CHEN, YANJIA; SUN, JIATENG; ZHANG, RUIYAN

    2016-01-01

    The transplantation of mesenchymal stem cells (MSCs) is considered to be a promising treatment for ischemic heart disease; however, the therapeutic effects and underlying mechanisms of action require further evaluation. Mitochondrial dysfunction is a key event in simulated ischemia/reperfusion (SI/R) injury. The purpose of the present study was to investigate the mechanism of mitochondrial transfer, which may be involved the antiapoptotic action of co-culture with MSCs. An in vitro model of simulated ischemia/reperfusion (SI/R) was used in the present study. The apoptotic indexes were significantly increased when H9c2 cardiomyocytes were induced in the SI/R group. Following co-culture with bone marrow-derived (BM)-MSCs, H9c2 cells exhibited marked resistance against the SI/R-induced apoptotic process. Besides, mitochondrial transfer via a tunneling nanotube (TNT) like structure was detected by confocal fluorescent microscopy. In addition, following pretreated with latrunculin-A (LatA), an inhibitor of TNT formation, the BM-MSCs were not able to rescue injured H9c2 cells from apoptosis, as previously observed. In conclusion, the anti-apoptotic ability of BM-MSCs may be partially attributed to the recovery of mitochondrial dysfunction in SI/R, and the formation of TNTs appears to be involved in this action of mitochondrial transfer between adjacent cells. PMID:26718099

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

    PubMed

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

    2016-02-01

    The transplantation of mesenchymal stem cells (MSCs) is considered to be a promising treatment for ischemic heart disease; however, the therapeutic effects and underlying mechanisms of action require further evaluation. Mitochondrial dysfunction is a key event in simulated ischemia/reperfusion (SI/R) injury. The purpose of the present study was to investigate the mechanism of mitochondrial transfer, which may be involved the antiapoptotic action of co-culture with MSCs. An invitro model of simulated ischemia/reperfusion (SI/R) was used in the present study. The apoptotic indexes were significantly increased when H9c2cardiomyocytes were induced in the SI/R group. Following co-culture with bone marrow-derived (BM)?MSCs, H9c2cells exhibited marked resistance against the SI/R-induced apoptotic process. Besides, mitochondrial transfer via a tunneling nanotube (TNT) like structure was detected by confocal fluorescent microscopy. In addition, following pretreated with latrunculin-A (LatA), an inhibitor of TNT formation, the BM-MSCs were not able to rescue injured H9c2 cells from apoptosis, as previously observed. In conclusion, the anti?apoptotic ability of BM?MSCs may be partially attributed to the recovery of mitochondrial dysfunction in SI/R, and the formation of TNTs appears to be involved in this action of mitochondrial transfer between adjacent cells. PMID:26718099

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

    PubMed

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

    2015-12-15

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

  1. 2,3,7,8-Tetrachlorodibenzo-p-dioxin and beta-naphthoflavone induce cellular hypertrophy in H9c2 cells by an aryl hydrocarbon receptor-dependant mechanism.

    PubMed

    Zordoky, Beshay N M; El-Kadi, Ayman O S

    2010-04-01

    Cigarette smoke is a major risk factor for cardiovascular diseases. It contains thousands of compounds that activate the aryl hydrocarbon receptor (AhR). In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR ligand, has been shown to cause cardiotoxic effects in several in vivo models. Although induction of CYP1 family is the most important effect of AhR activation, the role of CYP1 induction in mediating the cardiotoxic effect of TCDD is usually overlooked. Therefore, we investigated whether AhR activation causes a hypertrophic effect in H9c2 cells and we related this effect to changes in CYP gene expression. In the current study, the cardiac derived H9c2 cells were treated with two AhR ligands, TCDD and beta-naphthoflavone (BNF), for 24 and 48h. The expression of the hypertrophic markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), and several CYP genes were measured by real-time PCR. Treatment of H9c2 cells with TCDD or BNF for 24h caused a significant induction of CYP1A1, CYP1B1, and CYP4A1; however, there was no change in the expression of other genes. On the other hand, treatment of the cells with TCDD or BNF for 48h caused a significant induction of the hypertrophic markers, ANP and BNP, and several CYP genes such as CYP1A1, CYP1B1, CYP2E1, CYP2J3, and CYP4F4 parallel to a significant increase in the cell surface area. Neither TCDD nor BNF increased the oxidative stress in H9c2 cells at all concentrations tested. Interestingly, resveratrol, an AhR antagonist, protected the cells from TCDD-induced hypertrophy. In conclusion, AhR ligands caused a hypertrophic effect in H9c2 cells which was associated with induction of several CYP genes which can be prevented by resveratrol. PMID:19969063

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

    SciTech Connect

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

    2013-10-15

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

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

    SciTech Connect

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

    2014-07-18

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

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

    PubMed

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

    2016-02-01

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

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

    PubMed

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

    2004-12-01

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

  6. 17Beta-oestradiol regulates the expression of Na+/K+-ATPase beta1-subunit, sarcoplasmic reticulum Ca2+-ATPase and carbonic anhydrase iv in H9C2 cells.

    PubMed

    Liu, Chen-Geng; Xu, Ke-Qian; Xu, Xia; Huang, Jian-Jun; Xiao, Jing-Chuan; Zhang, Jie-Ping; Song, Hui-Ping

    2007-10-01

    1. It is necessary to improve our understanding of the effect of 17beta-oestradiol (E2) on the heart at a molecular and cellular level. In the present study, the effects of E2 on Na(+)/K(+)-ATPase, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and carbonic anhydrase IV (CAIV) in H9C2 cells were investigated. To identify the mechanism of action of E2 on these proteins, the oestrogen receptor (ER) antagonist tamoxifen was used. 2. The results indicated that 1 and 100 nmol/L E2 can enhance the activity of Na(+)/K(+)-ATPase and SERCA and upregulate the expression of the Na(+)/K(+)-ATPase beta1-subunit, SERCA2a and CAIV at both the mRNA and protein level compared with 0 and 0.01 nmol/L E2. 17beta-Oestradiol had the greatest effect at 100 nmol/L; 1 micromol/L E2 did not further protein expression compared with 100 nmol/L E2. 3. Tamoxifen (10 nmol/L) significantly decreased the activity of SERCA, as well as the expression of the Na(+)/K(+)-ATPase beta1-subunit and SERCA at the mRNA and protein level, in H9C2 cells cultured with 1 nmol/L E2. Tamoxifen alone had no significant effect on these proteins in H9C2 cells. 4. It may be hypothesized that a suitable E2 concentration has a protective effect on the heart and that the actual dose of E2 used in hormone-replacement therapy is important in menopausal women. PMID:17714085

  7. Arginine Vasopressin Enhances Cell Survival via a G Protein–Coupled Receptor Kinase 2/β-Arrestin1/Extracellular-Regulated Kinase 1/2–Dependent Pathway in H9c2 Cells

    PubMed Central

    Myers, Valerie D.; Coleman, Ryan C.; Feldman, Arthur M.

    2013-01-01

    Circulating levels of arginine vasopressin (AVP) are elevated during hypovolemia and during cardiac stress. AVP activates arginine vasopressin type 1A (V1A)/Gαq–coupled receptors in the heart and vasculature and V2/Gαs–coupled receptors in the kidney. However, little is known regarding the signaling pathways that influence the effects of V1A receptor (V1AR) activation during cellular injury. Using hypoxia-reoxygenation (H/R) as a cell injury model, we evaluated cell survival and caspase 3/7 activity in H9c2 myoblasts after treatment with AVP. Pretreatment of H9c2 cells with AVP significantly reduced H/R-induced cell death and caspase 3/7 activity, effects that were blocked via both selective V1AR inhibition and mitogen-activated protein kinase (MEK1/2) inhibition. AVP increased extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner that was sensitive to MEK1/2 inhibition and V1AR inhibition, but not V1BR or V2R inhibition. Discrete elements of the V1A/Gαq-protein kinase C (PKC) and V1A/G protein–coupled receptor kinase (GRK)/β-arrestin signaling cascades were inhibited to dissect the pathways responsible for the protective effects of V1AR signaling: Gαq (overexpression of Gq-I-ires-green fluorescent protein), PKC (administration of Ro 31-82425; 2-[8-(aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, HCl, bisindolylmaleimide X, HCl), GRK2 [C-terminal GRK2 peptide overexpression and small interfering RNA (siRNA) knockdown], GRK5 (siRNA knockdown), and β-arrestin1 (siRNA knockdown). These studies demonstrated that both Gαq/PKC- and GRK2/β-arrestin1–dependent V1AR signaling were capable of inducing ERK1/2 phosphorylation in response to AVP stimulation. However, AVP-mediated protection against H/R was elicited only via GRK2- and β-arrestin1–dependent signaling. These results suggest that activation of the V1AR in H9c2 cells mediates protective signaling via a GRK2/β−arrestin1/ERK1/2–dependent mechanism that leads to decreased caspase 3/7 activity and enhanced survival under conditions of ischemic stress. PMID:23690069

  8. Arginine vasopressin enhances cell survival via a G protein-coupled receptor kinase 2/?-arrestin1/extracellular-regulated kinase 1/2-dependent pathway in H9c2 cells.

    PubMed

    Zhu, Weizhong; Tilley, Douglas G; Myers, Valerie D; Coleman, Ryan C; Feldman, Arthur M

    2013-08-01

    Circulating levels of arginine vasopressin (AVP) are elevated during hypovolemia and during cardiac stress. AVP activates arginine vasopressin type 1A (V(1A))/G?(q)-coupled receptors in the heart and vasculature and V(2)/G?(s)-coupled receptors in the kidney. However, little is known regarding the signaling pathways that influence the effects of V(1A) receptor (V(1A)R) activation during cellular injury. Using hypoxia-reoxygenation (H/R) as a cell injury model, we evaluated cell survival and caspase 3/7 activity in H9c2 myoblasts after treatment with AVP. Pretreatment of H9c2 cells with AVP significantly reduced H/R-induced cell death and caspase 3/7 activity, effects that were blocked via both selective V(1A)R inhibition and mitogen-activated protein kinase (MEK1/2) inhibition. AVP increased extracellular-regulated kinase 1/2 (ERK1/2) phosphorylation in a concentration-dependent manner that was sensitive to MEK1/2 inhibition and V(1A)R inhibition, but not V(1B)R or V(2)R inhibition. Discrete elements of the V(1A)/G?(q)-protein kinase C (PKC) and V(1A)/G protein-coupled receptor kinase (GRK)/?-arrestin signaling cascades were inhibited to dissect the pathways responsible for the protective effects of V(1A)R signaling: G?(q) (overexpression of Gq-I-ires-green fluorescent protein), PKC (administration of Ro 31-82425; 2-[8-(aminomethyl)-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl]-3-(1-methyl-1H-indol-3-yl)maleimide, HCl, bisindolylmaleimide X, HCl), GRK2 [C-terminal GRK2 peptide overexpression and small interfering RNA (siRNA) knockdown], GRK5 (siRNA knockdown), and ?-arrestin1 (siRNA knockdown). These studies demonstrated that both G?(q)/PKC- and GRK2/?-arrestin1-dependent V(1A)R signaling were capable of inducing ERK1/2 phosphorylation in response to AVP stimulation. However, AVP-mediated protection against H/R was elicited only via GRK2- and ?-arrestin1-dependent signaling. These results suggest that activation of the V(1A)R in H9c2 cells mediates protective signaling via a GRK2/?-arrestin1/ERK1/2-dependent mechanism that leads to decreased caspase 3/7 activity and enhanced survival under conditions of ischemic stress. PMID:23690069

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

    PubMed

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

    2016-04-01

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

  10. Gene Expression Profiling of H9c2 Cells Subjected to H2O2-Induced Apoptosis with/without AF-HF001.

    PubMed

    Wang, Guping; Tang, Chunlei; Yan, Guijun; Feng, Bainian

    2016-02-01

    Heart failure represents a major health problem. The development of new drugs to treat this condition is essential. We previously discovered that AF-001 attenuates the cardiac defects caused by heart failure in zebrafish. In this paper, we report the identification of AF-HF001, an AF-001 derivative, and its effects on live cardiomyocytes subjected to oxidative damage. The in vitro results demonstrated that AF-HF001 attenuates the production of reactive oxygen species (ROS) and the myocardial cell apoptosis. A DNA microarray was performed to broadly analyze gene expression after H2O2 treatment with or without AF-HF001. Hierarchical clustering analysis revealed that AF-HF001 modifies the expression of certain genes (Ndufs2, Ndufb6, Ndufb8, Ndufa13, Ndufs3, Ndufs5, TPM1, MYH14, RyR1, and TIMP4) related to ROS production, cardiac contractility and extracellular matrix remodeling. AF-HF001 ameliorates oxidative damage, which may be related to the mitogen-activated protein kinase (MAPK) family and the intrinsic mitochondrial pathway. Altogether, this study suggests that AF-HF001 exhibits potential as a clinical drug candidate for the treatment of heart failure. PMID:26607605

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

    PubMed

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

    2015-11-01

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

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

    PubMed Central

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

    2015-01-01

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

  13. 17Beta-estradiol protects against oxidative stress-induced cell death through the glutathione/glutaredoxin-dependent redox regulation of Akt in myocardiac H9c2 cells.

    PubMed

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

    2006-05-12

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

  14. Overexpression of ornithine decarboxylase increases myogenic potential of H9c2 rat myoblasts.

    PubMed

    Govoni, Marco; Bonavita, Francesca; Shantz, Lisa M; Guarnieri, Carlo; Giordano, Emanuele

    2010-02-01

    Myoblast differentiation into multinuclear myotubes implies the slow-down of their proliferative drive and the expression of myogenin, an early marker of myogenic differentiation. Natural polyamines-such as putrescine, spermidine and spermine-are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. Many evidences in the literature point to their role in driving cellular differentiation processes. Here, we studied how polyamines may affect the differentiation of the myogenic cell line H9c2 into the muscle phenotype. Cell cultures were committed via a 7-day treatment with insulin which induced increase in the activity of ornithine decarboxylase, the first enzyme in the polyamine biosynthetic pathway, consistent with myogenic differentiation. To evaluate the role of polyamines in the differentiation process, cells were transfected with a plasmid overexpressing a stable ornithine decarboxylase, under control of a constitutive promoter. Overexpressing cells spontaneously differentiate into myotubes, without the need for induction with insulin; multinuclear myotubes and myogenin expression were apparent within 2 days of confluency of cultures. Polyamine depletion-by means of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase-abolished the differentiation process. These observations support the evidence that polyamines are a key step involved in differentiation of muscle cells. PMID:20013009

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

    PubMed

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

    2016-01-01

    Simvastatin is effective and well tolerated, with adverse reactions mainly affecting skeletal muscle. Important mechanisms for skeletal muscle toxicity include mitochondrial impairment and increased expression of atrogin-1. The aim was to study the mechanisms of toxicity of simvastatin on H9c2 cells (a rodent cardiomyocyte cell line) and on the heart of male C57BL/6 mice. After, exposure to 10?mol/L simvastatin for 24h, H9c2 cells showed impaired oxygen consumption, a reduction in the mitochondrial membrane potential and a decreased activity of several enzyme complexes of the mitochondrial electron transport chain (ETC). The cellular ATP level was also decreased, which was associated with phosphorylation of AMPK, dephosphorylation and nuclear translocation of FoxO3a as well as increased mRNA expression of atrogin-1. Markers of apoptosis were increased in simvastatin-treated H9c2 cells. Treatment of mice with 5mg/kg/day simvastatin for 21days was associated with a 5% drop in heart weight as well as impaired activity of several enzyme complexes of the ETC and increased mRNA expression of atrogin-1 and of markers of apoptosis in cardiac tissue. Cardiomyocytes exposed to simvastatin in vitro or in vivo sustain mitochondrial damage, which causes AMPK activation, dephosphorylation and nuclear transformation of FoxO3a as well as increased expression of atrogin-1. Mitochondrial damage and increased atrogin-1 expression are associated with apoptosis and increased protein breakdown, which may cause myocardial atrophy. PMID:25300705

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

    PubMed Central

    Behjati, Mohaddeseh; Moradi, Iman; Kazemi, Mohammad

    2015-01-01

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

  17. Phenylephrine preconditioning in embryonic heart H9c2 cells is mediated by up-regulation of SUR2B/Kir6.2: A first evidence for functional role of SUR2B in sarcolemmal KATP channels and cardioprotection

    PubMed Central

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

    2016-01-01

    ATP-sensitive K+ (KATP) channels were originally described in cardiomyocytes, where physiological levels of intracellular ATP keep them in a closed state. Structurally, these channels are composed of pore-forming inward rectifier, Kir6.1 or Kir6.2, and a regulatory, ATP-binding subunit, SUR1, SUR2A or SUR2B. SUR1 and Kir6.2 form pancreatic type of KATP channels, SUR2A and Kir6.2 form cardiac type of KATP channels, SUR2B and Kir6.1 form vascular smooth muscle type of KATP channels. The presence of SUR2B has been described in cardiomyocytes, but its functional significance and role has remained unknown. Pretreatment with phenylephrine (100 nM) for 24 h increased mRNA levels of SUR2B and Kir6.2, without affecting those levels of SUR1, SUR2A and Kir6.1 in embryonic heart H9c2 cells. Such increase was associated with increased K+ current through KATP channels and Kir6.2/SUR2B protein complexes as revealed by whole cell patch clamp electrophysiology and immunoprecipitation/Western blotting respectively. Pretreatment with phenylephrine (100 nM) generated a cellular phenotype that acquired resistance to chemical hypoxia induced by 2,4-dinitrophenol (DNP; 10 mM), which was accompanied by increased in K+ current in response to DNP (10 mM). Cytoprotection afforded by phenylephrine (100 nM) was abolished by infection of H9c2 cells with adenovirus containing Kir6.2AFA, a mutant form of Kir6.2 with largely reduced K+ conductance. Taking all together, the present findings demonstrate that the activation of α1-adrenoceptors up-regulates SUR2B/Kir6.2 to confer cardioprotection. This is the first account of possible physiological role of SUR2B in cardiomyocytes. PMID:26556311

  18. 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

  19. LKB1/AMPK pathway mediates resistin-induced cardiomyocyte hypertrophy in H9c2 embryonic rat cardiomyocytes

    PubMed Central

    LIU, PENG; CHENG, GUAN-CHANG; YE, QUN-HUI; DENG, YONG-ZHI; WU, LIN

    2016-01-01

    Resistin has been previously demonstrated to induce cardiac hypertrophy, however, the underlying molecular mechanisms of resistin-induced cardiac hypertrophy remain unclear. Using H9c2 cells, the present study investigated the liver kinase B1 (LKB1)/adenosine monophosphate-activated protein kinase (AMPK) signaling pathway for a potential role in mediating resistin-induced cardiomyocyte hypertrophy. Treatment of H9c2 cells with resistin increased cell surface area, protein synthesis, and expression of hypertrophic marker brain natriuretic peptide and β-myosin heavy chain. Treatment with metformine attenuated these effects of resistin. Furthermore, treatment with resistin decreased phosphorylation of LKB1 and AMPK, whereas pretreatment with metformin increased phosphorylation of LKB1 and AMPK that is reduced by resistin. These results suggest that resistin induces cardiac hypertrophy through the inactivation of the LKB1/AMPK cell signaling pathway. PMID:26998282

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

    PubMed

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

    2014-11-01

    Nanotechnology for the treatment and diagnosis has been emerging recently as a potential area of research and development. In the present study, selenium incorporated guar gum nanoparticles have been prepared by nanoprecipitation and characterized by transmission electron microscopy and particle size analysis. The nanoparticles were screened for antioxidant potential (metal chelation, total reducing power and hydroxyl radical scavenging activity) and were evaluated against the cell line based cardiac ischemia/reperfusion model with special emphasis on oxidative stress and mitochondrial parameters. The cell based cardiac ischemia model was employed using H9c2 cell lines. Investigations revealed that there was a significant alteration (P ? 0.05) in the innate antioxidant status (glutathione?, glutathione peroxidase?, thioredoxin reductase?, superoxide dismutase?, catalase?, lipid peroxidation?, protein carbonyl?, xanthine oxidase? and caspase 3 activity?), mitochondrial functions (reactive oxygen species generation, membrane potential, and pore opening) and calcium homeostasis (calcium ATPase and intracellular calcium overload) during both ischemia and reperfusion. For comparative evaluation, selenium, guar gum and selenium incorporated guar gum nanoparticles were evaluated for their protective properties against ischemia/reperfusion. The study reveals that selenium incorporated guar gum nanoparticles were better at protecting the cells from ischemia/reperfusion compared to selenium and guar gum nanoparticles. The potent antioxidant capability shown by the sample in in vitro assays may be the biochemical basis of its better biological activity. Further, the nanodimensions of the particle may be the additional factor responsible for its better effect. PMID:25307064

  1. 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

  2. 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

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

    PubMed

    Kazama, Kyosuke; Okada, Muneyoshi; Yamawaki, Hideyuki

    2015-02-20

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

  4. Exogenous NAD(+) supplementation protects H9c2 cardiac myoblasts against hypoxia/reoxygenation injury via Sirt1-p53 pathway.

    PubMed

    Liu, Ling; Wang, Ping; Liu, Xinwei; He, Dongwei; Liang, Canxin; Yu, Ying

    2014-04-01

    Nicotinamide adenine dinucleotide (NAD(+) ) not only transfers electrons in mitochondrial respiration, but also acts as an indispensable cosubstrate for Sirt1, the class III histone/nonhistone deacetylase. However, NAD(+) is depleted in myocardial ischemia/reperfusion (IR) injury. The objective of this study was to investigate the role of exogenous NAD(+) supplementation in hypoxia/reoxygenation (HR)-stressed H9c2 cardiac myoblasts. Firstly, the effects of distinct treating time points and doses of NAD(+) supplementation on the viability of HR-stressed H9c2 cells were detected. Secondly, intracellular NAD(+) levels in HR-stressed H9c2 cells at various extracellular NAD(+) concentrations were determined. Thirdly, the role of NAD(+) supplementation in HR-induced cell apoptosis and its relevance to Sirtuin 1-p53 pathway were investigated. Exogenous NAD(+) supplementation elevated intracellular NAD(+) level and reduced HR-induced cell death in both time- and concentration-dependent manners. It appeared that NAD(+) supplementation exerted the greatest protection when extracellular concentration ranged from 500 to 1000 μm and when NAD(+) was added immediately after reoxygenation began. NAD(+) replenishment restored Sirt1 activity, reduced the acetylation level of p53 (Lys373 & 382), and attenuated cell apoptosis in HR-stressed H9c2 cells, whereas inhibition of Sirt1 activity alleviated the effects of NAD(+) replenishment. These results indicated that exogenous NAD(+) supplementation attenuated HR-induced cell apoptosis, which was at least partly mediated by restoring Sirt1 activity and subsequently inhibiting p53 activity via deacetylating p53 at lysine 373 and 382. PMID:23384296

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

    PubMed

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

    2015-10-01

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

  6. Sirtuin-3 (SIRT3) Protein Attenuates Doxorubicin-induced Oxidative Stress and Improves Mitochondrial Respiration in H9c2 Cardiomyocytes.

    PubMed

    Cheung, Kyle G; Cole, Laura K; Xiang, Bo; Chen, Keyun; Ma, Xiuli; Myal, Yvonne; Hatch, Grant M; Tong, Qiang; Dolinsky, Vernon W

    2015-04-24

    Doxorubicin (DOX) is a chemotherapeutic agent effective in the treatment of many cancers. However, cardiac dysfunction caused by DOX limits its clinical use. DOX is believed to be harmful to cardiomyocytes by interfering with the mitochondrial phospholipid cardiolipin and causing inefficient electron transfer resulting in the production of reactive oxygen species (ROS). Sirtuin-3 (SIRT3) is a class III lysine deacetylase that is localized to the mitochondria and regulates mitochondrial respiration and oxidative stress resistance enzymes such as superoxide dismutase-2 (SOD2). The purpose of this study was to determine whether SIRT3 prevents DOX-induced mitochondrial ROS production. Administration of DOX to mice suppressed cardiac SIRT3 expression, and DOX induced a dose-dependent decrease in SIRT3 and SOD2 expression in H9c2 cardiomyocytes. SIRT3-null mouse embryonic fibroblasts produced significantly more ROS in the presence of DOX compared with wild-type cells. Overexpression of wild-type SIRT3 increased cardiolipin levels and rescued mitochondrial respiration and SOD2 expression in DOX-treated H9c2 cardiomyocytes and attenuated the amount of ROS produced following DOX treatment. These effects were absent when a deacetylase-deficient SIRT3 was expressed in H9c2 cells. Our results suggest that overexpression of SIRT3 attenuates DOX-induced ROS production, and this may involve increased SOD2 expression and improved mitochondrial bioenergetics. SIRT3 activation could be a potential therapy for DOX-induced cardiac dysfunction. PMID:25759382

  7. Levocarnitine Protects H9c2 Rat Cardiomyocytes from H2O2-induced Mitochondrial Dysfunction and Apoptosis

    PubMed Central

    Mao, Cui-Ying; Lu, Hai-Bin; Kong, Ning; Li, Jia-Yu; Liu, Miao; Yang, Chun-Yan; Yang, Ping

    2014-01-01

    Background: Although the protective effects of levocarnitine in patients with ischemic heart disease are related to the attenuation of oxidative stress injury, the exact mechanisms involved have yet to be fully understood. Our aim was to investigate the potential protective effects of levocarnitine pretreatment against oxidative stress in rat H9c2 cardiomyocytes. Methods: Cardiomyocytes were exposed to H2O2 to create an oxidative stress model. The cells were pretreated with 50, 100, or 200 μM levocarnitine for 1 hour before H2O2 exposure. Results: H2O2 exposure led to significant activation of oxidative stress in the cells, characterized by reduced viability, increased intracellular reactive oxygen species, lipid peroxidation, and reduced intracellular antioxidant activity. Mitochondrial dysfunction was also observed following H2O2 exposure, reflected by the loss of mitochondrial transmembrane potential and intracellular adenosine triphosphate. These pathophysiological processes led to cardiomyocyte apoptosis through activation of the intrinsic apoptotic pathway. More importantly, the levocarnitine pretreatment attenuated the H2O2-induced oxidative injury significantly, preserved mitochondrial function, and partially prevented cardiomyocyte apoptosis during the oxidative stress reaction. Western blotting analyses suggested that levocarnitine pretreatment increased plasma protein levels of Bcl-2, reduced Bax, and attenuated cytochrome C leakage from the mitochondria in the cells. Conclusion: Our in vitro study indicated that levocarnitine pretreatment may protect cardiomyocytes from oxidative stress-related damage. PMID:25170293

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

    PubMed

    Yun, J S; Kim, S Y

    2015-08-01

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

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

    PubMed

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

    2015-02-01

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

  10. Gui-ling-gao, a traditional Chinese functional food, prevents oxidative stress-induced apoptosis in H9c2 cardiomyocytes.

    PubMed

    Li, Fan; Wu, Jian-Hong; Wang, Qing-Hua; Shu, Yuan-Lan; Wan, Chun-Wai; Chan, Chi-On; Kam-Wah Mok, Daniel; Chan, Shun-Wan

    2013-04-30

    Functional foods have become an increasingly popular alternative to prevent diseases and maintain body health status. Gui-ling-gao (GLG, also known as turtle jelly) is a well-known traditional functional food popular in Southern China and Hong Kong. This study aimed to investigate the antioxidative and anti-apoptotic effects of GLG, a traditional Chinese functional food, on preventing oxidative stress-induced injury in H9c2 cardiomyocytes. In this study, the antioxidative capacities of GLG were measured by using both a cell-free assay [2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl assay] and biological methods [2,2'-azobis(2-amidinopropane)-induced haemolysis assay and H(2)O(2)-induced cell damage on H9c2 cardiomyocytes]. Additionally, the total phenolic content was measured using the Folin-Ciocalteu method. Furthermore, the anti-apoptotic effect of GLG was evaluated by nuclear staining and a DNA fragmentation assay. GLG was found to have good antioxidant activities and high total phenolic content. In H(2)O(2)-induced cell damage on H9c2 cells, GLG was demonstrated to ameliorate the apoptotic effects, such as nuclear condensations, increased intracellular caspase-3 activity and inter-nucleosomal DNA cleavage, induced by H(2)O(2). The present study demonstrated for the first time that GLG possesses anti-apoptotic potential in vitro and this effect may be mediated, in part, by its antioxidative function. Additionally, the antioxidative capacities of GLG were proved both chemically and biologically. This study provides scientific evidence to prove the anecdotal health-beneficial claim that the consumption of GLG could help the body to handle endogenous toxicants such as free radicals. PMID:23467630

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

    PubMed

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

    2016-04-01

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

  12. l-carnosine and verapamil inhibit hypoxia-induced expression of hypoxia inducible factor (HIF-1 alpha) in H9c2 cardiomyoblasts.

    PubMed

    Bharadwaj, Lalita A; Davies, Gerald F; Xavier, Ilungo J; Ovsenek, Nick

    2002-03-01

    Contractile failure of myocardial cells is a common cause of mortality in ischemic heart disease. In response to hypoxic conditions, cells upregulate the activity of hypoxia-inducible factor 1 (HIF-1) and express a number of genes encoding proteins that either enhance O (2)delivery or increase cellular ATP levels. HIF-1 is a heterodimer of bHLH-PAS proteins, HIF-1 alpha and HIF-1 beta. Both subunits are constitutively expressed under normoxic conditions, but HIF-1 alpha levels are kept low by proteolytic degradation, then stabilized under conditions of low O (2)by a mechanism that is poorly understood. Here we tested the hypothesis that expression of HIF-1 alpha in cardiac cells may be affected by two known cardioprotective agents. We tested l-carnosine, a naturally occurring dipeptide which has been shown to improve myocardial contractility during hypoxia, and verapamil, a calcium channel blocker frequently prescribed for the treatment of heart disease. The levels of HIF-1 alphamRNA remained relatively stable during time course hypoxia (1% O (2)) in H9c2 cardiomyoblasts, then increased slightly after 24 h. In cells pretreated with 1 microM carnosine, the levels of mRNA were transiently reduced, but then increased after 24 h similar to the controls. The levels of HIF-1 alpha protein increased rapidly in H9c2 cells within 30 min of hypoxia, but this induction was significantly reduced in cells treated with either carnosine or verapamil. In addition, treatment of cells with these agents further reduced the low levels of HIF-1 under normoxic conditions. These results suggest that l-carnosine and verapamil may affect the regulated proteolytic degradation of HIF-1 alpha in heart cells during hypoxia. PMID:11884212

  13. [Study on protective effect of Sijunzi decoction (red/white ginseng) on H2O2-induced H9c2 cardiomyocytes apoptosis].

    PubMed

    Sun, Na; Xu, Gang; Zhang, Fan; Xu, Shan; Liu, Peng-peng; Jia, Tian-zhu

    2015-05-01

    In this study, efforts were made to screen out the drug concentration of Sijunzi decoction (red ginseng) for in vitro intervention of H9c2 cardiomyocytes, select high, medium and low groups for subsequent experiments, establish the H2O2-induced myocardial cell apoptosis to investigate the protective effect of Sijunzi decoction (white/red ginseng), provide reference ginseng ingredients in Sijunzi decoction used to treat ischemic heart disease and reflect its curative effect, and observe its impacts on SOD, MAD, LDH and other indexes to preliminarily define the action mechanism. According to the results, red ginseng in Sijunzi decoction showed a better protective effect on H2O2-induced myocardial cell injury than that of white ginseng. Both of them could enhance SOD activity and reduce MDA production and LDH release, so as to significantly reduce the amount of apoptotic myocardial cells and play protective role. PMID:26390661

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

    PubMed Central

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

    2013-01-01

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

  15. The protective effect of lycopene on hypoxia/reoxygenation-induced endoplasmic reticulum stress in H9C2 cardiomyocytes.

    PubMed

    Gao, Yang; Jia, Pengyu; Shu, WenQi; Jia, Dalin

    2016-03-01

    Nowadays, drugs protecting ischemia/reperfusion (I/R) myocardium become more suitable for clinic. It has been confirmed lycopene has various protections, but lacking the observation of its effect on endoplasmic reticulum stress (ERS)-mediated apoptosis caused by hypoxia/reoxygenation (H/R). This study aims to clarify the protective effect of lycopene on ERS induced by H/R in H9C2 cardiomyocytes. Detect the survival rate, lactic dehydrogenase (LDH) activity, apoptosis ratio, glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP), c-Jun-N-terminal protein Kinase (JNK) and Caspase-12 mRNA and protein expression and phosphorylation of JNK (p-JNK) protein expression. LDH activity, apoptosis ratio and GRP78 protein expression increase in the H/R group, reduced by lycopene. The survival rate reduces in the H/R and thapsigargin (TG) groups; lycopene and 4-phenyl butyric acid (4-PBA) can improve it caused by H/R, lycopene also can improve it caused by TG. The apoptosis ratio, the expression of GRP78, CHOP and Caspase-12 mRNA and protein and p-JNK protein increase in the H/R and TG groups, weaken in the lycopene+H/R, 4-PBA+H/R and lycopene+TG groups. There is no obvious change in the expression of JNK mRNA or protein. Hence, our results provide the evidence that 10μM lycopene plays an obviously protective effect on H/R H9C2 cardiomyocytes, realized through reducing ERS and apoptosis. The possible mechanism may be related to CHOP, p-JNK and Caspase-12 pathways. PMID:26845695

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

    SciTech Connect

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

    2013-04-01

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

  17. 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

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

    PubMed

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

    2015-05-01

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

  19. Hydrogen sulfide attenuates doxorubicin-induced cardiotoxicity by inhibiting reactive oxygen species-activated extracellular signal-regulated kinase 1/2 in H9c2 cardiac myocytes.

    PubMed

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

    2015-11-01

    Doxorubicin (DOX) is a potent and available antitumor therapeutic agent; however, its clinical application is limited due to its cardiotoxicity. Preliminary evidence suggests that hydrogen sulfide (H2S) may exert protective effects on DOX‑induced cardiotoxicity. Therefore, the aim of the present study was to investigate whether the extracellular signal‑regulated kinase (ERK) 1/2 signaling pathway is involved in the cardioprotection of H2S against DOX‑induced cardiotoxicity. The present study demonstrated that pretreatment with sodium hydrosulfide (NaHS; a donor of H2S) prior to DOX exposure attenuated the decreased cell viability, the increased apoptosis rate and the intracellular accumulation of reactive oxygen species (ROS) in H9c2 cardiac myocytes. Exposure of H9c2 cardiac myocytes to DOX upregulated the expression levels of phosphorylated ERK1/2, which had been reduced by pretreatment with NaHS or N‑acetyl‑L‑cysteine, a ROS scavenger. In addition, H2S upregulated the anti‑apoptotic protein, Bcl‑2 and downregulated the pro‑apoptotic protein, Bax. Notably, U0126, a selective inhibitor of ERK1/2, was observed to mimic the above‑mentioned cytoprotective activity of H2S. In conclusion, these findings indicate that H2S attenuates DOX‑induced cardiotoxicity by inhibiting ROS-mediated activation of ERK1/2 in H9c2 cardiac myocytes. PMID:26299281

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

    PubMed

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

    2016-02-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-10-01

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

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

    PubMed

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

    2015-07-01

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

  4. Cell Lines

    PubMed Central

    Cherbas, Lucy; Gong, Lei

    2014-01-01

    We review the properties and uses of cell lines in Drosophila research, emphasizing the variety of lines, the large body of genomic and transcriptional data available for many of the lines, and the variety of ways the lines have been used to provide tools for and insights into the developmental, molecular, and cell biology of Drosophila and mammals. PMID:24434506

  5. Simultaneous expression of cardiac and skeletal muscle isoforms of the L-type Ca2+ channel in a rat heart muscle cell line.

    PubMed Central

    Meja-Alvarez, R; Tomaselli, G F; Marban, E

    1994-01-01

    1. We have investigated the identity of the L-type Ca2+ channels present in the H9c2 myoblast line derived from embryonic rat ventricle. To this end, we characterized macroscopic and unitary Ba2+ currents through Ca2+ channels, and looked for specific genetic messages encoding different L-type Ca2+ channel isoforms. 2. The macroscopic Ba2+ current (recorded in 10 mM BaCl2) revealed two components with different time courses of activation. The fast component (IBa,fast) activates with a time constant of 23 +/- 12 ms (at +10 mV), while the slow component activates with a time constant of 125 +/- 12 ms (at +10 mV). 3. Single-channel recordings revealed the presence of two independent channels with conductance values of 11 and 25 pS (in 70 mM Ba2+). These values are identical to those reported previously for skeletal muscle and cardiac Ca2+ channels, respectively. 4. The mean ensemble current from the 11 pS channel reproduced the time course of the slow component observed at the macroscopic level, while the 25 pS ensemble time course paralleled that of the fast component. 5. Reverse transcriptase polymerase chain reaction (PCR) with alpha 1-isoform-specific primers revealed the presence of two distinct transcripts in H9c2 cells. The sequences of the PCR products showed a high degree of homology with the corresponding segments of the rabbit cardiac and skeletal muscle L-type Ca2+ channel isoforms. Adult rat skeletal and cardiac muscle expressed only one type of transcript. 6. H9c2 cells appear to be unique in that they simultaneously express both skeletal muscle and cardiac isoforms of the L-type Ca2+ channel alpha 1-subunit. Thus, the H9c2 cell line may prove to be useful when studying the regulation of subtype-specific Ca2+ channel gene expression. Images Figure 8 PMID:7525945

  6. A novel highly selective adenosine A1 receptor agonist VCP28 reduces ischemia injury in a cardiac cell line and ischemia-reperfusion injury in isolated rat hearts at concentrations that do not affect heart rate.

    PubMed

    Urmaliya, Vijay B; Pouton, Colin W; Devine, Shane M; Haynes, John M; Warfe, Lyndon; Scammells, Peter J; White, Paul J

    2010-09-01

    The cardioprotective effects of a novel adenosine A1 receptor agonist N6-(2,2,5,5-tetramethylpyrrolidin-1-yloxyl-3-ylmethyl) adenosine (VCP28) were compared with the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) in a H9c2(2-1) cardiac cell line-simulated ischemia (SI) model (12 hours) and a global ischemia (30 minutes) and reperfusion (60 minutes) model in isolated rat heart model. H9c2(2-1) cells were treated with CPA and VCP28 at the start of ischemia for entire ischemic duration, whereas isolated rat hearts were treated at the onset of reperfusion for 15 minutes. In the H9c2(2-1) cells SI model, CPA and VCP28 (100 nM) significantly (P < 0.05, n = 5-6) reduced the proportion of nonviable cells (30.88% +/- 2.49% and 16.17% +/- 3.77% of SI group, respectively) and lactate dehydrogenase efflux. In isolated rat hearts, CPA and VCP28 significantly (n = 6-8, P < 0.05) improved post-ischemic contractility (dP/dt(max), 81.69% +/- 10.96%, 91.07% +/- 19.87% of baseline, respectively), left ventricular developed pressure, and end diastolic pressure and reduced infarct size. The adenosine A1 receptor antagonist abolished the cardioprotective effects of CPA and VCP28 in SI model and isolated rat hearts. In conclusion, the adenosine A1 receptor agonist VCP28 has equal cardioprotective effects to the prototype A1 agonist CPA at concentrations that have no effect on heart rate. PMID:20571427

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

    NASA Technical Reports Server (NTRS)

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

    1989-01-01

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

  8. Stable high level expression of a transfected human HSP70 gene protects a heart-derived muscle cell line against thermal stress.

    PubMed

    Heads, R J; Latchman, D S; Yellon, D M

    1994-06-01

    Heat shock protein 70 (HSP70) has been shown to play a fundamental role in the induction of thermotolerance in many biological systems. Elevated synthesis of HSP70 in response to diverse stresses such as heat, anoxia, ischaemia, ethanol and heavy metals has been correlated with protection against subsequent more severe stress and cross-tolerance to differing stresses. In this respect, exposure of the mammalian heart to sublethal heat treatment or ischaemia has been shown to protect against subsequent myocardial ischaemia with concomitant elevation of HSP70. However, direct demonstration that HSP70 can alone confer thermotolerance has until recently been restricted to transfection of fibroblasts with an HSP70 gene, although preliminary data from others suggests that transfection of H9c2 myocytes with an HSP70 gene can confer tolerance to substrate-free hypoxia. The purpose of this study was, therefore, to test directly whether a myocyte cell line which retains certain features of cardiac cells (as opposed to non-myocyte cells) can be protected against lethal thermal stress by transfection with a single HSP70 gene. Rat heart-derived H9c2 cells were transfected either with a plasmid from which high level expression of a human HSP70 gene is driven by a strong, heterologous (human) beta-actin promoter (APr-HS70), or with an analogous control plasmid containing no HSP70 gene (pAPr-1 neo). Following selection with the neomycin analogue G418, several clones were isolated which either expressed no HSP70 (control: pAPr-1 neo-derived) or stably expressed high constitutive levels of an inducible isoform of HSP70 (HSP70i) (APr-HS70-derived) as determined by Western blotting with a specific monoclonal antibody to HSP70i.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8089850

  9. CLO: The cell line ontology

    PubMed Central

    2014-01-01

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

  10. Available Lepidopteran Insect Cell Lines.

    PubMed

    Lynn, Dwight E; Harrison, Robert L

    2016-01-01

    This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. More than 320 lines from 65 species are listed. The official designation is given for each cell line as well as the species, tissue source, and, when known, the susceptibilities to baculoviruses. PMID:26820856

  11. Thyroid cell lines in research on goitrogenesis.

    PubMed

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

    1991-12-01

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

  12. Angiotensin II-induced p53-dependent cardiac apoptotic cell death: its prevention by metallothionein.

    PubMed

    Liu, Qiuju; Wang, Guanjun; Zhou, Guihua; Tan, Yi; Wang, Xiuli; Wei, Wei; Liu, Lucheng; Xue, Wanli; Feng, Wenke; Cai, Lu

    2009-12-15

    Apoptotic cell death was found to play a critical role in the development of diabetic cardiomyopathy. As one of pathogenic components of diabetes angiotensin II (Ang II) induced cardiac cell death in vitro and in vivo through induction of reactive oxygen and nitrogen species. However, Ang II-induced cell death signaling in the heart remains unclear. The present study was to investigate whether Ang II induces p53 expression and activation and if so, whether Ang II-induced cardiac cell death is p53-dependent, and whether a potent antioxidant metallothionein (MT) prevents Ang II-induced p53 expression, and associate apoptotic cell death signaling. A cardiac cell line (H9c2) was exposed to Ang II. We found that exposure of H9c2 cells to Ang II at 10, 50 and 100 nM for 24 h induced a significant apoptotic effect, measured by DNA fragmentation and cleaved caspase-3. Induction of apoptotic cell death by Ang II can be completely blocked by p53 inhibitor Pitithrin-alpha. Exposure of H9c2 cells to Ang II also significantly increased p53 phosphorylation, DNA double strand breaks and Bax/Bcl-2 ratio. All these effects were not observed in H9c2MT7 cells that forcedly overexpresses human MT-IIA gene, suggesting the preventive effect of antioxidant MT against Ang II-induced p53 activation and its apoptotic death signaling. Furthermore, the in vitro finding was validated in animal models by supplying Ang II to wild-type mice (WT) and MT-TG mice that has cardiac-specifically overexpressed MT gene. Ang II-induced significant up-regulation of p53 expression along with an increase in Bax/Bcl-2 ratio in the hearts of WT mice, but not MT-TG mice. These results suggest that Ang II-induced cardiac apoptotic cell death is mediated by p53 apoptotic signaling pathway, which is related to oxidative stress. Antioxidant MT can completely prevent Ang II-induced p53 activation and associated apoptotic effect in the heart. PMID:19808082

  13. Comparative Analysis of αB-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 αB-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 αB-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 αB-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 αB-crystallin is mobilized early after exposure to a high temperature to interact with damaged proteins but that the myocardial cells cannot produce sufficient αB-crystallin for protection against heat stress. Lower αB-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, αB-crystallin is a potential biomarker of heat stress. PMID:24466295

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

    PubMed Central

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

    2013-01-01

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

  15. Cell line: 2004-2014.

    PubMed

    2014-11-20

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

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

    PubMed

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

    2016-03-01

    Bis(indolyl)methane appended biaryls were designed, synthesized and evaluated in human cervical cancer cell lines (HeLa) for their anticancer activities and compared against normal rat cardiac myoblasts (H9C2) cells. Compounds 1-12 were synthesized, with variations in one of the phenyl unit, in a single step by condensation of biaryl-2-carbaldehydes with indole in the presence of para-toluenesulfonic acid. Compound 1 exhibited a GI50 value of 11.000.707?M and the derivatives, compounds 4 and 11 showed a GI50 value of 8.330.416?M and 9.130.177?M respectively in HeLa cells and was found to be non-toxic to H9C2 cells up to 20?M. Furthermore, compounds 1, 4 and 11 induced caspase dependent cellular apoptosis in a concentration-dependent manner, reduced mitochondrial membrane potential, inhibited the cell migration and downregulated the production of MMP-2 and MMP-9 in HeLa cells. PMID:26807764

  17. Refractory lining for electrochemical cell

    SciTech Connect

    Blander, M.; Cook, G.M.

    1987-08-18

    This patent describes an apparatus for processing a melt of molten iron in contact with a molten slag containing iron oxide, the apparatus consists of melt containing means including an electrically conductive refractory lining disposed for contact with an iron oxide containing melt, an anode in the melt containing means electrically separated from the refractory lining, and means for establishing a voltage between the refractory lining as cathode and the anode to reduce iron oxide to iron at the surface of the refractory lining in contact with the iron oxide containing melt, the refractory lining including a metal oxide selected from the group consisting of Mg chromites and MgO.

  18. Immortalization of Mouse Germ Line Stem Cells

    PubMed Central

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

    2011-01-01

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

  19. HLA expression in hepatocellular carcinoma cell lines.

    PubMed Central

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

    1994-01-01

    The present study undertook to investigate the biological significance of human leucocyte antigen expression in hepatocellular carcinoma and to elucidate the role of potential modulating agents on human leucocyte antigen expression. These studies used several hepatic tumour-derived cell lines as in vitro model systems. The cell lines included PLC/PRF/5 (Alexander cell line), Hep3B, HepG2, TONG PHC, HA22T/VGH, HA59T/VGH and Mahlavu. The cell lines K562 and Raji were used as negative and positive controls, respectively. K562, a B lymphoid-derived cell line, was shown to express negligible amounts of human leucocyte antigens, while Raji, an erythromyeloid-derived cell line, expressed both class I and class II human leucocyte antigens as well as their respective invariant chains, beta 2-microglobulin and Ii. Using an ELISA, experiments performed on these cell lines confirmed the natural expression of class I and class II antigens by the HA22T/VGH and HA59T/VGH cell lines, whereas PLC/PRF/5 displayed class II surface antigens only. The effects of modulating agents such as interferon-gamma sodium butyrate and clofazimine on human leucocyte antigen expression were investigated using the HA22T/VGH, HA59T/VGH and TONG PHC cell lines. These agents increased class II and class II human leucocyte antigen expression on HA22T/VGH and TONG PHC cells, but had no effect on the HA59T/VGH cell line. The results suggest a potential use for these agents as modulators of human leucocyte antigen expression by human heptocellular cell lines. PMID:8050184

  20. 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

  1. Biophysical Profiling of Tumor Cell Lines

    PubMed Central

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

    2011-01-01

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

  2. Refractory lining for electrochemical cell

    SciTech Connect

    Blander, Milton; Cook, Glenn M.

    1987-01-01

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

  3. Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

    PubMed

    Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

    2009-01-01

    The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines. PMID:18927105

  4. 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

  5. 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....

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

    SciTech Connect

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

    2011-04-01

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

  7. 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 μg/ml and that IC50 value on Hep 2 cell line was 112 μg whereas 94 μg for Vero cell line. Hence, T. divaricata has lesser significant action on Vero cell line. Conclusion: Medicinal plant drug discovery continues to provide new and important leads against various pharmacological targets including cancer. Our results clearly indicate the anticancer property of the medicinal plant T. divaricata against the human laryngeal carcinoma cell lines (Hep 2 cell line). PMID:26109773

  8. Chromosomal variation in lymphoblastoid cell lines

    PubMed Central

    Shirley, Matthew D.; Baugher, Joseph D.; Stevens, Eric L.; Tang, Zhenya; Gerry, Norman; Beiswanger, Christine M.; Berlin, Dorit S.; Pevsner, Jonathan

    2012-01-01

    Tens of thousands of lymphoblastoid cell lines (LCLs) have been established by the research community, providing nearly unlimited source material from samples of interest. LCLs are used to address questions in population genomics, mechanisms of disease, and pharmacogenomics. Thus, it is of fundamental importance to define the extent of chromosomal variation in LCLs. We measured variation in genotype and copy number in multiple LCLs derived from peripheral blood mononuclear cells (PBMCs) of single individuals as well as two comparison groups: (1) three types of differentiated cell lines (DCLs) and (2) triplicate HapMap samples. We then validated and extended our findings using data from a large study consisting of samples from blood or LCLs. We observed high concordances between genotypes and copy number estimates within all sample groups. While the genotypes of LCLs tended to faithfully reflect the genotypes of PBMCs, 13.7% (4 of 29) of immortalized cell lines harbored mosaic regions greater than 20 megabases which were not present in PBMCs, DCLs, or HapMap replicate samples. We created a list of putative LCL-specific changes (affecting regions such as immunoglobulin loci) that is available as a community resource. PMID:22374857

  9. A human gallbladder adenocarcinoma cell line.

    PubMed

    Johzaki, H; Iwasaki, H; Nishida, T; Isayama, T; Kikuchi, M

    1989-12-01

    A cell strain (FU-GBC-1) was established from cancerous ascites of a 68-year-old male patient with well-differentiated adenocarcinoma of the gallbladder. By light and electron microscopy, the cultured cells showed the morphologic features of adenocarcinoma characterized by gland-like structures, intracellular microcystic spaces, and mucous production. Immunoperoxidase stains showed that FU-GBC-1 cells expressed several epithelial tumor antigens including CA 19-9, carcinoembryonic antigen (CEA), and epithelial membrane antigen (EMA). The cell strain has been in continuous culture up to passage 44 for 1 1/2 years, with the population doubling time of 120 hours. The cytogenetic analysis by a G-band technique showed a constant loss of chromosome Y in FU-GBC-1 cells. The modal chromosome number at passage 12 was 82 with a range of 77 to 85. Flow cytometry with an ethidium bromide technique additionally confirmed aneuploid DNA content (4C) in the cultured cells at passage 12 and 35. Inoculation of FU-GBC-1 cells into the dermis of BALB/c nude mice produced transplantable adenocarcinoma identical to the original tumor. Because no continuous cell lines of the well-differentiated type of gallbladder adenocarcinoma have been reported in the literature currently, the newly established cell strain we report may yield a useful system for studying the morphologic and biologic characteristics of gallbladder adenocarcinoma. PMID:2680052

  10. Anomalous dystroglycan in carcinoma cell lines.

    PubMed

    Losasso, C; Di Tommaso, F; Sgambato, A; Ardito, R; Cittadini, A; Giardina, B; Petrucci, T C; Brancaccio, A

    2000-11-10

    Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non-tumorigenic mammary human cell lines, the expected beta-dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous approximately 31 kDa beta-dystroglycan band. alpha-Dystroglycan was udetectable in most of the cell lines in which beta-dystroglycan was found as a approximately 31 kDa species. An anomalous approximately 31 kDa beta-dystroglycan band was also observed in N-methyl-N-nitrosurea-induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low- and high-ionic strength, we demonstrated that both the 43 kDa and approximately 31 kDa beta-dystroglycan bands harbour their transmembrane segment. PMID:11078877

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

    PubMed

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

    2015-04-16

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

  12. Histone signature of metanephric mesenchyme cell lines

    PubMed Central

    McLaughlin, Nathan; Yao, Xiao; Li, Yuwen; Saifudeen, Zubaida; El-Dahr, Samir S

    2013-01-01

    The metanephric mesenchyme (MM) gives rise to nephrons, the filtering units of the mature kidney. The MM is composed of uninduced (Six2high/Lhx1low) and induced (Wnt-stimulated, Six2low/Lhx1high) cells. The global epigenetic state of MM cells is unknown, partly due to technical difficulty in isolating sufficient numbers of homogenous cell populations. We therefore took advantage of two mouse clonal cell lines representing the uninduced (mK3) and induced (mK4) metanephric mesenchyme (based on gene expression profiles and ability to induce branching of ureteric bud). ChIP-Seq revealed that whereas H3K4me3 active region peaks are enriched in metabolic genes, H3K27me3 peaks decorate mesenchyme and epithelial cell fate commitment genes. In uninduced mK3 cells, promoters of stemness genes (e.g., Six2, Osr1) are enriched with H3K4me3 peaks; these are lost in induced mK4 cells. ChIP-qPCR confirmed this finding and further demonstrated that G9a/H3K9me2 occupy the promoter region of Six2 in induced cells, consistent with the inactive state of transcription. Conversely, genes that mark the induced epithelialized state (e.g., Lhx1, Pax8), transition from a non-permissive to an active chromatin signature in mK3 vs. mK4 cells, respectively. Importantly, stimulation of Wnt signaling in uninduced mK3 cells provokes an active chromatin state (high H3K4me3, low H3K27me3), recruitment of ?-catenin, and loss of pre-bound histone methyltransferase Ezh2 in silent induced genes followed by activation of transcription. We conclude that the chromatin signature of uninduced and induced cells correlates strongly with their gene expression states, suggesting a role of chromatin-based mechanisms in MM cell fate. PMID:23867747

  13. Permanently Blocked Stem Cells Derived from Breast Cancer Cell Lines

    PubMed Central

    Sajithlal, Gangadharan B.; Rothermund, Kristi; Zhang, Fang; Dabbs, David J.; Latimer, Jean J.; Grant, Stephen G.; Prochownik, Edward V.

    2016-01-01

    Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. An unanticipated result, however, was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immuno-compromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs. PMID:20506227

  14. 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.

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

    PubMed

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

    2010-04-01

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

  16. Stable mammalian producer cell lines for structural biology.

    PubMed

    Bssow, Konrad

    2015-06-01

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

  17. In vitro radiosensitivity of human leukemia cell lines

    SciTech Connect

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (anti n, D/sub 0/) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL60 promyelocytic leukemia; K562 erythroleukemia; 45 acute lymphocytic leukemia; and 176 acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2006-01-01

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

  20. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

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

  1. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    1989-01-01

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

  2. 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...

  3. Culturing hybridoma cell lines for monoclonal antibody production.

    PubMed

    Winzeler, Alissa; Wang, Jack T

    2013-07-01

    This protocol describes how to culture hybridoma cell lines (e.g., Thy1.1) for monoclonal antibody production. Supernatants harvested from such cultures can be used to purify various rodent neural cell types by immunopanning. PMID:23818668

  4. Expression of liver functions in immortalised rat hepatocyte cell lines.

    PubMed

    MacDonald, C; Vass, M; Willett, B; Scott, A; Grant, H

    1994-06-01

    The differentiated hepatic function of two rat liver cell lines, P9 and SV40RH1, immortalised by transfection with SV40 DNA has been investigated in terms of the glutathione synthesis, and the activities of gamma-glutamyltransferase, glutathione-S-transferase and UDP-glucuronosyltransferase. SV40RH1 is a highly differentiated cell line at early passage, but the expression of some aspects of its differentiated phenotype is unstable and some functions are lost by passage 12-13. P9 is a less-well differentiated cell line, with relatively stable expression of functions between passages 4 and 13. In terms of differentiated function both cell lines represent a marked improvement over primary cultures of rat hepatocytes which de-differentiate rapidly within 24-48 h in culture. This retention of liver function in proliferating cell lines offers the opportunity to use such cells in in vitro toxicological studies. PMID:8086228

  5. Genotyping of Rhesus SCNT pluripotent stem cell lines.

    PubMed

    Cram, David S; Song, Bi; Trounson, Alan O

    2007-11-22

    Somatic cell nuclear transfer (SCNT) into enucleated oocytes has emerged as a technique that can be used to derive mouse embryonic stem cell lines with defined genotypes. In this issue Byrne et al. report the derivation of two SCNT Rhesus macaca male stem cell lines designated CRES-1 and CRES-2. Molecular studies detailed in their paper provides supporting evidence that the chromosome complement of CRES-1 and CRES-2 was genetically identical to the male cell donor nucleus and that the mitochondrial DNA originated from different recipient oocytes. In this validation paper, we independently confirm that both stem cell lines were indeed derived by SCNT. PMID:18004280

  6. The ultrastructure of lymphoblastoid cell lines from Marek's disease lymphomata.

    PubMed

    Frazier, J A; Powell, P C

    1975-01-01

    The ultrastructure of two lymphoblastoid cell lines derived from Marek's disease lymphomata has been studied. The cells varied from 5 to 12 mum in diameter and had large round or oval nuclei. A nucleolus was occasionally present and about 3% of cells showed projections of the nuclear envelope. The cytoplasm contained many ribosomes and several mitochondria but endoplasmic reticulum was sparse. A small number of cells contained annulate lamellae and crystalline structures were occasionally seen. Cells with immature intranuclear herpesvirus particles were rarely present. The cells had many ultrastructural features in common with Burkitt's lymphoma-derived cell lines. PMID:1156510

  7. Analysis of gene amplification in human tumor cell lines

    SciTech Connect

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

    1988-09-01

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

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

    SciTech Connect

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

    2012-01-15

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

  9. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ...The National Institute of Standards and Technology (NIST) Biochemical Science Division announces its intent to identify by short tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human Cell Lines Project. All data and corresponding information will be posted in a publically held database at the National Center For Biotechnology Information...

  10. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines.

    PubMed

    Eckerle, Isabella; Corman, Victor M; Mller, Marcel A; Lenk, Matthias; Ulrich, Rainer G; Drosten, Christian

    2014-02-01

    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

  11. Characterization of rainbow trout cell lines using microsatellite DNA profiling.

    PubMed

    Perry, G M; McDonald, G J; Ferguson, M M; Ganassin, R C; Bols, N C

    2001-11-01

    Ten microsatellite loci (Omy27DU,Omy325(A3)UoG, OmyFGT5TUF,OmyFGT14TUF, OmyFGT15TUF,OmyFGT23TUF, Omy77DU,Ssa20.19NUIG, Ots1BML, andOne18ASC) were amplified using the polymerase chain reaction to create genetic profiles for nine cell lines (RTG-2, RTH-149,RTL-W1,RTgill-W1, RTS-11, RTS-34st, RTP-2, RTP-91E and RTP-91F) from rainbow trout(Oncorhynchus mykiss) and one cell line (CHSE-214) from Chinook salmon (O. tschawytscha). A cell line (PHL) from anon-salmonid, the Pacific herring (Clupea harengus pallasi), was included as a control. The ten loci clearly revealed the uniqueness of each cell line, except for two cell lines (RTP-91E andRTP-91F) from the same fish. RTP-91E and RTP-91F were identical at all loci except Ssa20.19NUIG. The most useful locus for demonstrating uniqueness was Ots1BML. The information was used to demonstrate that an uncharacterized rainbow trout cell line (Clone 1A)was in fact CHSE-214, illustrating the usefulness of multiplexed microsatellites for the creation of genetic profiles for salmonid cell lines and for the testing of cell line cross-contamination. PMID:19002917

  12. The transcriptional diversity of 25 Drosophila cell lines

    SciTech Connect

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

    2010-11-15

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

  13. Phenotype and Genotype of Pancreatic Cancer Cell Lines

    PubMed Central

    Deer, Emily L.; Gonzalez-Hernandez, Jessica; Coursen, Jill D.; Shea, Jill E.; Ngatia, Josephat; Scaife, Courtney L.; Firpo, Matthew A.; Mulvihill, Sean J.

    2009-01-01

    The dismal prognosis of pancreatic adenocarcinoma (PA) is due in part due to a lack of molecular information regarding disease development. Established cell lines remain a useful tool for investigating these molecular events. Here we present a review of available information on commonly used PA cell lines as a resource to help investigators select the cell lines most appropriate for their particular research needs. Information on clinical history, in vitro and in vivo growth characteristics, phenotypic characteristics, such as adhesion, invasion, migration and tumorigenesis, and genotypic status of commonly altered genes (KRAS, p53, p16, and SMAD4) was evaluated. Identification of both consensus and discrepant information in the literature suggests careful evaluation before selection of cell lines and attention be given to cell line authentication. PMID:20418756

  14. Establishment and culture of leukemia-lymphoma cell lines.

    PubMed

    Drexler, Hans G

    2011-01-01

    The advent of continuous human leukemia-lymphoma cell lines as a rich resource of abundant, accessible, and manipulable living cells has contributed significantly to a better understanding of the pathophysiology of hematopoietic tumors. The first leukemia-lymphoma cell lines were established in 1963 and since then large numbers of new cell lines have been described. The major advantages of continuous leukemia-lymphoma cell lines are the unlimited supply and worldwide availability of identical cell material and the infinite viable storability in liquid nitrogen. These cell lines are characterized generally by monoclonal origin and differentiation arrest, sustained proliferation in vitro under preservation of most cellular features, and by specific genetic alterations. Here some of the more promising techniques for establishing new leukemia-lymphoma cell lines and the basic principles for culturing these cells are described. Several clinical and cell culture parameters might have some influence on the success rate, e.g., choice of culture medium and culture conditions, specimen site of the primary cells, and status of the patient at the time of sample collection. PMID:21516408

  15. Deriving cell lines from zebrafish embryos and tumors.

    PubMed

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

    2013-09-01

    Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb(-/-) embryos and an endothelial-like cell line from a tumor of an adult ptena(+/-)ptenb(-/-) zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 3-5 months. PMID:23672287

  16. Deriving Cell Lines from Zebrafish Embryos and Tumors

    PubMed Central

    Choorapoikayil, Suma; Overvoorde, John

    2013-01-01

    Abstract Over the last two decades the zebrafish has emerged as a powerful model organism in science. The experimental accessibility, the broad range of zebrafish mutants, and the highly conserved genetic and biochemical pathways between zebrafish and mammals lifted zebrafish to become one of the most attractive vertebrate models to study gene function and to model human diseases. Zebrafish cell lines are highly attractive to investigate cell biology and zebrafish cell lines complement the experimental tools that are available already. We established a straightforward method to culture cells from a single zebrafish embryo or a single tumor. Here we describe the generation of fibroblast-like cell lines from wild-type and ptenb?/? embryos and an endothelial-like cell line from a tumor of an adult ptena+/?ptenb?/? zebrafish. This protocol can easily be adapted to establish stable cell lines from any mutant or transgenic zebrafish line and the average time to obtain a pro-stable cell line is 35 months. PMID:23672287

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

    PubMed Central

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

    1990-01-01

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

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

    Cancer.gov

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

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

    PubMed Central

    Scharfmann, Raphal; Pechberty, Severine; Hazhouz, Yasmine; von Blow, 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 ? cellspecific 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

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

    PubMed

    Luviano, Axel; Aguiiga-Snchez, Itzen; Demare, Patricia; Tiburcio, Reynaldo; Ledesma-Martnez, Edgar; Santiago-Osorio, Edelmiro; Regla, Ignacio

    2014-05-01

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

  1. 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

  2. T-lymphoblastoid cell lines from Marek's disease lymphomas.

    PubMed

    Powell, P C; Payne, L N; Frazier, J A; Rennie, M

    1975-01-01

    The establishment and continuous culture of two lymphoblastoid cell lines derived from Marek's disease lymphomas is described. Although the cells carried T-lymphocyte surface antigens, they had many features in common with cultured Burkitt's lymphoma lymphoblasts, which carry B-cell determinants. A small proportion acted as infectious units in tissue culture, and a similarly small proportion contained intranuclear immature herpesvirus particles. The cells did not respond to phytohaemagglutinin. One cell line possessed some graft-versus-host capacity, as measured by the induction of splenomegaly. It is concluded that the development of acute Marek's disease involves the malignant transformation of thymus-dependent lymphocytes by Marek's disease virus. PMID:66182

  3. Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    PubMed Central

    Fausther, Michel; Goree, Jessica R.; Lavoie, lise G.; Graham, Alicia L.; Svigny, Jean; Dranoff, Jonathan A.

    2015-01-01

    The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC) and portal fibroblasts (PF). In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myo)fibroblasts and their contribution to the progression of liver fibrosis. PMID:25822334

  4. CHARACTERIZATION OF A SPONTANEOUSLY TRANSFORMED CHICKEN MONONUCLEAR CELL LINE

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  5. A Stable Cranial Neural Crest Cell Line from Mouse

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2002-09-01

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

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

    SciTech Connect

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

    2009-05-08

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

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

    Cancer.gov

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

  9. A comprehensive transcriptional portrait of human cancer cell lines.

    PubMed

    Klijn, Christiaan; Durinck, Steffen; Stawiski, Eric W; Haverty, Peter M; Jiang, Zhaoshi; Liu, Hanbin; Degenhardt, Jeremiah; Mayba, Oleg; Gnad, Florian; Liu, Jinfeng; Pau, Gregoire; Reeder, Jens; Cao, Yi; Mukhyala, Kiran; Selvaraj, Suresh K; Yu, Mamie; Zynda, Gregory J; Brauer, Matthew J; Wu, Thomas D; Gentleman, Robert C; Manning, Gerard; Yauch, Robert L; Bourgon, Richard; Stokoe, David; Modrusan, Zora; Neve, Richard M; de Sauvage, Frederic J; Settleman, Jeffrey; Seshagiri, Somasekar; Zhang, Zemin

    2015-03-01

    Tumor-derived cell lines have served as vital models to advance our understanding of oncogene function and therapeutic responses. Although substantial effort has been made to define the genomic constitution of cancer cell line panels, the transcriptome remains understudied. Here we describe RNA sequencing and single-nucleotide polymorphism (SNP) array analysis of 675 human cancer cell lines. We report comprehensive analyses of transcriptome features including gene expression, mutations, gene fusions and expression of non-human sequences. Of the 2,200 gene fusions catalogued, 1,435 consist of genes not previously found in fusions, providing many leads for further investigation. We combine multiple genome and transcriptome features in a pathway-based approach to enhance prediction of response to targeted therapeutics. Our results provide a valuable resource for studies that use cancer cell lines. PMID:25485619

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

    PubMed

    2015-12-01

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

  11. 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.

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

    SciTech Connect

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

    1991-01-01

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

  13. Reliable in vitro studies require appropriate ovarian cancer cell lines

    PubMed Central

    2014-01-01

    Ovarian cancer is the fifth most common cause of cancer death in women and the leading cause of death from gynaecological malignancies. Of the 75% women diagnosed with locally advanced or disseminated disease, only 30% will survive five years following treatment. This poor prognosis is due to the following reasons: limited understanding of the tumor origin, unclear initiating events and early developmental stages of ovarian cancer, lack of reliable ovarian cancer-specific biomarkers, and drug resistance in advanced cases. In the past, in vitro studies using cell line models have been an invaluable tool for basic, discovery-driven cancer research. However, numerous issues including misidentification and cross-contamination of cell lines have hindered research efforts. In this study we examined all ovarian cancer cell lines available from cell banks. Hereby, we identified inconsistencies in the reporting, difficulties in the identification of cell origin or clinical data of the donor patients, restricted ethnic and histological type representation, and a lack of tubal and peritoneal cancer cell lines. We recommend that all cell lines should be distributed via official cell banks only with strict guidelines regarding the minimal available information required to improve the quality of ovarian cancer research in future. PMID:24936210

  14. Supraphysiological thermal injury in different human bladder carcinoma cell lines.

    PubMed

    Jen, Chun-Ping; Huang, Ching-Te; Tsai, Cheng-Han

    2009-11-01

    Depending on the duration of exposure to supraphysiological temperatures, cellular proteins and organelles can suffer from structural alternations and irreversible denaturation, which may induce cell death. The thermotolerance of three human urinary bladder carcinoma cell lines, TSGH-8301, J82 and TCC-SUP (cytological grade 2, 3 and 4, respectively), was investigated in the present study. A home-made heating stage was used to provide a constant temperature for different cell lines of bladder carcinoma. The experimental data showed that the TCC-SUP and TSGH-8301 cells exhibited the lowest and highest thermotolerances, respectively, while J82 cells were intermediate. Moreover, the differences in the thermotolerances for the TSGH-8301 and J82 cells are significant when the supraphysiological temperature is higher than 43 degrees C. As for TSGH-8301 and TCC-SUP cells, the thermotolerances are significantly different for all of the thermal treatments tested. Furthermore, the thermotolerances of J82 and TCC-SUP are significantly different when the cells are exposed to a temperature less than 50 degrees C for longer than 2 min. Overall, the results suggest that the high cytological grade of the cell line of bladder cancer exhibits a low thermotolerance. The kinematic parameters of the activation energy and frequency factor for bladder cancer cell lines with different cytological grades were also quantitatively evaluated in this work. PMID:19657740

  15. 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

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

    PubMed Central

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

    2013-01-01

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

  17. Expressional patterns of chaperones in ten human tumor cell lines

    PubMed Central

    Myung, Jae-Kyung; Afjehi-Sadat, Leila; Felizardo-Cabatic, Maureen; Slavc, Irene; Lubec, Gert

    2004-01-01

    Background Chaperones (CH) play an important role in tumor biology but no systematic work on expressional patterns has been reported so far. The aim of the study was therefore to present an analytical method for the concomitant determination of several CH in human tumor cell lines, to generate expressional patterns in the individual cell lines and to search for tumor and non-tumor cell line specific CH expression. Human tumor cell lines of neuroblastoma, colorectal and adenocarcinoma of the ovary, osteosarcoma, rhabdomyosarcoma, malignant melanoma, lung, cervical and breast cancer, promyelocytic leukaemia were homogenised, proteins were separated on two-dimensional gel electrophoresis with in-gel digestion of proteins and MALDI-TOF/TOF analysis was carried out for the identification of CH. Results A series of CH was identified including the main CH groups as HSP90/HATPas_C, HSP70, Cpn60_TCP1, DnaJ, Thioredoxin, TPR, Pro_isomerase, HSP20, ERP29_C, KE2, Prefoldin, DUF704, BAG, GrpE and DcpS. Conclusions The ten individual tumor cell lines showed different expression patterns, which are important for the design of CH studies in tumor cell lines. The results can serve as a reference map and form the basis of a concomitant determination of CH by a protein chemical rather than an immunochemical method, independent of antibody availability or specificity. PMID:15598346

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

    PubMed Central

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

    1996-01-01

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

  19. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

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

    2016-01-01

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

  20. In vitro radiosensitivity of human leukemia cell lines

    SciTech Connect

    Weichselbaum, R.R.; Greenberger, J.S.; Schmidt, A.; Karpas, A.; Moloney, W.C.; Little, J.B.

    1981-05-01

    The in vitro radiobiologic survival values (n, D0) of four tumor lines derived from human hematopoietic tumors were studied. These cell lines were HL50 (n . 1.3, D0 . 117 rad(1.17 Gy)), promyelocytic leukemia; K562 (n . 1.4, D0 . 165 rad(1.65 Gy)), erythroleukemia; 45 (n . 1.1, D0 . 147 rad(1.47 Gy)), acute lymphocyte leukemia; and 176 (n . 4.0, D0 . 76 rad(0.76 Gy)), acute monomyelogenous leukemia. More cell lines must be examined before the exact relationship between in vitro radiosensitivity and clinical radiocurability is firmly established.

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

    Cancer.gov

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  4. 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

  5. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

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

  6. 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 period from March 1, 2010 through March 31, 2011. As a result of program completion, the Stack Block Test System was ready to support installation and electrical operation of a RRFCS solid oxide fuel cell (SOFC) stack block in the second quarter of 2011.

  7. Steroid hormone secretion in inflammatory breast cancer cell lines.

    PubMed

    Illera, Juan Carlos; Caceres, Sara; Peña, Laura; de Andres, Paloma J; Monsalve, Beatriz; Illera, Maria J; Woodward, Wendy A; Reuben, James M; Silvan, Gema

    2015-12-01

    Inflammatory breast carcinoma (IBC) is a special type of breast cancer with a poor survival rate. Though several IBC cell lines have been established, recently a first IMC cell line was established. The aims of this study were: (1) to validate a highly sensitive, reliable, accurate and direct amplified enzyme immunoassay (EIA) to measure several cell-secreted steroid hormones: progesterone (P4), androstenedione (A4), testosterone (T), 17β-estradiol (E2) and estrone sulfate (SO4E1) in the culture medium. (2) To assess whether hormone production profile by IPC-366 cells validates the IMC model for human IBC. We validated a non-competitive amplified EIA for inflammatory breast cancer cell lines based on the results of accuracy, precision, sensitivity and parallelism. The low detection limits of the technique were: P4=13.2 pg/well, A4=2.3 pg/well, T=11.4 pg/well, E2=1.9 pg/well and SO4E1=4.5 pg/well. Intra- and inter-assay coefficient of variation percentages were <10%. The mean recovery rate of hormone added to the culture medium was >90%. In all hormones studied SUM149 have higher levels (1.4 times, but not significant) than IPC-366, and the correlation index between SUM149 and IPC-366 concentrations were >97%. We can coclude that cells of both cell lines, IPC-366 and SUM149, are capable to produce steroid hormone in culture media. The presented EIA methodology is very valuable for the detection of steroid production in culture media and could be used in hormone regulation studies and therapeutic agents in cell lines of inflammatory and non-inflammatory mammary carcinoma or other cancer cell lines in preclinical studies. PMID:26495931

  8. 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 ...

  9. Evaluation of dendrimer safety and efficacy through cell line studies.

    PubMed

    Kesharwani, Prashant; Gajbhiye, Virendra; Tekade, Rakesh K; Jain, Narendra K

    2011-09-01

    Dendrimers, by virtue of their therapeutic value, have recently generated enormous interest among biomedical scientists. Advancement of dendrimeric nano-architecture with well defined size, shape and controlled exterior functionality embraces promise in biomedical and pharmaceutical applications such as drug delivery, solubilization, DNA transfection and diagnosis. Highly branched, monodisperse, stable molecular level and low polydispersity with micelle-like behavior possessing nano-scale container property distinguish these structures as inimitable and optimum carrier for those applications. Dendrimers has been evaluated for delivery of different types of bioactives inside the cells. Different types of techniques are being used for exploring dendrimer safety and efficacy via cell cytotoxicity assays, cell uptake studies by fluorescent microscopy, cell line studies, flow cytometry, gamma scintigraphy and confocal microscopy; are being used over a decade. Among these, cell line studies are widely used for ex vivo characterization of dendrimers and other nanocarriers. Cell lines are homogeneous population of cells, stable after mitosis, and have an unlimited capacity for division. This review focuses on the use of different cell line studies including anticancer drugs, anti-HIV drugs, anti-inflammatory drugs, anti-tubercular-drugs, photodynamic therapy, hormonal therapy employing dendritic nanocarrier. PMID:21443471

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

    PubMed

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

    1994-01-01

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

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

    SciTech Connect

    Sherwood, J.B.; Shouval, D.

    1986-01-01

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

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

    PubMed

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

    2014-09-01

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

  13. 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

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

    PubMed

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

    1988-04-01

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

  15. JNK Inhibition Inhibits Lateral Line Neuromast Hair Cell Development

    PubMed Central

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

    2016-01-01

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

  16. 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

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

    PubMed

    Uphoff, Cord C; Drexler, Hans G

    2002-02-01

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

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

    PubMed

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

    2015-12-01

    We describe an adaptation of ?C31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu(2+)-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays-a major emphasis of cell-based studies. PMID:26450921

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

    PubMed Central

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

    2015-01-01

    We describe an adaptation of φC31 integrase–mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking-site lines carries a single mapped copy of one of the docking platforms. Vectors for targeted substitution contain a cloning cassette flanked by attB sites. Targeted substitution occurs by integrase-mediated substitution between the attP sites (integrated) and the attB sites (vector). We describe procedures for isolating cells carrying the substitutions and for eliminating the products of secondary off-target events. We demonstrate the technology by integrating a cassette containing a Cu2+-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression, and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays—a major emphasis of cell-based studies. PMID:26450921

  20. 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.

  1. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

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

  2. 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.

  3. In Vitro Growth of Thymic Tumor Cell Lines from Xenopus

    PubMed Central

    Pasquier, Louis du; Robert, Jacques

    1992-01-01

    A spontaneous lymphoid thymus tumor was discovered in a male Xenopus of the MHC ff genotype. The tumor cell can be transplanted in histocompatible larval ff hosts, but not in ff adults unless irradiated (3000 rad). The tumor is rejected by allogeneic hosts. The tumor cells express neither markers of the B-cell lineage nor MHC encoded molecules; they express only markers of the T-cell lineage. Its lymphoid population is clonal as revealed by the existence of a stable rearrangement pattern of the immunoglobulin genes. Cell lines growing continuously in vitro have been derived from the tumor. PMID:1343098

  4. Comparative proteomic profiling of Hodgkin lymphoma cell lines.

    PubMed

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

    2015-12-15

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

  5. Establishment of a human malignant fibrous mesothelioma cell line and the biological characteristics compared with malignant epithelial mesothelioma cell line.

    PubMed

    Ishiwata, Iwao; Ishiwata, Emiko; Hirano, Takashi

    2008-08-01

    Two human malignant mesothelioma cell lines, which we designated "epithelial mesothelioma cells" and "fibrous mesothelioma cells", were established from the pleural fluid containing malignant mesothelial cells of a 72-year-old Japanese man. These cell lines were separated by the colonial techniques from the initiation of the primary cultures and grew well without interruption for 12 years. They were characterized as producing hyaluronic acid. These cell lines displayed different biological characteristics, including morphology, heterotransplantability and genetics using with BAC array CGH. The epithelial mesothelioma cells were epithelial in shape and transplantable into the subcutis of nude mice, while the cells of the fibrous mesothelioma line were fibroblast-like and transplantable into the submucosa of Hamster's cheek pouches but not into the subcutis of nude mice. The mesotheliomas are classified into three types: epithelial mesothelioma, fibrous mesothelioma and mixed type. The gene copy number losses observed on 9p21.3, 9p21.2, 9p21.1, among others may be a major mechanism of malignant mesothelioma carcinogenesis. We considered and supported the combination theory for the histogenesis of malignant mesothelioma. PMID:18667024

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

    PubMed

    Ezawa, K; Minato, K; Dobashi, K

    1996-01-01

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

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

    PubMed

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

    2016-05-01

    Factor IX (FIX) is a vitamin K-dependent protein, and it has become a valuable pharmaceutical in the Hemophilia B treatment. We evaluated the potential of recombinant human FIX (rhFIX) expression in 293T and SK-Hep-1 human cell lines. SK-Hep-1-FIX cells produced higher levels of biologically active protein. The growth profile of 293T-FIX cells was not influenced by lentiviral integration number into the cellular genome. SK-Hep-1-FIX cells showed a significantly lower growth rate than SK-Hep-1 cells. γ-carboxylation process is significant to FIX biological activity, thus we performed a expression analysis of genes involved in this process. The 293T gene expression suggests that this cell line could efficiently carboxylate FIX, however only 28% of the total secreted protein is active. SK-Hep-1 cells did not express high amounts of VKORC1 and carboxylase, but this cell line secreted large amounts of active protein. Enrichment of culture medium with Ca(+2) and Mg(+2) ions did not affect positively rhFIX expression in SK-Hep-1 cells. In 293T cells, the addition of 0.5 mM Ca(+2) and 1 mM Mg(+2) resulted in higher rhFIX concentration. SK-Hep-1 cell line proved to be very effective in rhFIX production, and it can be used as a novel biotechnological platform for the production of recombinant proteins. PMID:26802680

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  9. Glutathione peroxidase isoenzymes in human tumor cell lines.

    PubMed

    Paukert, T; Sailer, R; Strauss, W S L; Schubert-Zsilavecz, M; Zimmer, A

    2011-11-01

    A set of human tumor cell lines was characterized in terms of the GPx isoenzymes GPx1, -2, -3 and -4. Semiquantitative PCR was used to investigate the GPx mRNA transcripts and the GPx activity was determined photometrically. As a result of culturing under standard conditions, diverse distribution of GPx mRNA and basic GPx activity was found in the investigated cell lines. PCR results showed nearly ubiquitous existence of the isoenzymes GPx1 and GPx4. GPx2 mRNA transcript was only detected in the colonic cell line CaCo-2. After detection of the GPx3 mRNA transcripts in most of the tested cell lines, an ELISA was performed to investigate if the GPx3 protein is present as well. However, the GPx3 protein could not be detected. Glutathione peroxidases contain the amino acid selenocysteine in their active centre. Selenocysteine contains selenium instead of sulfur in cysteine. Therefore, the influence of selenium on GPx activity and GPx isoenzyme distribution was investigated. Cell culturing with additional selenium showed a clear elevation of GPx activity in Mono Mac 6 cells but no gain of mRNA transcripts or any change in the isoenzyme's distribution. PMID:22204137

  10. Expression of the somatostatin gene in human astrocytoma cell lines.

    PubMed

    Mercure, L; Tannenbaum, G S; Schipper, H M; Phaneuf, D; Wainberg, M A

    1996-03-01

    Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system. PMID:8991628

  11. Molecular cytogenetic analysis of breast cancer cell lines.

    PubMed

    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

    2000-11-01

    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/ approximately pawefish. PMID:11044355

  12. Derivation of ductlike cell lines from a transplantable acinar cell carcinoma of the rat pancreas.

    PubMed Central

    Pettengill, O. S.; Faris, R. A.; Bell, R. H.; Kuhlmann, E. T.; Longnecker, D. S.

    1993-01-01

    Two cell lines were derived from a transplantable acinar cell carcinoma that had been established from a primary carcinoma of the pancreas in an azaserine-treated Lewis rat. The cultured tumor cells initially produced amylase, but production of exocrine enzymes ceased after 1-2 weeks in culture. The cultured cells were tumorigenic in Lewis rats, and one line produced solid tumors composed of ductlike structures surrounded by dense fibrous tissue. The second cell line produced partially solid and partially cystic tumors with a mixed phenotype of squamous, mucinous, and glandular areas when it grew in vivo following regrafting. Both cell lines lost structural and immunohistochemical acinar cell markers while acquiring duct cell markers during culture and regrafting. These studies provide strong support for the hypothesis that ductlike carcinomas can arise from neoplastic pancreatic acinar cells in rats. Images Figure 2 Figure 3 Figure 4 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:8391218

  13. Thyroid hormone transport in a human glioma cell line.

    PubMed

    Goncalves, E; Lakshmanan, M; Pontecorvi, A; Robbins, J

    1990-03-01

    The uptake of 3,5,3'-triiodothyronine (T3) and thyroxine (T4) was studied in human glioma cells (Hs 683) and compared with that in several other neural cell lines. At 25 degrees C or 37 degrees C, total cell uptake rose rapidly and reached equilibrium within 60 min. The glioma cells had the highest uptake: 47.6 fmol of L-T3 and 43.4 fmol of L-T4 per 10(6) cells at 37 degrees C. These were inhibited 77% and 72%, respectively, by excess unlabeled hormone. Uptake in the nuclei reached equilibrium between 90 and 120 min and was also highest in glioma cells: 1.46 fmol of L-T3 and 0.49 fmol of L-T4 per 10(6) cells. When expressed as percent of total cell uptake, however, glioma cells had the lowest values (3.1% for L-T3 and 1.1% for L-T4). Also in contrast to other cell lines, glioma cells transported L-T4 almost as effectively as L-T3. D-T3 and D-T4 total cell uptake was 86% and 96% lower than that of the respective L-isomers, and the nuclear uptake as a fraction of the cell uptake was similar. Kinetic analysis of the initial rate of cell uptake gave Vmax values for D-T3 and D-T4 that were 97% and 98% lower than for the L-isomers. Antimycin and monodansylcadaverine decreased the Vmax as well as the equilibrium cell and nuclear uptake of the L-isomers. The apparent nuclear affinity constant for L-T4 in intact cells was inhibited 90% in the presence of antimycin, whereas no effect was observed in isolated nuclei.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2328825

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

    PubMed

    Dutt, Kamla; Cao, Yang

    2009-06-01

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

  15. LINEing germ and embryonic stem cells silencing of retrotransposons

    PubMed Central

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-01-01

    Almost half of our genome is occupied by transposable elements. Although most of them are inactive, one type of non-long terminal repeat (LTR) retrotransposon, long interspersed nuclear element 1 (LINE1), is capable of retrotransposition. Two studies in this issue, Pezic and colleagues (pp. 14101428) and Castro-Diaz and colleagues (pp. 13971409), provide novel insight into the regulation of LINE1s in human embryonic stem cells and mouse germ cells and shed new light on the conservation of complex mechanisms to ensure silencing of transposable elements in mammals. PMID:24990961

  16. 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

  17. VR09 Cell Line: An EBV-Positive Lymphoblastoid Cell Line with In Vivo Characteristics of Diffuse Large B Cell Lymphoma of Activated B-Cell Type

    PubMed Central

    Nichele, Ilaria; Zam, Alberto; Bertolaso, Anna; Bifari, Francesco; Tinelli, Martina; Franchini, Marta; Stradoni, Roberta; Aprili, Fiorenza; Pizzolo, Giovanni; Krampera, Mauro

    2012-01-01

    Background small B-cell neoplasms can show plasmacytic differentiation and may potentially progress to aggressive lymphoma (DLBCL). Epstein-Barr virus (EBV) infection may cause the transformation of malignant cells in vitro. Design and Method we established VR09 cell line with plasmacytic differentiation, obtained from a case of atypical, non-CLL B-cell chronic lymphoproliferative disease with plasmacytic features. We used flow cytometry, immunohistochemistry, polymerase chain reaction, cytogenetic analysis and florescence in situ hybridization in the attempt at thoroughly characterizing the cell line. We showed VR09 tumorigenic potential in vivo, leading to the development of activated DLBCL with plasmacytic features. Results VR09 cells displayed plasmacytic appearance and grew as spherical tumors when inoculated subcutaneously into immunodeficient Rag2?/? ?-chain?/? mice. VR09 cell line and tumors displayed the phenotype of activated stage of B cell maturation, with secretory differentiation (CD19+ CD20+ CD79a+ CD79b+/? CD138+ cyclin D1- Ki67 80% IgM+ IgD+ MUM1+ MNDA+ CD10- CD22+ CD23+ CD43+ K+, ?- Bcl2+ Bcl6-) and they presented episomal EBV genome, chromosome 12 trisomy, lack of c-MYC rearrangement and Myd88 gene mutation, presence of somatic hypermutation in the VH region, and wild-type p53. Conclusion This new EBV-positive cell line may be useful to further characterize in vivo activated DLBCL with plasmacytic features. PMID:23285191

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

    PubMed

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

    2001-01-01

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

  19. 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

  20. ATP modulates PTEN subcellular localization in multiple cancer cell lines

    PubMed Central

    Lobo, Glenn P.; Waite, Kristin A.; Planchon, Sarah M.; Romigh, Todd; Houghton, Janet A.; Eng, Charis

    2008-01-01

    The tumour suppressor gene PTEN plays an important somatic role in both hereditary and sporadic breast carcinogenesis. While the role of PTEN's lipid phosphatase activity, as a negative regulator of the cytoplasmic phosphatidylinositol-3-kinase/Akt pathway is well known, it is now well established that PTEN exists and functions in the nucleus. Multiple mechanisms of regulating PTEN's subcellular localization have been reported. However none are ubiquitous across multiple cancer cell lines and tissue types. We show here that adenosine triphosphate (ATP) regulates PTEN subcellular localization in a variety of different cancer cell lines, including those derived from breast, colon and thyroid carcinomas. Cells deficient in ATP show an increased level of nuclear PTEN protein. This increase in PTEN is reversed when cells are supplemented with ATP, ADP or AMP. In contrast, the addition of the non-hydrolyzable analogue ATP?S, did not reverse nuclear PTEN protein levels in all the cell types tested. To our knowledge, this is the first report that describes a regulation of PTEN subcellular localization that is not specific to one cell line or tissue type, but appears to be common across a variety of cell lineages. PMID:18579579

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

    PubMed

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

    2016-01-01

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

  2. A cell line that can induce thymocyte positive selection.

    PubMed

    Hugo, P; Kappler, J W; Godfrey, D I; Marrack, P C

    1992-12-17

    The thymus positively selects thymocytes that bear T-cell receptors which recognize antigen presented by self major histocompatibility complex (MHC) proteins. Positive selection is usually driven by MHC products on radiation-resistant cortical epithelial cells. It is unknown whether positive selection is mediated by all thymic epithelial cells or by some specialized subsets. Here we introduce an H-2b-expressing thymic epithelial cell line into the thymuses of lethally irradiated H-2k animals reconstituted with H-2b/k F1 BM or fetal liver cells. I-Ab-restricted T cells are found in these animals, demonstrating that selection occurs on the introduced epithelial cells. PMID:1465132

  3. Radiation-induced apoptosis and cell cycle alterations in human carcinoma cell lines with different radiosensitivities.

    PubMed

    Hunkov, L; Chorvth, M; Duraj, J; Bartosov, Z; Sevckov, L; Sulikov, M; Chovancov, J; Sedlk, J; Chorvth, B; Boljeskov, E

    2000-01-01

    Radiosensitivity of examined human neoplastic cell lines was assessed with the aid of MTT assay. Differences between radiosensitive and radioresistant human neoplastic cell lines were as follow: a) radiation-induced apoptosis detected by flow cytometry was apparent in the most radiosensitive (i.e. CH-1 ovarian carcinoma cell line), but not in the radioresistant (i.e. SKOV-3 ovarian carcinoma) cell lines, b) radiation-induced G2/M arrest appeared early after irradiation (6 hours) in both the radioresistant SKOV-3 cells and in the radiosensitive CH-1 human ovarian carcinoma cell line, but a different pattern was observed 24 hours after irradiation with 2 Gy dose with G2/M arrest only in radiosensitive cell line. The radiosensitivity and resistance to radiation-induced apoptosis in the radioresistant human breast carcinoma MDA-MB-231 cell line were similar to those observed in SKOV-3 cells. These data suggest that radiation-induced apoptosis and cell cycle alterations can predict radiosensitivity at least in some examined human malignant cells in vitro. PMID:10870683

  4. Establishment and characterization of human B cell precursor-leukemia cell lines.

    PubMed

    Matsuo, Y; Drexler, H G

    1998-07-01

    A large number of continuous human leukemia cell lines have been established over the last three decades. Clearly, leukemia cell lines have become important research tools. Here, we have summarized the immunological, molecular and standard cytogenetic features of a panel of well characterized B cell precursor (BCP)-leukemia cell lines which were derived from patients with acute lymphoblastic/undifferentiated leukemia (ALL/AUL) or chronic myeloid leukemia (CML) in blast crisis. Following the recently proposed immunological EGIL classification, we assigned our panel of 27 BCP-cell lines to one of the following categories: B-I pro-B cell line; B-II common-B cell line; and B-III pre-B cell line. All cell lines express general B-lineage associated surface markers (HLA-DR, CD22, CD79a) being negative for surface immunoglobulin (Ig); the differences between the subgroups reside in expression of CD10 and cytoplasmic Ig. Several BCP-cell lines show the myelomonocytic cell-associated markers CD13 and/or CD33. These immunologically 'biphenotypic' BCP-cell lines are generally TdT+ CD10+ CD13+ CD19+ CD22+ CD34+ and carry the Philadelphia (Ph) translocation. The BCP-cell lines display surface receptors for interferon-gamma (CD119), interleukin-7 (CD127) and FLT-3 ligand (CD135). All BCP-cell lines examined have complex numerical and structural chromosomal alterations including translocations commonly seen in BCP-ALL such as t(4;11), t(9;22), t(11;19), t(12;21), and t(17;19) involving the fusion genes MLL-AF4, BCR-ABL, ENL-MLL, TEL/ETV6-AML1 and E2A-HLF, respectively. Besides the expected rearrangement of the Ig heavy chain receptor gene, several cell lines also have rearrangements of the T cell receptor genes beta, gamma or delta. While some BCP-cell lines express (aberrantly) myeloperoxidase at the mRNA level, most lines are negative in the immunological or cytochemical staining. Several large series documented the difficulty in establishing such BCP cell lines with success rates in the range of 10-20% (on average 15%). Still, since the establishment of the first bonafide BCP-cell line in 1974 (cell line REH), some 150 cell lines have been established of which, however, only a small percentage have been sufficiently well characterized and described. A higher success rate for immortalizing any given leukemia cell might depend on a closer emulation of the physiological in vivo microenvironment. The possibility to grow in vitro leukemia cells at will would represent ideal experimental systems permitting basic research and patient-specific investigations. In summary, the use of well-characterized BCP-cell lines provide unprecedented opportunities for studying a multitude of biological aspects related to normal and neoplastic B-lymphocytes. PMID:9680106

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

    SciTech Connect

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

    2012-01-01

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

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

    PubMed

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

    1998-11-01

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

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

    PubMed

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

    2016-01-01

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

  8. A Novel Cell Line from Spontaneously Immortalized Murine Microglia

    PubMed Central

    Kulas, Joshua; Combs, Colin K.

    2014-01-01

    Background Purified microglia cultures are useful tools to study microglial behavior in vitro. Microglial cell lines serve as an attractive alternative to primary microglia culture, circumventing the costly and lengthy preparation of the latter. However, immortalization by genetic or pharmacologic manipulations may show altered physiology from primary microglia. New Method A novel microglial cell line was isolated from a primary glial culture of postnatal murine cerebral cortices. The culture contained a population of spontaneously transformed microglia that continued to divide without genetic or pharmacological manipulations. After several clones were isolated, one particular clone, SIM-A9, was analyzed for its microglial characteristics. Results SIM-A9 cells expressed macrophage/microglia-specific proteins, CD68 and Iba1. SIM-A9 cells were responsive to exogenous inflammatory stimulation with lipopolysaccharide and ?-amyloid, triggering tyrosine kinase-based and NF?B signaling cascades as well as TNF? secretion. SIM-A9 cells also exhibited phagocytic uptake of fluorescent labeled ?-amyloid and bacterial bioparticles. Furthermore, lipopolysaccharide increased the levels of inducible nitric oxide synthase and cyclooxygenase-2, whereas IL-4 stimulation increased arginase-1 levels demonstrating that SIM-A9 cells are capable of switching their profiles to pro- or anti-inflammatory phenotypes, respectively. Comparison with Existing Methods The use of SIM-A9 cells avoids expensive and lengthy procedures required for the preparation of primary microglia. Spontaneously immortalized SIM-A9 cells are expected to behave more comparably to primary microglia than virally transformed or pharmacologically induced microglial cell lines. Conclusions SIM-A9 cells exhibit key characteristics of cultured primary microglia and may serve as a valuable model system for the investigation of microglial behavior in vitro. PMID:24975292

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

    PubMed

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

    1989-07-01

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

  10. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

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

    Methylation of Arsenite by Some Mammalian Cell Lines.

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

  11. 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...

  12. Use of Cell Lines in the Investigation of Pharmacogenetic Loci

    PubMed Central

    Zhang, Wei; Dolan, M. Eileen

    2009-01-01

    Drug response and toxicity, complex traits that are often highly varied among individuals, likely involve multiple genetic and non-genetic factors. Pharmacogenomic research aims to individualize therapy in an effort to maximize efficacy and minimize toxicity for each patient. Cell lines can be used as a model system for cellular pharmacologic effects, which include, but are not limited to, drug-induced cytotoxicity or apoptosis, biochemical effects and enzymatic reactions. Because severe toxicities may be associated with drugs such as chemotherapeutics, cell lines derived from healthy individuals or patients provide a convenient model to study how human genetic variation alters response to these drugs that would be unsafe or unethical to administer to human volunteers. In addition to the traditional candidate gene approaches that focus on well-understood candidate genes and pathways, the availability of extensive genotypic and phenotypic data on some cell line models has begun to allow genome-wide association (GWA) studies to simultaneously test the entire human genome for associations with drug response and toxicity. Though with some important limitations, the use of these cell lines in pharmacogenomic discovery demonstrates the promise of constructing a more comprehensive model that may ultimately integrate both genetic and non-genetic factors to predict individual response and toxicity to anticancer drugs. PMID:19925429

  13. 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...

  14. Establishment and characterization of a human cholangiocarcinoma cell line.

    PubMed

    Ma, Stephanie; Hu, Liang; Huang, Xiao-Hui; Cao, Liang-Qi; Chan, Kwok Wah; Wang, Qian; Guan, Xin-Yuan

    2007-11-01

    Cholangiocarcinoma (CC) is a rare malignant tumor arising from the biliary tract. The disease is notoriously difficult to diagnose and is usually fatal due to its late clinical presentation and the lack of effective non-surgical therapeutic strategies. To date, little is known about the cancer biology of the disease and the establishment and characterization of only a few CC cell lines have been reported. We report here the establishment of a new human cancer cell line, HKGZ-CC, from a moderate to poorly differentiated intrahepatic bile duct carcinoma from a Chinese patient. Morphological characteristics, growth kinetics, ability to grow on anchorage-independent soft agar, tumorigenicity in nude mice and cytogenetic features of the cell line were investigated. Chromosome banding karyotype and comparative genomic hybridization analyses revealed chromosomal changes in 1pter-p31, 1q31-qter, 3q, 8q21-qter, 9pter-9q34, 10, 13q21-qter and X. This newly established cell line should serve as a useful model for studying the molecular pathogenesis of CC. PMID:17914572

  15. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

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

  16. Glucocorticoid inhibition of cellular proliferation in rat hepatoma cell lines

    SciTech Connect

    Cook, P.W.

    1987-01-01

    Glucocorticoids were shown to inhibit the growth rate of Fu5 rat hepatoma cells cultured in the presence or absence of serum and thus, induced a more stringent dependence on serum for growth in this cell line. Fu5 cells, made quiescent at low cell density by continuous exposure to glucocorticoid in the absence of serum, were induced with serum and insulin, which subsequently caused a rapid reinitiation of cellular proliferation. Analysis of total RNA isolated from hormone treated Fu5 cells undergoing serum/insulin induction of DNA synthesis revealed a sequential expression of cellular proto-oncogene products in the absence of any immediate changes in intracellular Ca{sup ++} levels. Introduction of functional glucocorticoid receptor genes into both classes of dexamethasone resistant variants restored glucocorticoid responsiveness and suppression of cell growth. The BDS1 rat hepatoma cell line, an Fu5 derived subclone hypersensitive to the antiprofliferation effects of glucocorticoid, was observed to externalize a glucocorticoid suppressible mitogen (GSM) activity capable of mimicking EGF and insulin induced stimulation of ({sup 3}H)thymidine incorporation into serum starved, competant Balb/c 3T3 cells.

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

    PubMed Central

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

    1987-01-01

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

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

    TOXLINE Toxicology Bibliographic Information

    Nagy K; Sung HK; Zhang P; Laflamme S; Vincent P; Agha-Mohammadi S; Woltjen K; Monetti C; Michael IP; Smith LC; Nagy A

    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.

  19. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

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

    1992-06-01

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

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

    PubMed

    Santoro, Anna; Conde, Javier; Scotece, Morena; Abella, Vanessa; Lpez, Vernica; Pino, Jess; Gmez, Rodolfo; Gmez-Reino, Juan Jess; Gualillo, Oreste

    2015-12-01

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

  1. Characterization of the camel skin cell line Dubca.

    PubMed

    Klopries, M; Wernery, U; Kaaden, O R

    1995-01-01

    A skin fibroblast cell culture was established from a 2-month-old dromedary foetus. The cells were transformed by infection with SV40 and cloned in soft agar. The established cell line is now designated Dubca cells (Dubai camel) and has been in permanent culture for 95 passages. The cell culture was examined morphologically, chromosome preparations made and DNA fingerprinting performed by hybridization with the oligonucleotide probe (GTG)5. SV40 large T antigen was detected by western blotting. The viral host range was determined by infection with viruses of different families. Camelpox virus (CaPV) bovine herpesvirus-1 (BHV-1), vesicular stomatitis virus (VSV) and border disease virus (BDV) could be propagated in these cells. PMID:8556315

  2. 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

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

    PubMed Central

    2014-01-01

    Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as aa or juara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Aa is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 ?g/mL of bark, seed, and total aa fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnetts or Tukeys post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to aa treatment. The extracts caused significant reduction (p?cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that aa possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the anticancer potential of aa may help in the development of chemopreventive drugs and may have therapeutic effects in the treatment of breast cancer. PMID:24886139

  4. The interaction of normal lymphocytes and cells from lymphoid cell lines

    PubMed Central

    Mackintosh, Pauline; Wallin, Josephine; Hardy, D. A.; Ling, N. R.; Steel, C. M.

    1973-01-01

    Cells from thirty-three human lymphoid cell lines and sublines have been typed for HL-A antigens by a microcytotoxicity test. Similar patterns of HL-A antigens were found for the cell line cells and for the fresh lymphocytes of the donor of the line (eleven cases). However, certain typing sera gave positive reactions with the cell line cells which were not found with the fresh lymphocytes. No correlation was noted between the pattern of these `extra' reactions and the HL-A typing of the cells. These same typing sera often gave positive reactions with blood lymphocytes cultured for several days in conventional media. These positive reactions were quantitatively more pronounced and sometimes quantitatively different if the cells had been stimulated. A range of normal sera failed to react with cell line cells suggesting that the HL-A typing sera giving `extra' reactions are detecting antigens in some way related to a histocompatibility system. Absorption studies performed with two of the lines confirmed the HL-A typing by the direct cytotoxicity test. Two sera giving `extra' reactions were also tested in the absorption experiments. The results indicated that antibodies other than those of the HL-A specificity designated for these sera were responsible for the `extra' reactions. It is suggested that `extra' reactions indicate a change in the apparent antigenic expression of lymphoid cells reflecting altered membrane characteristics as they adapt to a culture environment. PMID:4123471

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

    PubMed

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

    2016-01-01

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. 2016 by John Wiley & Sons, Inc. PMID:26840224

  6. Optimized Sleeping Beauty transposons rapidly generate stable transgenic cell lines.

    PubMed

    Kowarz, Eric; Lscher, Denise; Marschalek, Rolf

    2015-04-01

    Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of retro-/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium-throughput setting (three to five days). Cell lines robustly express any gene-of-interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner. PMID:25650551

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

    PubMed Central

    Piskareva, Olga; Muniyappa, Mohan

    2007-01-01

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

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

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

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

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

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

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

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

    PubMed Central

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

    2011-01-01

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

  11. Cytotoxicity evaluation of silica nanoparticles using fish cell lines.

    PubMed

    Vo, Nguyen T K; Bufalino, Mary R; Hartlen, Kurtis D; Kitaev, Vladimir; Lee, Lucy E J

    2014-01-01

    Nanoparticles (NPs) have extensive industrial, biotechnological, and biomedical/pharmaceutical applications, leading to concerns over health risks to humans and biota. Among various types of nanoparticles, silica nanoparticles (SiO2 NPs) have become popular as nanostructuring, drug delivery, and optical imaging agents. SiO2 NPs are highly stable and could bioaccumulate in the environment. Although toxicity studies of SiO2 NPs to human and mammalian cells have been reported, their effects on aquatic biota, especially fish, have not been significantly studied. Twelve adherent fish cell lines derived from six species (rainbow trout, fathead minnow, zebrafish, goldfish, haddock, and American eel) were used to comparatively evaluate viability of cells by measuring metabolic impairment using Alamar Blue. Toxicity of SiO2 NPs appeared to be size-, time-, temperature-, and dose-dependent as well as tissue-specific. However, dosages greater than 100?g/mL were needed to achieve 24h EC50 values (effective concentrations needed to reduce cell viability by 50%). Smaller SiO2 NPs (16nm) were relatively more toxic than larger sized ones (24 and 44nm) and external lining epithelial tissue (skin, gills)-derived cells were more sensitive than cells derived from internal tissues (liver, brain, intestine, gonads) or embryos. Higher EC50 values were achieved when toxicity assessment was performed at higher incubation temperatures. These findings are in overall agreement with similar human and mouse cell studies reported to date. Thus, fish cell lines could be valuable for screening emerging contaminants in aquatic environments including NPs through rapid high-throughput cytotoxicity bioassays. PMID:24357037

  12. The genomic landscape of epithelioid sarcoma cell lines and tumours.

    PubMed

    Jamshidi, Farzad; Bashashati, Ali; Shumansky, Karey; Dickson, Brendan; Gokgoz, Nalan; Wunder, Jay S; Andrulis, Irene L; Lazar, Alexander J; Shah, Sohrab P; Huntsman, David G; Nielsen, Torsten O

    2016-01-01

    We carried out whole genome and transcriptome sequencing on four tumour/normal pairs of epithelioid sarcoma. These index cases were supplemented with whole transcriptome sequencing of three additional tumours and three cell lines. Unlike rhabdoid tumour (the other major group of SMARCB1-negative cancers), epithelioid sarcoma shows a complex genome with a higher mutational rate, comparable to that of ovarian carcinoma. Despite this mutational burden, SMARCB1 mutations remain the most frequently recurring event and are probably critical drivers of tumour formation. Several cases show heterozygous SMARCB1 mutations without inactivation of the second allele, and we explore this further in vitro. Finding CDKN2A deletions in our discovery cohort, we evaluated CDKN2A protein expression in a tissue microarray. Six out of 16 cases had lost CDKN2A in greater than or equal to 90% of cells, while the remaining cases had retained the protein. Expression analysis of epithelioid sarcoma cell lines by transcriptome sequencing shows a unique profile that does not cluster with any particular tissue type or with other SWI/SNF-aberrant lines. Evaluation of the levels of members of the SWI/SNF complex other than SMARCB1 revealed that these proteins are expressed as part of a residual complex, similarly to previously studied rhabdoid tumour lines. This residual SWI/SNF is susceptible to synthetic lethality and may therefore indicate a therapeutic opportunity. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:26365879

  13. Comparative Proteomic Profiling of Pancreatic Ductal Adenocarcinoma Cell Lines

    PubMed Central

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

    2014-01-01

    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

  14. 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

  15. Niclosamide inhibits the proliferation of human osteosarcoma cell lines by inducing apoptosis and cell cycle arrest.

    PubMed

    Li, Zonghuan; Yu, Yifeng; Sun, Shaoxing; Qi, Baiwen; Wang, Weiyang; Yu, Aixi

    2015-04-01

    Niclosamide, used as an antihelminthic, has demonstrated some properties of anticancer effects. However, its role in osteosarcoma remains to be determined. The aim of this study was to determine the effect of niclosamide on human osteosarcoma cell lines. The human MG-63 and U2OS osteosarcoma cell lines were treated with different concentrations of niclosamide. The cell inhibitory rate was calculated by CCK-8 assay. Cell cycle was detected by flow cytometry. Cell apoptosis was determined by Hoechst33324 staining, flow cytometry and fluorescence microscope, respectively. The expression of bcl-2, bax and pro-caspase-3 were measured by western blotting. Niclosamide exerted an inhibitory effect on the two cell lines in a time- and dose-dependent manner. Niclosamide was found to induce the arrest of S and G2/M phase in U2OS cells and G2/M in MG-63 cells. Moreover, niclosamide induced apoptosis in MG-63 and U2OS cells. The bax/bcl-2 ratio increased while the expression of pro?caspase-3 decreased significantly in the two cell lines. The results indicated that niclosamide inhibits proliferation, and induces apoptosis and cell cycle arrest in human osteosarcoma cell lines. PMID:25634333

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

    PubMed

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

    1990-01-01

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

  17. Study of adenovirus reproduction in different lymphoblastoid cell lines.

    PubMed

    Biliavska, L O; Povnitsa, O Yu; Nesterova, N V; Zagorodnya, S D; Bobko, T M; Golovan, A V; Voychuk, S I

    2014-01-01

    The comparative characteristic of the reproduction of adenovirus serotypes 2 and 5 (HAdV-C2 and -C5) in the various lymphoblastoid cell lines were studied. Rapid formation of infectious viruses in Raji, MP-1, Namalwa, BJAB, MT4 and Jurkat cells was marked and it was found to be close to the level of viruses during reproduction in permissive Hep-2 epithelial cells. Yield of infective adenovirus was low in B95-8 cells, which were chronically infected with Epstein-Barr virus (EBV). This may indicate the interference of Ad with EBV during super-infection. The CEM cells produced chronically low amounts of human adenovirus serotype 2. PMID:25639041

  18. 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,...

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

    PubMed

    Moraes, Karen C M; Diniz, Lívia F; Bahia, Maria Terezinha

    2015-04-01

    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

  20. 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

  1. Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts.

    PubMed

    Li, Ping-Chun; Tien, Yun-Chen; Day, Cecilia Hsuan; Pai, Peiying; Kuo, Wei-Wen; Chen, Tung-Sheng; Kuo, Chia-Hua; Tsai, Chang-Hai; Ju, Da-Tong; Huang, Chih-Yang

    2015-04-01

    Dilong is an earthworm extract with a dense nutritional content, widely used in Chinese herbal medicine to remove stasis and stimulate wound healing. Earthworm extracts are traditionally used by indigenous people throughout the world. How this Dilong inhibits Lipopolysaccharide (LPS)-induced cardiomyoblast cell apoptosis is still unclear. This study investigates the Dilong extract effect on LPS-induced apoptosis in H9c2 cardiomyoblast cells. LPS (1 ?g/ml) administration for 24 h induced apoptosis in H9c2 cells. Cell apoptosis was detected using MTT, LDH, TUNEL assay and JC-1 staining. Western blot analysis was used to detect pro-apoptotic and anti-apoptotic proteins. Dilong extract totally blocked the LPS impact, leading to the activation of anti-apoptotic proteins, Bcl-2 and Bcl-xL, stabilized the mitochondria membrane and down-regulated the extrinsic and intrinsic pro-apoptotic proteins, TNF-?, active caspase-8, t-Bid, Bax, active caspase-9 and active caspase-3. Dilong could potentially serve as a cardio protective agent against LPS-induced H9c2 cardiomyoblast cell apoptosis. PMID:25249212

  2. Epitope tagging of endogenous genes in diverse human cell lines.

    PubMed

    Kim, Jung-Sik; Bonifant, Challice; Bunz, Fred; Lane, William S; Waldman, Todd

    2008-11-01

    Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with epitope-tagged alleles of two cancer-related genes in a variety of untransformed and transformed human cell lines. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest. PMID:18784188

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

    PubMed

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

    1999-12-01

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

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

    PubMed Central

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

    1994-01-01

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

  5. Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

    PubMed

    Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

    2013-08-01

    Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages. PMID:23782837

  6. 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; OBrien, 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

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

    PubMed

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

    2015-01-01

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

  8. 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

  9. 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.

  10. Modulation of protectin (CD59 antigen) cell surface expression on human neoplastic cell lines.

    PubMed

    Sedlk, J; Hunkov, L; Duraj, J; Grfov, M; Chorvth, B

    1993-01-01

    The ability of cytokines (IFN alpha, IFN gamma, TNF alpha, IL-1 alpha, IL-6), all-trans retinoic acid, 1,25(OH)2-vitamin D3 and the tumor promoting phorbol ester TPA to regulate cell surface expression of protectin (CD59 antigen) on human hematopoietic and non-hematopoietic neoplastic cell lines was examined with the aid of immunocytofluorometric measurements. The tumor promoting phorbol ester TPA induced a marked up-regulation of protectin in all examined cell lines with the exception of promyelocytic leukemia HL-60, where TPA significantly decreased protectin cell surface expression. All-trans retinoic acid weakly down-regulated cell surface protectin on K-562, while 1,25(OH)2-vitamin D3 produced such effect on HL-60 cells. None of the examined cytokines induced a significant protectin down-regulation in the examined cell lines. PMID:7507222

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

    PubMed

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

    2014-01-01

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

  12. A cell line from an anaplastic transitional cell carcinoma of human urinary bladder.

    PubMed Central

    Nayak, S. K.; O'Toole, C.; Price, Z. H.

    1977-01-01

    A cell line, TCCSUP, derived from an undifferentiated, Grade IV transitional cell carcinoma is described. The karyotype showed an abnormal distribution of chromosomes, with no obvious modal number. Distinct marker chromosomes were observed in both early and late in vitro passages. These cells have been subcultured over 50 times during a 20-month period. TCCSUP differs in certain morphological and immunological features from other cell lines from transitional cell carcinomas. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:836756

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

    PubMed Central

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

    2015-01-01

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

  14. 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

  15. Hepatitis C virus infection of cholangiocarcinoma cell lines

    PubMed Central

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

    2015-01-01

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

  16. 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 invitro 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 thathybrid cellular pseudoislets exhibited an oval shape, with sizes ranging from 590 to 1200 ?m. Their morphology was similar to nave 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

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

    PubMed

    Kniss, Douglas A; Summerfield, Taryn L

    2014-02-11

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

  18. Diethyldithiocarbamate-induced cytotoxicity and apoptosis in leukemia cell lines.

    PubMed

    Kanno, Syu-ichi; Matsukawa, Emi; Miura, Ai; Shouji, Ai; Asou, Keiko; Ishikawa, Masaaki

    2003-07-01

    Diethyldithiocarbamate (DDTC) has been shown to induce cytotoxicity in several different systems. We examined whether the DDTC-induced cytotoxicity was via apoptosis, or in relation to intracellular glutathione (GSH) in various murine and human leukemia cell lines. The cells most sensitive to DDTC-induced cytotoxicity were P388 lymphoid neoplasma cells and NALM-6, a B cell line of acute lymphocytic leukemia (ALL). The next level of susceptible cells included J774.1, having a macrophage function, HL-60 premyelocytic leukemia cells, MOLT-4, an acute lymphoblastic leukemia cell, and Jurkat, a T-cell leukemia. U937 (expressing many monocyte-like characteristics), K562 erythroleukemia and K562/DXR (a multidrug-resistant clone derived from K562) were almost unaffected by DDTC. P388 was also highly susceptible to H(2)O(2), a most useful exogenous reactive oxygen species generator, and was lower in intracellular total GSH content than other leukemia cells. DDTC-induced cytotoxicity was closely related to intracellular GSH, but the level of cellular GSH did not always correlate with H(2)O(2)-induced cytotoxicity in this experiment. K562 had a higher intracellular total GSH content and showed lower susceptibility to DDTC and H(2)O(2), but with the combination of DDTC and DL-buthionine-(S,R)-sulfoximine (BSO), cytotoxicity increased significantly. The ratio of GSH/GSSG in P388 was reduced by DDTC or H(2)O(2). H(2)O(2)-induced cytotoxicity was completely blocked by catalase (CAT), while it was enhanced by superoxide dismutase (SOD). CAT or SOD did not affect DDTC-induced cytotoxicity. N-Acetylcysteine (NAC: 1 mM), a vanguard substance of GSH, and aurintricarboxylic acid (ATA: 100 microM), an endonuclease inhibitor, ameliorated DDTC-induced cytotoxicity and apoptosis. In conclusion, we suggest that DDTC-induced cytotoxicity was via an oxidative shift in the intracellular redox state, and accompanied the activation of endonuclease through apoptosis in leukemia cell lines. PMID:12843619

  19. Role of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 in myocardial cells in diabetes

    PubMed Central

    ZHOU, WENZHONG; YANG, JIAN; ZHANG, DI; LI, FENGYIN; LI, GUO; GU, YANYUN; LUO, MIN

    2015-01-01

    Cardiovascular complications are the major causes of morbidity and mortality associated with Type 2 Diabetes. Among the macrovascular complications, diabetic cardiomyopathy (DCM) is generally considered to be inadequately recognized and managed. Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3), is known to play a key role in the initiation of the mitochondrial pathway of apoptosis induced by hypoxia and acidosis in the heart. It is unknown whether BNIP3 is also important for cardiac cell survival or adaption in response to hyperglycemia. Based on the previous finding that BNIP3 was significantly induced in the diabetic rat heart, BNIP3 was transfected in primary rat cardiomyocytes and the H9c2 cell line in the present study. Overexpressed BNIP3 decreased the mitochondrial membrane potential and induced cell apoptosis. When BNIP3 was knocked down, the effect on cell apoptosis was reversed. Transcriptome analysis showed that the genes regulating mitochondrial metabolism, such as carnitine palmitoyltransferase 1b, cytochrome c oxidase subunit VIIIb and creatine kinase (brain), and those regulating cardiac fibrosis, such as matrix metallopeptidase 9, could be the targets of BNIP3 in rat cardiomyocytes. In conclusion, hyperglycemia-induced BNIP3 expression may compromise cardiac cell survival and function. Under the diabetic condition, BNIP3 could be involved in the regulation of mitochondrial function, lipid metabolism and fibrosis. BNIP3 could therefore serve as a potential drug target against diabetic macrovascular complications and, in particular, DCM. PMID:26170914

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

    SciTech Connect

    Wenjun Xi

    2008-08-18

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

  1. Characterization of butyrate uptake by nontransformed intestinal epithelial cell lines.

    PubMed

    Gonçalves, Pedro; Araújo, João R; Martel, Fátima

    2011-03-01

    Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon. Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells. Uptake of ¹⁴C-BT by IEC-6 cells was (1) time- and concentration-dependent; (2) pH-dependent; (3) Na+-, Cl⁻- and energy-dependent; (4) inhibited by BT structural analogues; (5) sensitive to monocarboxylate transporter 1 (MCT1) inhibitors; and (6) insensitive to DIDS and amiloride. IEC-6 cells express both MCT1 and Na+-coupled monocarboxylate transporter 1 (SMCT1) mRNA. We conclude that ¹⁴C-BT uptake by IEC-6 cells mainly involves MCT1, with a small contribution of SMCT1. Acute exposure to ethanol, acetaldehyde, indomethacin, resveratrol and quercetin reduced ¹⁴C-BT uptake. Chronic exposure to resveratrol and quercetin reduced ¹⁴C-BT uptake but had no effect on either MCT1 or SMCT1 mRNA levels. Uptake of ¹⁴C-BT by FHC cells was time- and concentration-dependent but pH-, Na+-, Cl⁻- and energy-independent and insensitive to BT structural analogues and MCT1 inhibitors. Although MCT1 (but not SMCT1) mRNA expression was found in FHC cells, the characteristics of ¹⁴C-BT uptake by FHC cells did not support either MCT1 or SMCT1 involvement. In conclusion, uptake characteristics of ¹⁴C-BT differ between IEC-6 and FHC cells. IEC-6 cells demonstrate MCT1- and SMCT1-mediated transport, while FHC cells do not. PMID:21286694

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

    SciTech Connect

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

    2012-07-20

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

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

    PubMed

    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

  4. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

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

    2010-09-03

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

  5. Growth of fish cell lines in glutamine-free media.

    PubMed

    Bols, N C; Ganassin, R C; Tom, D J; Lee, L E

    1994-01-01

    The glutamine requirement for the in vitro proliferation of fish cells was investigated with cell lines from four different species and three tissues: goldfish skin (GFSk-S1), Chinook salmon embryo (CHSE-214), and rainbow trout liver (RTL-W1) and spleen (RTSp-W1). With a supplement of fetal bovine serum, the basal medium, Leibovitz's L-15, without glutamine supported the proliferation of all four cell lines as well, or nearly as well, as L-15 with 2 mM glutamine. This was true over short term assays of two to four weeks and for continuous propagation. CHSE-214 also grew as well with or without 2 mM glutamine in Minimum Essential Medium with fetal bovine serum. However, when the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in L-15 without glutamine. Therefore, glutamine was not required for growth in L-15, and in fact, was inhibitory in the absence of the dialyzable fraction of serum. By contrast, glutamine appeared to be important for growth in Minimum Essential Medium. When the supplement was dialyzed fetal bovine serum, CHSE-214 grew much better in Minimum Essential Medium with 2 mM glutamine. These results suggest that the glutamine requirement for the in vitro proliferation of fish cells is conditional and depends on the basal medium and serum supplement. PMID:7766144

  6. Interactions of Streptococcus iniae with phagocytic cell line.

    PubMed

    El Aamri, Fatima; Remuzgo-Martnez, S; Acosta, Flix; Real, Fernando; Ramos-Vivas, Jos; Icardo, Jos M; Padilla, Daniel

    2015-04-01

    Streptococcus iniae has become one of the most serious aquatic pathogens in the last decade, causing large losses in wild and farmed fish worldwide. There is clear evidence that this pathogen is capable not only of causing serious disease in fish but also of being transferred to and infecting humans. In this study, we investigate the interaction of S. iniae with two murine macrophage cell lines, J774-A1 and RAW 264.7. Cytotoxicity assay demonstrated significant differences between live and UV-light killed IUSA-1 strains. The burst respiratory activity decreased to baseline after 1 and 4 h of exposure for J774-A1 and RAW 264.7, respectively. Immunofluorescent and ultrastructural study of infected cells confirmed the intracellular localization of bacteria at 1 h and 24 h post-infection. Using qRT-PCR arrays, we investigated the changes in the gene expression of immune relevant genes associated with macrophage activation. In this screening, we identified 11 of 84 genes up-regulated, we observed over-expression of pro-inflammatory response as IL-1?, IL-1?, and TNF-?, without a good anti-inflammatory response. Present findings suggest a capacity of S. iniae to modulate a mammalian macrophages cell lines to their survival and replication intracellular, which makes this cell type as a reservoir for continued infection. PMID:24956597

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

    SciTech Connect

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

    2012-10-19

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

  8. Gene transfer to human cells using retrovirus vectors produced by a new polytropic packaging cell line.

    PubMed Central

    Loiler, S A; DiFronzo, N L; Holland, C A

    1997-01-01

    We report here the construction of a new packaging cell line, called MPAC, that packages defective retroviral vectors in viral particles with envelope proteins derived from a Moloney mink cell focus-inducing (MCF) polytropic virus. We characterized the tropism of MPAC-packaged retroviral vectors and show that some human cell lines can be infected with these vectors while others cannot. In addition, we show that some human cells fully support MCF virus replication while others either partially or fully restrict MCF virus replication. PMID:9151879

  9. The quantitative proteome of a human cell line

    PubMed Central

    Beck, Martin; Schmidt, Alexander; Malmstroem, Johan; Claassen, Manfred; Ori, Alessandro; Szymborska, Anna; Herzog, Franz; Rinner, Oliver; Ellenberg, Jan; Aebersold, Ruedi

    2011-01-01

    The generation of mathematical models of biological processes, the simulation of these processes under different conditions, and the comparison and integration of multiple data sets are explicit goals of systems biology that require the knowledge of the absolute quantity of the system's components. To date, systematic estimates of cellular protein concentrations have been exceptionally scarce. Here, we provide a quantitative description of the proteome of a commonly used human cell line in two functional states, interphase and mitosis. We show that these human cultured cells express at least ?10 000 proteins and that the quantified proteins span a concentration range of seven orders of magnitude up to 20 000 000 copies per cell. We discuss how protein abundance is linked to function and evolution. PMID:22068332

  10. Flow cytometric DNA analysis of human cancers and cell lines.

    PubMed

    Krueger, Sarah A; Wilson, George D

    2011-01-01

    Measurement of DNA content was one of the first applications to be developed in the use of flow cytometry and is still used routinely in many experimental and, to a lesser extent, clinical studies. The goal of this technique is to produce a high quality DNA profiles for accurate analysis of DNA content and cell cycle distribution. In this chapter, we describe three DNA measurement methods that satisfy this requirement in different situations. It is widely accepted that the Vindelov method produces the highest quality DNA profiles in nuclei from solid tumours or cell lines. However, in many situations, DNA content is combined with another marker, so we describe a method which produces high quality DNA profiles in intact cells. Third, because the Vindelov technique requires prompt processing of fresh tumours, so we also describe a technique that derives nuclei from ethanol fixed tumours providing the convenience of storage before processing. PMID:21516421

  11. Heterogeneity in the radiation survival curves and biochemical properties of human lung cancer cell lines

    SciTech Connect

    Morstyn, G.; Russo, A.; Carney, D.N.; Karawya, E.; Wilson, S.H.; Mitchell, J.B.

    1984-10-01

    Human lung cancers of distinct histology exhibit different responses to radiation therapy in vivo. For examination of the basis of this phenomenon, the radiation survival curves and levels of relevant enzymes were determined in 16 lung cancer cell lines derived from tumors of different histology. These included lines from 5 adenocarcinomas, 7 small cell tumors, 3 variant small cell tumors, and 1 large cell tumor. These findings were compared to those obtained with the use of a normal skin fibroblast cell line. Whether cloned in liquid culture or soft agarose, cell lines had similar radiation survival curves. These curves were consistent with the apparent in vivo radiation responsiveness of the tumors. Although considerable heterogeneity in radiation survival curves was observed among the cell lines, cells from large cell lines and small variant lines had pronounced shoulders and extrapolation numbers (n) from 5.6 to 14. In contrast, cells from small cell lines and adenocarcinoma cell lines were more sensitive (-n values of 1-3.3). In these cell lines, levels of DNA polymerase beta, glutathione (GSH), GSH transferase, GSH reductase (NAD(P)H), gamma-glutamyltransferase did not correlate with radiation parameters of sensitivity. DNA polymerase beta and GSH levels were, however, higher than those in a line of normal skin fibroblasts. These cell lines may be useful in identifying the basis of the variable responsiveness of human lung cancer cells to ionizing radiation.

  12. Cell-cycle synchronization reverses Taxol resistance of human ovarian cancer cell lines

    PubMed Central

    2013-01-01

    Background Taxol is a powerful chemotherapy agent leading to mitotic arrest and cell death; however, its clinical efficacy has been hampered due to the development of drug resistance. Taxol specifically targets the cell cycle. Progress through mitosis (M stage) is an absolute requirement for drug-induced death because cell death is markedly reduced in cells blocked at the G1-S transition. The measured doubling time for ovarian cancer cells is about 27 h. As such, during treatment with Taxol most of the cells are not in the M stage of the cell cycle. Thus, the effect of cell-cycle synchronization was investigated in regard to reversing Taxol resistance in ovarian cancer cells. Methods Giemsa-Wright staining was used for assessing the morphology of the cells. The doubling time of the cells was calculated using formula as follows: Td?=?In2/slope. The resistant index and cell cycle were measured via MTT assays and flow cytometry. Thymidine was used to induce cell-cycle synchronization, and cell apoptosis rates following exposure to Taxol were measured using a flow cytometer. Results The growth doubling time of two Taxol-resistant cell lines were longer than that of Taxol-sensitive cells. Apoptotic rates in Taxol-sensitive and -resistant cell lines after synchronization and exposure to Taxol were all higher compared to unsynchronized controls (p <0.05). Conclusions Synchronization of the cell-cycle resulted in an increased effectiveness of Taxol toward ovarian cancer cell lines. We speculated that formation of drug resistance toward Taxol in ovarian cancer could be partly attributed to the longer doubling time of these cells. PMID:23899403

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

    PubMed

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

    2016-01-01

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

  14. 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; Mhlendick, 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

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

    PubMed

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

    2016-01-22

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

  16. Targeting mitochondrial citrate transport in breast cancer cell lines.

    PubMed

    Ozkaya, Ali Burak; Ak, Handan; Atay, Sevcan; Aydin, Hikmet Hakan

    2015-01-01

    Lipogenesis is considered to be a very important aspect of cancer metabolism and targeting de novo lipid synthesis or related pathways are among novel approaches to treat cancer. Many targets of the pathway including ATP-citrate lyase (ACLY), acetyl-CoA carboxylase and fatty acid synthase have been evaluated for their potential in cancer treatment. However the role of citrate transport protein (CTP), another important component of lipogenesis pathway, is not well known for cancer metabolism and cell survival. Here we report that while chemical inhibition of CTP reduces cytoplasmic citrate levels and limits breast cancer cell viability effectively, siRNA based inhibition had little effect on both. We also compared the effects of CTP inhibition with ACLY and found that the inhibition of ACLY reduced cytoplasmic citrate levels and limited cell viability more effectively than CTP inhibition. Finally we have demonstrated that neither cell cycle arrest nor autophagy was induced in cells treated with CTP or ACLY siRNA. Inhibitions triggered apoptosis but only slightly. Growth inhibitory effects do not occur in normal mammary epithelial MCF-10A cell line. PMID:25511512

  17. Functional somatostatin receptors on a rat pancreatic acinar cell line

    SciTech Connect

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.; Clerc, P.; Logsdon, C.; Svoboda, M.; Susini, C.; Vaysse, N.; Ribet, A. Mount Zion Hospital and Medical Center, San Francisco, CA Universite Libre de Bruxelles, Brussels )

    1988-07-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.

  18. Complementation analysis of the murine scid cell line

    SciTech Connect

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

    1995-09-01

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

  19. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation

    PubMed Central

    Taylor, AW; Dixit, S; Yu, J

    2015-01-01

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

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

    PubMed Central

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

    2010-01-01

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

  1. Differential epigenetic regulation of Aiolos expression in human tumoral cell lines and primary cells.

    PubMed

    Duhamel, Marianne; Navarro, Pablo; Cario-Andre, Muriel; Billot, Katy; Arrouss, Issam; Rebollo, Angelita

    2008-02-01

    In order to investigate the epigenetic component of Aiolos regulation, we analyzed the methylation status of its 5' CpG island in relation to histone modifications. Inhibition of CpG methylation restores Aiolos expression, as well as euchromatin-associated markers, in U937 and 1106 mel cell lines. DNA methylation and low levels of euchromatin-associated signatures are observed in U937 and 1106 mel cell lines, while the opposite characterizes Daudi, Jurkat, T and B cells. CpG methylation is not necessary to repress transcription in monocytes and melanocytes where silencing mechanism involves heterochromatin-associated signature. We show that DNA methylation directs Aiolos silencing and chromatin status in tumor cell lines, while in primary cells is mainly regulated by histone modifications. PMID:18206652

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

    PubMed

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

    2015-11-16

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

  3. Establishment and characterisation of two cell lines derived from a primary adenocarcinoma of the duodenum

    PubMed Central

    Golding, M; Stamp, G W H; Oates, T; Lalani, E-N

    1996-01-01

    AimsTo establish two cell lines from a primary duodenal adenocarcinoma; to describe the morphological, growth, ploidy, and immunophenotypic characteristics of these cell lines. MethodsThe cell lines, designated DAC/S and DAC/E, were characterised using both in vitro and in vivo cell culture techniques, light and electron microscopy, immunocytochemistry, and FACS analyses. ResultsBoth cell lines have an epithelial origin, are aneuploid and display characteristics of transformed cells. The cell lines differ from each other in morphology, doubling time and serum requirements. These cell lines are anchorage dependent and do not grow in nude mice. ConclusionsDAC/S and DAC/E cell lines are derived from neoplastic epithelium and could provide in vitro model systems for future investigations of the cell and molecular biology of duodenal neoplasia. Images PMID:16696042

  4. 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.

  5. Ultrastructure, karyology and immunology of a cell line originated from a human transitional-cell carcinoma.

    PubMed Central

    O'Toole, C.; Price, Z. H.; Ohnuki, Y.; Unsgaard, B.

    1978-01-01

    A cell line (J82) was derived from a poorly differentiated, invasive, transitional-cell carcinoma, Stage T3. The cells have been propagated in vitro for 5 years and showed 100% aneuploidy and a mixed epithelial-fibroblastic morphology. The majority of cells contained 2Y chromosomes and several distinctive markers. Peripheral-blood lymphocytes from the donor of the J82 cells were tested sequentially for cytotoxicity toward autologous and allogeneic tumour cells. Autologous cytotoxicity was detected against J82 cells in early in vitro passage. Allogeneic lymphocytes from some patients with transitional-cell carcinoma were also cytotoxic to J82 cells in primary culture. However, selective cytotoxicity by lymphoid cells from bladder-carcinoma patients was not detected against J82 cells in long-term tissue culture. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 8 Fig. 9 PMID:687519

  6. Specific human cytotoxic T cells recognize B-cell lines persistently infected with respiratory syncytial virus.

    PubMed Central

    Bangham, C R; McMichael, A J

    1986-01-01

    The T-lymphocyte response to respiratory syncytial (RS) virus has been invoked to explain the bronchiolitis and pneumonia caused by RS virus in human infants. However, T cells also appear to play a role in protection against RS virus infection. Although RS virus-specific human lymphocytes have been demonstrated, neither the phenotype nor the function of the lymphocytes was characterized. We describe here the induction of anti-RS virus cytotoxic T lymphocytes, in both bulk culture and restimulated cell lines, from human peripheral blood. Infection of Epstein-Barr virus-transformed human B-cell lines with RS virus in vitro readily caused a persistent infection; these cells continued to synthesize RS viral proteins and secrete infectious RS virus 4 months after infection. The persistently infected cells were used both to restimulate cytotoxic-T-cell precursors and as targets for RS virus-specific cytotoxic T cells. Images PMID:3097646

  7. Verification and unmasking of widely used human esophageal adenocarcinoma cell lines.

    PubMed

    Boonstra, Jurjen J; van Marion, Ronald; Beer, David G; Lin, Lin; Chaves, Paula; Ribeiro, Catarina; Pereira, A Dias; Roque, Lcia; Darnton, S Jane; Altorki, Nasser K; Schrump, David S; Klimstra, David S; Tang, Laura H; Eshleman, James R; Alvarez, Hector; Shimada, Yutaka; van Dekken, Herman; Tilanus, Hugo W; Dinjens, Winand N M

    2010-02-24

    For decades, hundreds of different human tumor type-specific cell lines have been used in experimental cancer research as models for their respective tumors. The veracity of experimental results for a specific tumor type relies on the correct derivation of the cell line. In a worldwide effort, we verified the authenticity of all available esophageal adenocarcinoma (EAC) cell lines. We proved that the frequently used cell lines SEG-1 and BIC-1 and the SK-GT-5 cell line are in fact cell lines from other tumor types. Experimental results based on these contaminated cell lines have led to ongoing clinical trials recruiting EAC patients, to more than 100 scientific publications, and to at least three National Institutes of Health cancer research grants and 11 US patents, which emphasizes the importance of our findings. Widespread use of contaminated cell lines threatens the development of treatment strategies for EAC. PMID:20075370

  8. Is parainfluenza virus a threatening virus for human cancer cell lines?

    PubMed

    Danjoh, Inaho; Sone, Hiyori; Noda, Nahomi; Iimura, Emi; Nagayoshi, Mariko; Saijo, Kaoru; Hiroyama, Takashi; Nakamura, Yukio

    2009-08-01

    Immortalized cell lines, such as human cancer cell lines, are an indispensable experimental resource for many types of biological and medical research. However, unless the cell line has been authenticated prior to use, interpretation of experimental results may be problematic. The potential problems this may cause are illustrated by studies in which authentication of cell lines has not been carried out. For example, immortalized cell lines may unknowingly be infected with viruses that alter their characteristics. In fact, parainfluenza virus type 5 (PIV5) poses a threat to the use of immortalized cell lines in biological and medical research; PIV5 infection significantly alters cellular physiology associated with the response to interferon. If PIV5 infection is widespread in immortalized cell lines, then a very large number of published studies might have to be re-evaluated. Fortunately, analyses of a large number of immortalized cell lines indicate that PIV5 infection is not widespread. PMID:19624309

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

    PubMed

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

    1988-01-01

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

  10. Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.

    PubMed

    Kaszuba-Zwoinska, J; Wojcik, K; Bereta, M; Ziomber, A; Pierzchalski, P; Rokita, E; Marcinkiewicz, J; Zaraska, W; Thor, P

    2010-04-01

    Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin. PMID:20436221

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

    PubMed

    Mehta, Rajvi H

    2014-11-01

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

  12. 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

  13. 5-Aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines.

    PubMed

    Chibazakura, Taku; Toriyabe, Yui; Fujii, Hiroshi; Takahashi, Kiwamu; Kawakami, Mariko; Kuwamura, Haruna; Haga, Hazuki; Ogura, Shun-ichiro; Abe, Fuminori; Nakajima, Motowo; Yoshikawa, Hirofumi; Tanaka, Tohru

    2015-01-01

    5-aminolevulinic acid (5-ALA) is contained in all organisms and a starting substrate for heme biosynthesis. Since administration of 5-ALA specifically leads cancer cells to accumulate protoporphyrin IX (PpIX), a potent photosensitizer, we tested if 5-ALA also serves as a thermosensitizer. 5-ALA enhanced heat-induced cell death of cancer cell lines such as HepG2, Caco-2, and Kato III, but not other cancer cell lines including U2-OS and normal cell lines including WI-38. Those 5-ALA-sensitive cancer cells, but neither U2-OS nor WI-38, accumulated intracellular PpIX and exhibited an increased reactive oxygen species (ROS) generation under thermal stress with 5-ALA treatment. In addition, blocking the PpIX-exporting transporter ABCG2 in U2-OS and WI-38 cells enhanced their cell death under thermal stress with 5-ALA. Finally, a ROS scavenger compromised the cell death enhancement by 5-ALA. These suggest that 5-ALA can sensitize certain cancer cells, but not normal cells, to thermal stress via accumulation of PpIX and increase of ROS generation. PMID:25346276

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

    PubMed

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

    2009-03-01

    Biological experiments are most often performed with immortalized cell lines because they are readily available and can be expanded without limitation. However, cell lines may differ from the in vivo situation in important aspects. Here we introduce a straightforward methodology to compare cell lines to their cognate primary cells and to derive a comparative functional phenotype. We used SILAC (stable isotope labeling by amino acids in cell culture) for quantitative, mass spectrometry-based comparison of the hepatoma cell line Hepa1-6 with primary hepatocytes. The resulting quantitative proteome of 4,063 proteins had an asymmetric distribution, with many proteins down-regulated in the cell line. Bioinformatic analysis of the quantitative proteomics phenotypes revealed that Hepa1-6 cells were deficient in mitochondria, reflecting re-arrangement of metabolic pathways, drastically up-regulate cell cycle-associated functions and largely shut down drug metabolizing enzymes characteristic for the liver. This quantitative knowledge of changes provides an important basis to adapt cell lines to more closely resemble physiological conditions. PMID:18952599

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

    PubMed Central

    Palacios, R; Golunski, E; Samaridis, J

    1995-01-01

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

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

    PubMed

    Klingbeil, Ma Ftima G; Xavier, Flvia C A; Sardinha, Luiz R; Severino, Patricia; Mathor, Monica B; Rodrigues, Rodrigo V; Pinto, Dcio S

    2013-11-01

    Head and neck squamous cell carcinoma is a complex disease with several etiologic factors and different molecular changes that may trigger certain events; it is also globally one of the most common malignancies in this topography. Extracts from ViscumalbumL. (VA) (mistletoe) have been used as adjuvant therapies with promising results in several types of cancer, mainly in European countries. Invitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells, activating the apoptotic cascade or leading cells to necrosis. This study aimed to verify the effects of three types of VA extracts (Iscador Qu Spezial, IscadorP and IscadorM) in squamous cell carcinoma of the tongue cell lines SCC9 and SCC25, not previously studied. A concentration of 0.3mg/ml (IC50) of the drugs induced apoptosis, affecting gene expression and protein levels of AKT, PTEN and CYCLIND1. It was concluded that VA extracts have a cytotoxic effect on SCC9 and SCC25 cell lines, but while SCC9 cell line was more resistant to the action of the drugs, IscadorQu Spezial and IscadorM have higher cytotoxic potential in both cell lines compared to IscadorP. PMID:24026291

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

    PubMed

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

    2013-06-01

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

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

    PubMed Central

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

    2013-01-01

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

  19. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    SciTech Connect

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

    2010-05-28

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

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

    NASA Technical Reports Server (NTRS)

    Passini, Cheryl A.; Goochee, Charles F.

    1989-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Dulbecco, Renato; Bologna, Mauro; Unger, Michael

    1980-03-01

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

  2. Viral carcinogenesis in a pronephric cell line. An ultrastructural study.

    PubMed Central

    Wong, W. Y.; Tweedell, K. S.

    1975-01-01

    Herpesvirus recovered from cell fractions of the spontaneous Lucké renal tumor of adult Rana pipiens were used to infect a cell line derived from pronephroi of the same species. Viruses and virus-associated structures previously found in the primary renal tumor were observed, including nuclear inclusions of capsids with single or double membranes and capsids with nucleoids often within nuclear sacs. Embedded within the clumped and marginated chromatin were 55-nm tubular elements and associated unit membrane structures. Virus-associated, 35-nm tubular elements were also seen. The cytoplasm contained single, enveloped nucleoid virus and clusters of virus within cytoplasmic vesicles. Other cytoplasmic inclusions were dense, virus-associated, 25-nm filaments, virus particles within myeloid bodies, and possible viral budding from tubular organelles. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:1155580

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Effects of Notch2 and Notch3 on Cell Proliferation and Apoptosis of Trophoblast Cell Lines

    PubMed Central

    Zhao, Wei-Xiu; Zhuang, Xu; Huang, Tao-Tao; Feng, Ran; Lin, Jian-Hua

    2015-01-01

    Aims: To investigate the effect of Notch2 and Notch3 on cell proliferation and apoptosis of two trophoblast cell lines, BeWo and JAR. Methods: Notch2 and Notch3 expression in BeWo and JAR cells was upregulated or downregulated using lentivirus-mediated overexpression or RNA interference. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. The effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V-PE Apoptosis kit. Lentivirus-based overexpression vectors were constructed by cloning the full-length coding sequences of human Notch2 and Notch3 C-terminally tagged with GFP or GFP alone (control) into a lentivirus-based expression vector. Lentivirus-based gene silencing vectors were prepared by cloning small interfering sequences targeting human Notch2 and Notch3 and scrambled control RNA sequence into a lentivirus-based gene knockdown vector. The effect of Notch2 and Notch3 on cell proliferation was assessed by the CCK-8 assay. And the effect of Notch2 and Notch3 on the apoptosis of BeWo and JAR cells was evaluated by flow cytometry using the Annexin V PE Apoptosis kit. Results: We found that the downregulation of Notch2 and Notch3 gene expression in BeWo and JAR cells resulted in an increase in cell proliferation, while upregulation of Notch3 and Notch2 expression led to a decrease in cell proliferation. Moreover, the overexpression of Notch3 and Notch2 in BeWo and JAR cells reduced apoptosis in these trophoblast cell lines, whereas apoptosis was increased in the cells in which the expression of Notch3 and Notch2 was downregulated. Conclusions: Notch2 and Notch3 inhibited both cell proliferation and cell apoptosis in BeWo and JAR trophoblast cell lines. PMID:26640406

  5. Characterization of proteoglycans synthesized by a rat parathyroid cell line

    SciTech Connect

    Yanagishita, M.; Brandi, M.L.; Sakaguchi, K. )

    1989-09-15

    The structure, biosynthesis, and distribution of cell-associated proteoglycans in a clonal line of parathyroid cells, which exhibit differentiated characteristics such as calcium-regulated hormone secretion and cell growth, were studied by metabolic labeling with (3H) glucosamine and (35S)sulfate as precursors. Proteoglycans were isolated by two consecutive ion exchange chromatography steps and then analyzed by gel filtration, polyacrylamide gel electrophoresis, and specific enzyme and chemical reactions. The cells synthesize almost exclusively (greater than 95%) heparan sulfate (HS) proteoglycans with a glycosaminoglycan synthesis rate of approximately 0.5 micrograms/10(6) cells/24 h. Two major HS proteoglycan species were identified. HS proteoglycan-I has a mass of approximately kDa with a single HS chain (approximately 12 kDa) and a core protein of approximately 150 kDa including oligosaccharides. HS proteoglycan-II has a mass of approximately 170 kDa with 3-4 HS chains (approximately 30 kDa) and a core protein of 70-80 kDa including oligosaccharides. In the medium with low ionized calcium (0.05 mM), HS proteoglycan-I is synthesized at approximately 1.6 times the rate and HS proteoglycan-II at a similar rate as for cells cultured in the medium with high ionized calcium (2.1 mM). The distribution of proteoglycans, examined by the accessibility of the molecules to trypsin, was dramatically influenced by environmental calcium concentration; at low calcium levels 70-80% of the HS proteoglycans are trypsin-accessible while only 20-30% are accessible at high calcium levels. This suggests that the proteoglycans are primarily on the cell surface in low calcium and in trypsin-inaccessible compartments in high calcium conditions.

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

    SciTech Connect

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

    1989-07-01

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

  7. Lineage infidelity of MDA-MB-435 cells: expression of melanocyte proteins in a breast cancer cell line.

    PubMed

    Sellappan, Shankar; Grijalva, Rebecca; Zhou, Xiaoyan; Yang, Wentao; Eli, Menashe Bar; Mills, Gordon B; Yu, Dihua

    2004-05-15

    The origin of cell lines is critical in defining cell type-specific biological functions. Several reports suggested that the MDA-MB-435 cell line, a cell line extensively used for studying breast cancer biology, has a gene expression pattern most compatible with melanocyte origin. However, we demonstrate that MDA-MB-435 cells express breast-specific or epithelial-specific markers. Also, MDA-MB-435 cells were induced to express breast differentiation-specific proteins and secrete milk lipids as observed in other well-established breast cancer cell lines. Notably, MDA-MB-435 cells also expressed melanocyte-specific proteins as did another highly aggressive breast cancer cell line. MDA-MB-435 xenograft tissue sections stained entirely positive for epithelium-specific markers but only partially positive for melanocyte-specific markers. Thus, MDA-MB-435 is most likely a breast epithelial cell line that has undergone lineage infidelity. PMID:15150101

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

    PubMed

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

    2015-11-10

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Vuorinen, T; Vainionp, R; Vanharanta, R; Hyypi, T

    1996-01-01

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

  11. Nucleotide composition analysis of tRNA from leukemia patient cell samples and human cell lines.

    PubMed Central

    Agris, P F

    1975-01-01

    A technique developed for analysis of less than microgram quantities of tRNA has been applied to the study of human leukemia. Leucocytes from peripheal blood and bone marrow samples of six, untreated leukemia patients and cells of five different established human cell lines were maintained for 18 hours in media containing (32P)-phosphate. Incorporation of radioactive phosphate into the cells from the patient samples was slightly less than that of the cell lines. Likewise, incorporation of (32P)-phosphate into the tRNA of the patient samples (approximately 5 x 106 DPM/mug tRNA) was also less then that incorporated into the tRNA of the cell lines. The major and minor nucleotide compositions of the unfractionated tRNA preparations from each patient sample and each cell line were determined and compared. Similarities and differences in the major and minor nucleotide compositions of the tRNA preparations are discussed with reference to types of leukemia and the importance of patient sample analysis versus analysis of cultured human cells. PMID:1057159

  12. Establishment and characterization of two embryonic cell lines of Bombyx mori.

    PubMed

    Pan, Min-Hui; Xiao, Shi-Quan; Chen, Min; Hong, Xi-Jun; Lu, Cheng

    2007-02-01

    Two cell lines, i.e., BmE-SWU1 and BmE-SWU2, were established from silkworm embryonic tissues of the reversion phase through primary culture in Grace's medium supplemented with 20% fetal bovine serum. The BmE-SWU1 cell line mainly included diploid spindle cells and round cells, which were large and had severe heteroploidy karyotypes. The population doubling time of the 30th passage of the cell line was 58.7 hr. BmE-SWU2 cells were oblong or round, and small. The population doubling time for the 30th passage of the cell line was 46.6 hr. Of BmE-SWU2 cells 89.9% were diploid (2n=56). Both strains were attached to epithelial-like cell lines and were susceptible to Bombyx mori nucleopolyhedroviruse (BmNPV). Inter simple sequence repeat (ISSR) fingerprinting of silkworm embryonic cell line was obtained. PMID:17570024

  13. Use of an agar culture technique for establishing lymphoid cell lines from Marek's disease lymphomas.

    PubMed

    Payne, L N; Howes, K; Rennie, M; Bumstead, J M; Kidd, A W

    1981-12-01

    Lymphoblastoid cell lines derived from Marek's disease (MD) lymphomas have been established with difficulty by a number of workers. We have compared with conventional liquid culture methods the efficiency of a new technique for establishing lymphoid cell lines in which lymphoma cells were cultured initially in agar medium. Cells from 39/79 lymphomas gave rise to loose lymphoid colonies in the seeded agar after 7 days' incubation at 41 degrees C. Two types of macrophage colony also developed. When lymphoid colonies in agar were transferred to liquid culture, 23/39 gave rise to permanent lymphoid cell lines, compared with 8/33 comparable cultures initiated in liquid medium. Twenty-nine new cell lines have been developed from Rhode Island Red, line 6 and line 7 chickens. All carry T-cell markers and the MD tumour-associated surface antigen (MATSA) and showed variable but low responsiveness to lectin mitogens. The new cell lines, when first established, consisted mainly of small, lymphocytoid cells, but, after varying times, these changed into typical lymphoblastoid lines and an increased expression of an embryonic antigen was associated with this change. The lymphocytoid line cells were more slowly growing and density-dependent than were the lymphoblastoid cells, and lymphocytoid lines grew better at 41 degrees C and lymphoblastoid lines better at 37 degrees C. MD virus could be rescued from some of the lines but others appeared to be true non-producers. PMID:7199514

  14. Lung Cancer Cell Lines as Tools for Biomedical Discovery and Research

    PubMed Central

    Girard, Luc; Lockwood, William W.; Lam, Wan L.; Minna, John D.

    2010-01-01

    Lung cancer cell lines have made a substantial contribution to lung cancer translational research and biomedical discovery. A systematic approach to initiating and characterizing cell lines from small cell and nonsmall cell lung carcinomas has led to the current collection of more than 200 lung cancer cell lines, a number that exceeds those for other common epithelial cancers combined. The ready availability and widespread dissemination of the lines to investigators worldwide have resulted in more than 9000 citations, including multiple examples of important biomedical discoveries. The high (but not perfect) genomic similarities between lung cancer cell lines and the lung tumor type from which they were derived provide evidence of the relevance of their use. However, major problems including misidentification or cell line contamination remain. Ongoing studies and new approaches are expected to reveal the full potential of the lung cancer cell line panel. PMID:20679594

  15. Inhibitory effects of xanthohumol from hops (Humulus lupulus L.) on human hepatocellular carcinoma cell lines.

    PubMed

    Ho, Yi-Chien; Liu, Chi-Hsien; Chen, Chien-Nan; Duan, Kow-Jen; Lin, Ming-Tse

    2008-11-01

    Xanthohumol is one of the main flavonoids in hop extracts and in beer. Very few investigations of xanthohumol have studied hepatocellular carcinoma. In this study, the inhibitory effects of xanthohumol on human hepatocellular carcinoma cell lines were investigated. The IC(50) values of xanthohumol for two hepatocellular carcinoma cell lines and one normal hepatocyte cell line were 108, 166 and 211 microm, respectively. Normal murine hepatocyte cell line had more resistance to xanthohumol than hepatocellular carcinoma cell lines. Besides, the inhibitory effects of xanthohumol on human hepatocellular carcinoma cell lines were attributed to apoptosis as indicated in the results of flow cytometry, fluorescent nuclear staining and electrophoresis of oligonucleosomal DNA fragments. Hop xanthohumol was more efficient in the growth inhibition of hepatocellular carcinoma cell lines than the flavonoids silibinin and naringin from thistle and citrus. It was shown for the first time that xanthohumol from hops effectively inhibits proliferation of human hepatocellular carcinoma cells in vitro. PMID:18814205

  16. 'Fluorescent Cell Chip' for immunotoxicity testing: Development of the c-fos expression reporter cell lines

    SciTech Connect

    Trzaska, Dominika; Zembek, Patrycja; Olszewski, Maciej; Adamczewska, Violetta; Ulleras, Erik; Dastych, JarosIaw . E-mail: jdastych@cbm.pan.pl

    2005-09-01

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

  17. PACAP protects against TNFα-induced cell death in olfactory epithelium and olfactory placodal cell lines

    PubMed Central

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

    2010-01-01

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

  18. Gliotoxin-induced cytotoxicity in three salmonid cell lines: cell death by apoptosis and necrosis.

    PubMed

    DeWitte-Orr, S J; Bols, N C

    2005-06-01

    Epithelial (CHSE-214), fibroblast (RTG-2) and macrophage (RTS11) cell lines from Chinook salmon and rainbow trout were tested for their sensitivity to gliotoxin, a fungal metabolite. Gliotoxin treatment for 6 or 24 h caused cell viability to decrease in a dose-dependent manner, with effective concentrations (EC50s) being similar for the three cell lines but varying with exposure time. Under some exposure conditions, hallmarks of apoptosis were detected. Apoptosis was evaluated by the appearance of fragmented nuclei upon H33258 staining and of genomic DNA laddering into 180 bp oligomers. Gliotoxin induced cell detachment in RTG-2 and CHSE-214 cultures, under some conditions. These were the only cultures of these two cell lines in which apoptosis was detected, and apoptotic cells appeared more frequent in the detached population. At the highest concentration, 15 microM, the cells died by an alternative mode, likely necrosis. By contrast, in RTS11 cultures cell detachment was not observed, and apoptosis occurred over a wider concentration range, even 15 microM, reaching levels of over 90%. The preferential death by necrosis for epithelial cells (CHSE-214) and by apoptosis for macrophages (RTS11) could be a beneficial host response to gliotoxin-producing fungi, leading respectively to the development and then resolution of inflammation. PMID:16046190

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2016-02-01

    Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2(+) cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for drug screening. PMID:26659251

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

    PubMed Central

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

    2016-01-01

    ABSTRACT Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for drug screening. PMID:26659251

  2. Effect of Docosahexaenoic Acid on Cell Cycle Pathways in Breast Cell Lines With Different Transformation Degree.

    PubMed

    Rescigno, Tania; Capasso, Anna; Tecce, Mario Felice

    2016-06-01

    n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), abundant in fish, have been shown to affect development and progression of some types of cancer, including breast cancer. The aim of our study was to further analyze and clarify the effects of these nutrients on the molecular mechanisms underlying breast cancer. Following treatments with DHA we examined cell viability, death, cell cycle, and some molecular effects in breast cell lines with different transformation, phenotypic, and biochemical characteristics (MCF-10A, MCF-7, SK-BR-3, ZR-75-1). These investigations showed that DHA is able to affect cell viability, proliferation, and cell cycle progression in a different way in each assayed breast cell line. The activation of ERK1/2 and STAT3 pathways and the expression and/or activation of molecules involved in cell cycle regulation such as p21(Waf1/Cip1) and p53, are very differently regulated by DHA treatments in each cell model. DHA selectively: (i) arrests non tumoral MCF-10A breast cells in G0 /G1 cycle phase, activating p21(Waf1/Cip1) , and p53, (ii) induces to death highly transformed breast cells SK-BR-3, reducing ERK1/2 and STAT3 phosphorylation and (iii) only slightly affects each analyzed process in MCF-7 breast cell line with transformation degree lower than SK-BR-3 cells. These findings suggest a more relevant inhibitory role of DHA within early development and late progression of breast cancer cell transformation and a variable effect in the other phases, depending on individual molecular properties and degree of malignancy of each clinical case. J. Cell. Physiol. 231: 1226-1236, 2016. © 2015 Wiley Periodicals, Inc. PMID:26480024

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

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

  5. In vitro acute cytotoxicity of abamectin to the Gill Cell Line of Flounder Paralichthy olivaceus

    NASA Astrophysics Data System (ADS)

    Xu, Yuyan; Guo, Huarong; Xiao, Qin; Su, Feng; Yin, Licheng

    2007-10-01

    The cytotoxicity of abamectin to the Gill Cell Line of Flounder (FG cell line) was examined in this study. It was found that the exposure of FG cells to abamectin caused the decreases of both cell growth rate and antioxidant enzyme activities, and the increase of intracellular O2 - content. It was proposed that the reduction of antioxidant enzyme activities in FG cells caused the accumulation of O2 - content in FG cells, leading to the change of cell morphology and even the death of cells. The results showed that FG cell line is suitable for the evaluation of the acute toxicity of abamectin.

  6. Synthesis of immunoglobulins by biopsied tissues and cell lines from Burkitt's lymphoma

    PubMed Central

    Van Furth, R.; Gorter, H.; Nadkarni, J. S.; Nadkarni, J. J.; Klein, E.; Clifford, P.

    1972-01-01

    Cell suspensions of Burkitt's lymphomas and cell lines derived from the same tumours were compared for immunoglobulin synthesis by analysis of the culture fluid. Thirty-one out of fifty tumour cell suspensions (i.e. twenty-one out of thirty-five patients) synthesized IgG (γ-chains) with type κ and/or type λ light chains; IgM synthesis was found in only five cases. Of the Burkitt's lymphoma cell lines, twelve out of nineteen synthesized IgG (γ-chains); type κ light chains were produced by ten of these cell lines and type λ light chains by three. Only three cell lines synthesized μ chains. IgA synthesis was not detected in any of the biopsied tissues or cell lines. Comparison of the immunoglobulin synthesis by the cells of the biopsied tissue and the derived cell line showed very good agreement. This leads to the conclusion that the pattern of immunoglobulins synthesized by Burkitt's lymphoma cell lines is representative of the original tumour. Investigation of cells from repeat biopsies, and serial testing of the derived cell lines showed that the capacity to synthesize particular immunoglobulins and chains remained constant. The fact that many of the biopsied tumour tissues and cell lines synthesized more than one immunoglobulin, or different classes of heavy chains and types of light chains, raises the question whether these immunoglobulin-producing cells originate from one or more cells. ImagesFIG. 1FIG. 2FIG. 3 PMID:5021706

  7. Proposals for the characterization and description of new human leukemia-lymphoma cell lines.

    PubMed

    Drexler, H G; Matsuo, Y; Minowada, J

    1998-03-01

    Continuous human leukemia-lymphoma cell lines have become invaluable tools for hematological research as they provide an unlimited amount of cellular material. The first human lymphoma cell line Raji was established in 1963; since then several hundred leukemia-lymphoma cell lines spanning almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) have been described. The cardinal features of leukemia-lymphoma cell lines are their monoclonal origin, arrest of differentiation, and (growth factor-independent or -dependent) unlimited proliferation. Categorization of cell lines usually follows the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, a detailed characterization of both primary and cultured cells in absolutely necessary. New cell lines, in particular, must be adequately, characterized; while cell culture data and immunological and cytogenetic features are essential, cell lines should be described in as much detail as possible. In addition to this recommended multiparameter characterization and the obligatory immortality of the culture, authentication of the true origin of the cells, novelty, scientific significance and availability of the cell line for other investigators are of utmost importance. It is still extremely difficult to establish new leukemia-lymphoma cell lines (except for some subtypes), and most attempts fail. Paramount to the lack of our understanding as to why certain cells start to proliferate in culture and others do not (thus implying a random process), is probably the difficulty of mimicking in vitro the physiological in vivo microenvironment. Attempts to improve the efficiency of cell line establishment should focus on examining the appropriateness of the in vitro culture conditions; these conditions should emulate as closely as possible the in vivo situation. In summary, leukemia-lymphoma cell lines have the potential to greatly facilitate diverse studies of normal and malignant hematopoiesis; to that end, these cell lines must be extensively characterized and adequately described. PMID:9710721

  8. A Cell-Permeable Fluorescent Polymeric Thermometer for Intracellular Temperature Mapping in Mammalian Cell Lines

    PubMed Central

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

    2015-01-01

    Changes in intracellular temperatures reflect the activity of the cell. Thus, the tool to measure intracellular temperatures could provide valuable information about cellular status. We previously reported a method to analyze the intracellular temperature distribution using a fluorescent polymeric thermometer (FPT) in combination with fluorescence lifetime imaging microscopy (FLIM). Intracellular delivery of the FPT used in the previous study required microinjection. We now report a novel FPT that is cell permeable and highly photostable, and we describe the application of this FPT to the imaging of intracellular temperature distributions in various types of mammalian cell lines. This cell-permeable FPT displayed a temperature resolution of 0.05C to 0.54C within the range from 28C to 38C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature. PMID:25692871

  9. Novel non-viral method for transfection of primary leukemia cells and cell lines.

    PubMed

    Schakowski, Frank; Buttgereit, Peter; Mazur, Martin; Mrten, Angela; Schttker, Bjrn; Gorschlter, Marcus; Schmidt-Wolf, Ingo GH

    2004-01-12

    BACKGROUND: Tumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods. METHODS: The aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions. RESULTS: Using this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP. CONCLUSION: The non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy. PMID:14715084

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

    PubMed

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

    2008-09-01

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

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

    PubMed

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

    2016-01-01

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly. PMID:26772746

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

    PubMed Central

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

    2016-01-01

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly. PMID:26772746

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    PubMed Central

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

    1998-01-01

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

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

    SciTech Connect

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

    1997-07-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  17. Cell death induced by Bothrops asper snake venom metalloproteinase on endothelial and other cell lines.

    PubMed

    Brenes, Oscar; Muz, Eduardo; Roldn-Rodrguez, Raquel; Daz, Cecilia

    2010-06-01

    Two adherent cell lines, BAEC and HeLa, and non-adherent Jurkat, were treated with snake venom metalloproteinase BaP1 to determine whether cytotoxicity, previously reported for this toxin, could be mediated by the process of anoikis. It was observed that there was no correlation between the ability of this toxin to induce loss of adherence, and the cytotoxic effect, since concentrations that do not induce loss of adherence (3-6 microg/mL), were able to trigger 50% of cytotoxicity in BAEC. In the case of HeLa, where toxicity was very low (less than 20% at maximun concentrations and times of exposure), significant detachment and no toxicity was observed at concentrations of 1.5 microg/mL, showing also no correlation between both events. We also observed differences between BAEC toxicity measured by XTT reduction and DNA fragmentation determined by flow cytometry (as an indicator of apoptosis), since concentrations that induce 100% of cytotoxicity barely showed any DNA fragmentation (12% at 24h), suggesting that if apoptosis was involved, DNA damage is still not present, although chromatin condensation, another indicator of apoptosis, is observed in 40% of the cells. Inhibition of BAEC cytotoxicity by caspase inhibitors indicate that apoptosis is playing a role in this process, but other mechanisms of cell death could be participating also. Another way to determine whether the mechanism of cell death was related to anoikis was using a non-adherent cell line, which should show substrate independence. We determined by TUNEL that at 50 microg/ml BaP1 triggered 50% of apoptosis at 96 h, an effect that was seen earlier, suggesting also that if this toxin was inducing apoptosis in a non-adherent cell line, the mechanism could not be related to loss of attachment. Cell cycle arrest in S phase was also observed in Jurkat cells, an effect that could be leading to apoptosis. In conclusion, since there was no correlation between cell detachment and cytotoxicity (and apoptosis) in adherent cell lines and due to the ability of BaP1 to induce apoptosis in a non-adherent cell line, we suggest that this enzyme is toxic by a mechanism not related to anoikis, and that in the case of Jurkat cells, it is likely to be related to its ability to induce cell cycle arrest. Processes other than apoptosis could be also involved in the cell death mechanism mediated by BaP1 on BAEC. PMID:20219457

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  19. 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

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

    PubMed

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

    2014-12-01

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

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

    NASA Astrophysics Data System (ADS)

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

    1993-06-01

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

  2. Establishment of Marek's disease lymphoblastoid cell lines from transplantable versus primary lymphomas.

    PubMed

    Calnek, B W; Murthy, K K; Schat, K A

    1978-01-15

    Six new Marek's disease (MD) lymphoblastoid cell lines were established in vitro by cultivation in a medium containing 2-mercaptoethanol (2-ME). Attempts using primary lymphoma cells were generally unsuccessful; only one of 28 lymphomas yielded a cell line and that one came from an experimentally immunosuppressed chicken. In contrast, two of seven low-passage, and two of two established MD transplantable lymphomas grew readily in vitro. A sixth line was obtained using buffy coat cells from a leukemic chicken. It was concluded that the use of transplantable tumor cells and a medium containing 2-ME provided a combination highly suited to the establishment of cell lines from MD. PMID:624595

  3. [Use of continuous human and animal cell lines for the production of viral vaccines].

    PubMed

    Grachev, V P; Khapchaev, Iu Kh

    2008-01-01

    History of development of safety criteria for continuous human and animal cell lines approved for manufacture of immunobiologic preparations. It was noted that current WHO documents recommend mandatory use of respective WHO's reference cell cultures (Vero-10-87 for continuous cell lines, and Wi-38 or MRC-5 for diploid cell lines) during attestation of new cell cultures proposed for the manufacturing of immunobiologic preparations. Examples of practical use of continuous cell lines (CCLs) for production of viral vaccines on industrial scale are described. On the basis of modern data most important principles were formulated which should be considered to provide safety and efficacy of vaccines produced on the CCLs. PMID:18368760

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

    PubMed

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

    2008-08-01

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

  5. Arsenic trioxide induced endoplasmic reticulum stress in laryngeal squamous cell line Hep-2 cells.

    PubMed

    Yang, Xinxin; An, Liangxiang; Li, Xiaoyu

    2014-02-01

    Arsenic trioxide (As2O3) has been used in the treatment of acute promyelocytic leukemia (APL) and many malignant solid tumors. Recently, endoplasmic reticulum (ER) stress plays an important role in As2O3-treated laryngeal squamous cell line Hep-2 cells. In the present work, the expression of ER stress-related proteins was investigated in As2O3-treated Hep-2 cells. The results showed that As2O3 increased the expression of GRP78, CHOP, phosphorylated eIF2? and ATF4, all of which are the molecule of ER stress. Therefore, As2O3 induced ER stress in Hep-2 cells. PMID:23880367

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

    PubMed Central

    Hamilton, Gerhard

    2014-01-01

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

  7. Expression of the Cell Adhesion Molecule CD44 in Human Lung Tumors and Cell Lines.

    PubMed

    Resnick; Clarke; Siegfried; Landreneau; Asman; Ge; Kierstead; Dougherty; Cooper

    1998-06-01

    Background: The purpose of this study was to examine the expression of the cell adhesion molecule CD44 in normal lung, primary and metastatic lung tumors, and cell lines derived from primary lung carcinomas. Methods and Results: A total of 68 lung specimens including normal tissue and primary and metastatic tumors, as well as 28 cell lines cultured from primary lung tumors with high recurrence, were examined for CD44 expression by semiquantitative reverse transcription polymerase chain reaction. Variant exon expression was confirmed by Southern blotting and hybridization of particular samples. In tumor tissues, loss of CD44 variant expression correlated with increasing tumor stage; a smaller percentage of more aggressive and poorly differentiated tumors expressed CD44v. Tumors metastatic to the lung were negative for CD44 variant expression. In primary lung cell lines, as in tumor tissue, tumors of higher histologic grade were characterized by loss of CD44 variant expression. Conclusion: CD44 isoform expression in normal lung and tumor tissues and cell lines revealed an overall decrease in CD44 alternative splicing in lung neoplasms of increased malignancy. PMID:10029660

  8. Decreased T cell reactivity to EpsteinBarr virus infected lymphoblastoid cell lines in multiple sclerosis

    PubMed Central

    Pender, M P; Csurhes, P A; Lenarczyk, A; Pfluger, C M M; Burrows, S R

    2009-01-01

    Objective: To investigate T cell and antibody immunity to EpsteinBarr virus (EBV) in multiple sclerosis (MS). Methods: Immunoglobulin G (IgG) immunity to EBV nuclear antigen 1 (EBNA1) and viral capsid antigen was measured by enzyme linked immunosorbent assays, and T cell immunity was assessed using enzyme linked immunospot assays to measure the frequency of peripheral blood mononuclear cells (PBMC) producing interferon ? in response to autologous EBV infected B cell lymphoblastoid cell lines (LCL) in 34 EBV seropositive healthy subjects and 34 EBV seropositive patients with MS who had not received immunomodulatory therapy in the previous 3 months. Results: Patients with MS had increased levels of anti-EBNA1 IgG but a decreased frequency of LCL specific T cells compared with healthy subjects. Using purified populations of CD4+ T cells and CD8+ T cells, we showed that the LCL specific response resides predominantly in the CD8+ population, with a frequency 57-fold higher than in the CD4+ population. The decreased CD8+ T cell response to LCL in MS was not caused by decreased HLA class I expression by LCL, and LCL from MS patients could be killed normally by HLA matched EBV specific cytotoxic CD8+ T cell clones from healthy subjects. Furthermore, the decreased CD8+ T cell immunity to EBV was not due to a primary defect in the function of CD8+ T cells because EBV specific cytotoxic CD8+ T cell lines could be generated normally from the PBMC of patients with MS. Conclusion: This quantitative deficiency in CD8+ T cell immunity to EBV might be responsible for the accumulation of EBV infected B cells in the brains of patients with MS. PMID:19015225

  9. Mutations and polymorphisms in mitochondrial DNA in head and neck cancer cell lines

    PubMed Central

    Allegra, E; Garozzo, A; Lombardo, N; De Clemente, M; Carey, TE

    2006-01-01

    Summary Changes in mitochondrial DNA have been reported in cancer cells. Since little information exists regarding mt DNA mutations in head and neck, the present study focused on ten head and neck cancer cell lines in the attempt to detect alterations in the ND4 gene sequence. DNA was extracted from 10 head and neck squamous cell carcinoma lines from 9 patients. MtDNA sequences were compared in normal and tumour cell line DNA. In ten head and neck squamous cell carcinoma cell lines, 8 somatic mutations and 5 polymorphisms of the mitochondrial gene for ND4 were found. All 5 polymorphisms were silent. Of the 8 somatic mutations, 3 altered the amino acid sequence suggesting a possible effect on enzyme function. The mitochondrial mutations and polymorphisms found demonstrated that these can serve as clonal markers for individual cell lines and demonstrate that the mitochondrial genome remains stable in the cell lines during in vitro culture. PMID:18236634

  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. 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

  12. Establishment and characterization of an ovarian cell line of the silkworm, Bombyx mori.

    PubMed

    Pan, Min-Hui; Cai, Xiu-Juan; Liu, Min; Lv, Jun; Tang, Hui; Tan, Juan; Lu, Cheng

    2010-02-01

    A cell line BmN-SWU1 was established from the ovarian tissues of 3-day-old fourth instar Bombyx mori larvae of the 21-872nlw variety by performing primary cultures in Grace's medium supplemented with 20% fetal bovine serum (FBS). The cell line primarily consisted of short spindle cells and round cells. The frequency of cells with chromosome number 2n=56 was 80.5%; therefore, the cell line was considered to be a diploid cell line. The population-doubling time (PDT) at 45th passage line was 57.7h. This cell line was susceptible to the B. mori nuclear polyhedrovirus (BmNPV), and the median tissue culture infective dose (TCID(50)) at a cell density of 10(5) cells/ml was 16.3 OBs/ml. The transient expression efficiency of the green fluorescent protein (GFP) gene in this cell line was 54.8%. We used the BmN-SWU1 cell line to select and establish a GFP transgenic cell line. PMID:19665160

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

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

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

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

    PubMed

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

    2010-11-15

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

  15. Establishment and characterization of a new canine B-cell leukemia cell line.

    PubMed

    Nakaichi, M; Taura, Y; Kanki, M; Mamba, K; Momoi, Y; Tsujimoto, H; Nakama, S

    1996-05-01

    A new cell line derived from a spontaneous canine leukemia was established and designated GL-1. The cells have been cultured in a floating fashion and passaged for over two years. They were round with rich cytoplasm containing many rough endoplasmic reticula and mitochondria. Peroxidase staining was negative. The nuclei of many cells were round, but segmented nuclei were seen frequently. The doubling time of the cells was 27.3 hr and they had 78 chromosomes. Surface marker analysis using monoclonal antibodies (MABs) and flowcytometry revealed that GL-1 possessed CD45 and surface IgG. However, the cells did not react with MABs detecting T-cell markers. These results indicate that GL-1 has a lymphocytic lineage and is derived from a B-cell leukemia. PMID:8741612

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

    PubMed

    Patil, Shankargouda; Rao, Roopa S; Raj, A Thirumal

    2015-01-01

    Oral cell lines have provided valuable insights into the various molecular pathways in oral carcinogenesis. Several landmark studies in oral oncology have utilized commercially available normal, dysplastic and cancer cell lines to decode the genetic alterations leading to the development of oral cancer. Most of these studies have shown a significant degree of variation in their mutation landscapes. These variations were thought to represent the heterogeneity of oral cancer.(1) But in a recent study, Dickman et al have shown that normal and dysplastic cell lines carry specific genetic alterations within the parent cell line, thus questioning the authenticity of several published mutation profiles. These genetic alterations in the commercial cell lines have been attributed to several factors, the most common being immortalization. Normal and dysplastic cell lines unlike cancer cell lines attain senescence following limited number of replication. Immortalization of the normal and dysplastic cell lines would aid the researcher in maintaining a viable population of cells for further studies. Ideally, the immortalized cell line must possess potential for indefinite replication and must retain the genetic makeup of its parent cell line.(2). PMID:26718303

  17. Sprouty4 interferes with cell proliferation and migration of breast cancer-derived cell lines.

    PubMed

    Vanas, Vanita; Mhlbacher, Elsa; Kral, Rosana; Sutterlty-Fall, Hedwig

    2014-05-01

    Sprouty proteins are modulators of mitogen-induced signal transduction processes and therefore can influence the process of cancerogenesis. In particular, Sprouty2 has been shown to have an important role in cancer development of many tumor entities including breast cancer. In this report, we investigated the role of Sprouty4 in breast cancer-derived cell lines. We have found that ectopic Sprouty4 expression inhibits cell proliferation of breast cancer cell lines independently of their endogenous expression levels. Corroborating Sprouty4 downregulation causes accelerated growth. Furthermore, we demonstrate that an increase in Sprouty4 content interferes with serum-induced activation of mitogen-activated protein kinase pathway. Additionally, Sprouty4 expression negatively influences cell migration. These data suggest that Sprouty4 is a possible candidate for a tumor suppressor in breast cancer. PMID:24402575

  18. Studies on the phenotype and karyotype of immortalized rabbit kidney epithelial cell lines.

    PubMed

    MacDonald, C; Watts, P; Stuart, B; Kreuzburg-Duffy, U; Scott, D M; Kinne, R K

    1991-08-01

    Differentiated mammalian cell lines can be isolated by immortalizing primary cells by transfection with DNA from plasmids containing sequences from SV40 early region. These cell lines show cytogenetic abnormalities but the degree of aneuploidy is considerably less than that observed in other established cell lines. No correlation was observed between the degree of differentiation of a clone and the extent of chromosomal damage. PMID:2070827

  19. Immune suppressor factor confers stromal cell line with enhanced supporting activity for hematopoietic stem cells

    SciTech Connect

    Nakajima, Hideaki . E-mail: hnakajim@ims.u-tokyo.ac.jp; Shibata, Fumi; Fukuchi, Yumi; Goto-Koshino, Yuko; Ito, Miyuki; Urano, Atsushi; Nakahata, Tatsutoshi; Aburatani, Hiroyuki; Kitamura, Toshio

    2006-02-03

    Immune suppressor factor (ISF) is a subunit of the vacuolar ATPase proton pump. We earlier identified a short form of ISF (ShIF) as a stroma-derived factor that supports cytokine-independent growth of mutant Ba/F3 cells. Here, we report that ISF/ShIF supports self-renewal and expansion of primary hematopoietic stem cells (HSCs). Co-culture of murine bone marrow cells with a stromal cell line overexpressing ISF or ShIF (MS10/ISF or MS10/ShIF) not only enhanced their colony-forming activity and the numbers of long-term culture initiating cells, but also maintained the competitive repopulating activity of HSC. This stem cell supporting activity depended on the proton-transfer function of ISF/ShIF. Gene expression analysis of ISF/ShIF-transfected cell lines revealed down-regulation of secreted frizzled-related protein-1 and tissue inhibitor of metalloproteinase-3, and the restoration of their expressions in MS10/ISF cells partially reversed its enhanced LTC-IC supporting activity to a normal level. These results suggest that ISF/ShIF confers stromal cells with enhanced supporting activities for HSCs by modulating Wnt-activity and the extracellular matrix.

  20. Establishment and Characterization of 7 Novel Hepatocellular Carcinoma Cell Lines from Patient-Derived Tumor Xenografts

    PubMed Central

    Hu, Gang; Xie, Fubo; Ouyang, Kedong; Tang, Xuzhen; Wang, Minjun; Wen, Danyi; Zhu, Yizhun; Qin, Xiaoran

    2014-01-01

    Hepatocellular carcinoma (HCC) is a common cancer with poor prognosis worldwide and the molecular mechanism is not well understood. This study aimed to establish a collection of human HCC cell lines from patient-derived xenograft (PDX) models. From the 20 surgical HCC sample collections, 7 tumors were successfully developed in immunodeficient mice and further established 7 novel HCC cell lines (LIXC002, LIXC003, LIXC004, LIXC006, LIXC011, LIXC012 and CPL0903) by primary culture. The characterization of cell lines was defined by morphology, growth kinetics, cell cycle, chromosome analysis, short tandem repeat (STR) analysis, molecular profile, and tumorigenicity. Additionally, response to clinical chemotherapeutics was validated both in vitro and in vivo. STR analysis indicated that all cell lines were unique cells different from known cell lines and free of contamination by bacteria or mycoplasma. The other findings were quite heterogeneous between individual lines. Chromosome aberration could be found in all cell lines. Alpha-fetoprotein was overexpressed only in 3 out of 7 cell lines. 4 cell lines expressed high level of vimentin. Ki67 was strongly stained in all cell lines. mRNA level of retinoic acid induced protein 3 (RAI3) was decreased in all cell lines. The 7 novel cell lines showed variable sensitivity to 8 tested compounds. LIXC011 and CPL0903 possessed multiple drug resistance property. Sorafenib inhibited xenograft tumor growth of LIXC006, but not of LIXC012. Our results indicated that the 7 novel cell lines with low passage maintaining their clinical and pathological characters could be good tools for further exploring the molecular mechanism of HCC and anti-cancer drug screening. PMID:24416385

  1. Alterations in radiation induced cell cycle perturbations by 2-deoxy-D-glucose in human tumor cell lines.

    PubMed

    Adhikari, J S; Dwarakanath, B S; Mathur, Rohit; Ravindranath, T

    2003-12-01

    In the present studies, effects of glucose analogue, 2-deoxy-D-glucose (2-DG) on radiation-induced cell cycle perturbations were investigated in human tumor cell lines. In unirradiated cells, the levels of cyclin B1 in G2 phase were significantly higher in both the glioma cell lines as compared to squamous carcinoma cells. Upon irradiation with Co60 gamma-rays (2 Gy), the cyclin B1 levels were reduced in U87 cells, while no significant changes could be observed in other cell lines, which correlated well with the transient G2 delay observed under these conditions by the BrdU pulse chase measurements. 2-DG (5 mM, 2 hr) induced accumulation of cells in the G2 phase and a time-dependent increase in the levels of cyclin B1 in both the glioma cell lines, while significant changes could not be observed in any of the squamous carcinoma cell lines. 2-DG enhanced the cyclin B1 level further in all the cell lines following irradiation, albeit to different extents. Interestingly, an increase in the unscheduled expression of B1 levels in G1 phase 48 hr after irradiation was observed in all the cell lines investigated. 2-DG also increased the levels of cyclin D1 at 24 hr in BMG-1 cell line. These observations imply that 2-DG-induced alterations in the cell cycle progression are partly responsible for its radiomodifying effects. PMID:15320491

  2. Chemical Degradation of Cathode Linings in Hall-Hroult CellsAn Autopsy Study of Three Spent Pot Linings

    NASA Astrophysics Data System (ADS)

    Tschpe, Kati; Schning, Cristian; Rutlin, Jrn; Grande, Tor

    2012-04-01

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

  3. 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.

  4. BST-1, a surface molecule of bone marrow stromal cell lines that facilitates pre-B-cell growth.

    PubMed Central

    Kaisho, T; Ishikawa, J; Oritani, K; Inazawa, J; Tomizawa, H; Muraoka, O; Ochi, T; Hirano, T

    1994-01-01

    Bone marrow stromal cells are essential for B-lymphocyte development. However, how stromal cells regulate B lymphopoiesis is not clear. In this paper, we report the molecular cloning of a stromal cell line-derived glycosyl-phosphatidylinositol-anchored molecule, BST-1, that facilitates pre-B-cell growth. The deduced amino acid sequence of BST-1 exhibited 33% identity with CD38. BST-1 was expressed in a wide range of tissues and in umbilical vein endothelial cells, whereas it was scarcely expressed in a variety of hematopoietic cell lines. The gene for BST-1 was assigned to chromosome 14q32.3, where immunoglobulin heavy-chain genes are clustered. BST-1 expression was enhanced in rheumatoid arthritis patient-derived bone marrow stromal cell lines that were previously shown to have an enhanced ability to support the growth of a pre-B-cell line as compared with stromal cell lines derived from healthy donors. Images PMID:8202488

  5. Metallothionein turnover in mammalian cell lines: implications in drug resistance

    SciTech Connect

    Monia, B.P.; Butt, T.R.; Ecker, D.J.; Mirabelli, C.K.; Crooke, S.T.

    1986-05-01

    Metallothioneins (MT) are low molecular weight, cysteine-rich proteins believed to participate in metal detoxification. A wide variety of cells in culture have been shown to accumulate MT in response to metal administration. These metal-induced increases in MT levels result from an increased rate of MT gene transcription, MT mRNA accumulation, and MT synthesis. Turnover of Cd-, Zn- and Au-induced MT was studied in a Chinese Hamster Ovary (CHO) cell line which was resistant to Cd and the Au-containing drug Auranofin (AF). Cd, Zn and Au were potent inducers of MT mRNA and accumulated approximately equal amounts of mRNA under the conditions employed in this study. Pulse-chase studies utilizing (/sup 35/S)cysteine revealed that the half-life of Au-, Zn- and Cd-induced MT was 0.75, 10 and 24 hrs. respectively. The reported differences in the tertiary structure of Au-MT from that of Cd-MT lead us to propose that the differences in half-lives observed reflect differences in subceptibility to intracellular proteolysis, which in turn, may effect the ability of MT to confer resistance to various metals.

  6. The endocannabinoid system in the human granulosa cell line KGN.

    PubMed

    Ernst, Jana; Grabiec, Urszula; Greither, Thomas; Fischer, Bernd; Dehghani, Faramarz

    2016-03-01

    Ovarian steroidogenesis is embedded in a sensitive network of regulatory mechanisms crucial for human fertility. The endocannabinoid system (ECS) represents an intrinsic modulating system involved in the regulation of endocrine functions. In the present study we characterized the ECS in the human granulosa cell line KGN and its impact on gonadotropin sensitivity and steroid hormone synthesis under basal and FSH-stimulated conditions. Expression studies were performed and estradiol was measured. CB1, CB2, DAGL, FAAH, GPR55, MAGL, NAPE-PLD and TRPV1 were expressed without FSH-dependent effects. Treatment with selective cannabinoid receptor agonists reduced basal but not FSH-stimulated estradiol and CYP19. Progesterone was not altered by ECS manipulation. CB1 agonist changed the expression of miRNAs associated with granulosa cell function, e.g. miR-23a, miR-24, miR-181a and miR-320a. Present data indicate a modulating role of the intrinsic ovarian ECS in the regulation of estradiol synthesis. PMID:26773729

  7. 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

  8. 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.

  9. The Human Lung Adenocarcinoma Cell Line EKVX Produces an Infectious Xenotropic Murine Leukemia Virus

    PubMed Central

    Cmarik, Joan L.; Troxler, Jami A.; Hanson, Charlotte A.; Zhang, Xiang; Ruscetti, Sandra K.

    2011-01-01

    The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells. PMID:22355448

  10. The human lung adenocarcinoma cell line EKVX produces an infectious xenotropic murine leukemia virus.

    PubMed

    Cmarik, Joan L; Troxler, Jami A; Hanson, Charlotte A; Zhang, Xiang; Ruscetti, Sandra K

    2011-12-01

    The cell lines of the NCI-60 panel represent different cancer types and have been widely utilized for drug screening and molecular target identification. Screening these cell lines for envelope proteins or gene sequences related to xenotropic murine leukemia viruses (X-MLVs) revealed that one cell line, EKVX, was a candidate for production of an infectious gammaretrovirus. The presence of a retrovirus infectious to human cells was confirmed by the cell-free transmission of infection to the human prostate cancer cell line LNCaP. Amplification and sequencing of additional proviral sequences from EKVX confirmed a high degree of similarity to X-MLV. The cell line EKVX was established following passage of the original tumor cells through nude mice, providing a possible source of the X-MLV found in the EKVX cells. PMID:22355448

  11. Alteration of protein phosphorylation patterns in cell lines morphologically transformed by human cytomegalovirus.

    PubMed

    Muganda-Ojiaku, P M; Huang, E S

    1987-05-01

    Human fibroblastic cell lines morphologically transformed by either live virus or DNA fragments of human cytomegalovirus had altered plasma membrane protein composition; quantitative changes, and gains and losses in protein composition in comparison to normal parent cell lines were detected. These transformed cell lines showed altered total cell protein phosphorylation patterns when compared to parent cell lines. A two to four fold increase in in vivo protein phosphorylation at serine and threonine residues was observed; no increase in phosphorylation at total cell tyrosine residues was detected. Analysis of the in vivo phosphorylated protein by two dimensional gel electrophoresis revealed some similarities as well as differences in the types of polypeptides phosphorylated between transformed and control cell lines. Increased (two-to sixfold over parent cell extracts) casein kinase and polyamine dependent casein kinase activities were detected in HCMV transformed cell extracts. PMID:2441849

  12. 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

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

    PubMed

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

    2015-10-01

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

  14. Increased sensitivity of a xeroderma pigmentosum lymphoblastoid cell line to serum deprivation in vitro.

    PubMed

    Lambert, W C; Lambert, M W

    1983-08-01

    Two human lymphoblastoid cell lines, GM 1989, from a normal individual, and GM 2345, from a patient with xeroderma pigmentosum, complementation group A, were selected for comparative biochemical studies because they both grow rapidly and at virtually identical rates in sealed flasks in RPMI 1640 medium buffered to physiological pH with HEPES buffer, supplemented with 12% heat-inactivated fetal bovine serum. Although the two cell lines showed no difference in growth parameters assayed by standard methods, further studies showed that the GM 2345 cell line was markedly more sensitive to diminution of the serum concentration of the culture medium than was the normal cell line. These results indicate that lymphoblastoid cell lines, particularly those from individuals with certain genetic or metabolic diseases, may be growing under marginal or limiting circumstances, different from those of control cell lines, which are not detected by standard techniques used to monitor mammalian cell cultures. PMID:6885101

  15. Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

    PubMed Central

    Robin, Jerome D; Wright, Woody; Zou, Yaqun; Cossette, Stacy; Lawlor, Michael

    2015-01-01

    The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patients diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications. PMID:25651101

  16. The differentiation antigen Ly-6E.1 is expressed in mouse metastatic tumor cell lines.

    PubMed

    Cohn, M A; Kramerov, D; Hulgaard, E F; Lukanidin, E M

    1997-02-17

    We report the cloning of the mouse surface GPI-anchored Ly-6E.1 protein from a highly metastatic mouse adenocarcinoma cell line CSML-100 by differential display. The expression is specific for the metastatic cell line as the closely related, non-metastatic mouse adenocarcinoma cell line CSML-0 does not express Ly-6E.1. Northern blot analysis reveals expression in a number of mouse tumour cell lines, exclusively metastatic ones. To date, active Ly-6A/E has only been described in lymphoid cells. The correlation between Ly-6E.1 expression, and the ability to metastasize, is discussed. PMID:9042962

  17. Differentiation potential of germ line stem cells derived from the postnatal mouse ovary.

    PubMed

    Pacchiarotti, Jason; Maki, Chad; Ramos, Thomas; Marh, Joel; Howerton, Kyle; Wong, Jadelind; Pham, Jane; Anorve, Sandra; Chow, Yung-Chiong; Izadyar, Fariborz

    2010-03-01

    General belief in reproductive biology is that in most mammals female germ line stem cells are differentiated to primary oocytes during fetal development and oogenesis starts from a pool of primordial follicles after birth. This idea has been challenged previously by using follicle kinetics studies and demonstration of mitotically active germ cells in the postnatal mouse ovary (Johnson et al., 2004; Kerr et al., 2006; Zhang et al., 2008). However, the existence of a population of self-renewing ovarian germ line stem cells in postnatal mammals is still controversial (Eggan et al., 2006; Telfer et al., 2005; Gosden, 2004). Recently, production of offspring from a germ line stem cell line derived from the neonatal mouse ovary was reported (Zou et al., 2009). This report strongly supports the existence of germ line stem cells and their ability to expand in vitro. Recently, using a transgenic mouse model in which GFP is expressed under a germ cell-specific Oct-4 promoter, we isolated and generated multipotent cell lines from male germ line stem cells (Izadyar et al., 2008). Using the same strategy we isolated and derived cell lines from postnatal mouse ovary. Interestingly, ovarian germ line stem cells expanded in the same culture conditions as the male suggesting that they have similar requirements for their self-renewal. After 1 year of culture and many passages, ovarian germ line stem cells maintained their characteristics and telomerase activity, expressed germ cell and stem cell markers and revealed normal karyotype. As standard protocol for differentiation induction, these cells were aggregated and their ability to form embryoid bodies (EBs) was investigated. EBs generated in the presence of growth factors showed classical morphology and expressed specific markers for three germ layers. However, in the absence of growth promoting factors EBs were smaller and large cells with the morphological and molecular characteristics of oocytes were formed. This study shows the existence of a population of germ line stem cell in postnatal mouse ovary with multipotent characteristics. PMID:20138422

  18. Monochloramine-induced cell growth inhibition and apoptosis in a rat gastric mucosal cell line.

    PubMed

    Naito, Y; Yoshikawa, T; Fujii, T; Boku, Y; Yagi, N; Dao, S; Yoshida, N; Kondo, M; Matsui, H; Ohtani-Fujita, N; Sakai, T

    1997-01-01

    Recent studies have indicated that monochloramine (NH2Cl), a reaction product of NH3 and hypochlorous acid, is involved in the pathogenesis of Helicobacter pylori-associated gastric mucosal damage, but how NH2Cl contributes to lesions is unclear. In the present study, the effects of NH2Cl on mucosal cell growth and the cell cycle were evaluated in vitro using a normal rat gastric mucosal cell line RGM-1. Cell viability was assessed by the Trypan Blue dye exclusion test and cell cycle patterns were determined by DNA labeling with propidium iodide and flow cytometric quantification. NH2Cl inhibited the growth of RGM-1 cells in a concentration-dependent manner. Exposure of cells to NH2Cl caused a time- and dose-dependent loss of G1-phase cells with accumulation of G2/M-phase cells, and produced a fraction of subdiploid cells with oligonucleosomal DNA degradation characteristic of apoptosis. NH2Cl-induced apoptosis was confirmed by fluorescent microscopy with Hoechst 33342 and propidium iodide. These results suggest that NH2Cl inhibits gastric mucosal cell growth and induces apoptosis in RGM-1 cells, events that may be important in gastric mucosal damage or atrophy induced by H. pylori infection. PMID:9479646

  19. Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines

    PubMed Central

    Swaminathan, Vinay; Mythreye, Karthikeyan; OBrien, E Tim; Berchuck, Andrew; Blobe, Gerard C; Superfine, Richard

    2011-01-01

    Cancer cells are defined by their ability to invade through the basement membrane, a critical step during metastasis. While increased secretion of proteases, which facilitates degradation of the basement membrane, and alterations in the cytoskeletal architecture of cancer cells have been previously studied, the contribution of the mechanical properties of cells in invasion is unclear. Here we apply a magnetic tweezer system to establish that stiffness of patient tumor cells and cancer cell lines inversely correlates with migration and invasion through three-dimensional basement membranes, a correlation known as a power law. We found that cancer cells with the highest migratory and invasive potential are five times less stiff than cells with the lowest migration and invasion potential. Moreover, decreasing cell stiffness by pharmacological inhibition of myosin II increases invasiveness, while increasing cell stiffness by restoring expression of the metastasis suppressor T?RIII/betaglycan decreases invasiveness. These findings are the first demonstration of the power law relation between the stiffness and the invasiveness of cancer cells and show that mechanical phenotypes can be used to grade the metastatic potential of cell populations with the potential for single cell grading. The measurement of a mechanical phenotype, taking minutes rather than hours needed for invasion assays, is promising as a quantitative diagnostic method and as a discovery tool for therapeutics. By demonstrating that altering stiffness predictably alters invasiveness, our results indicate that pathways regulating these mechanical phenotypes are novel targets for molecular therapy of cancer. PMID:21642375

  20. Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: identification of tumor origin by idiotypic analysis.

    PubMed Central

    Hurley, J N; Fu, S M; Kunkel, H G; McKenna, G; Scharff, M D

    1978-01-01

    Multiple lymphoblastoid cell lines have been derived from two patients with chronic lymphocytic leukemia with an associated monoclonal immunoglobulin (Ig) band. Idiotypic antisera raised against the monoclonal serum Ig bands were shown to be specific for the membrane Ig of the patients' leukemic cells. The idiotypic determinants in these patients thereby constitute tumor-specific antigens. Surface and intracellular immunofluorescence studies utilizing these idiotypic antisera were used to identify the cell lines of leukemic origin. These studies showed that certain cell lines from each patient were derived from the leukemic cells while other cell lines were derived from residual normal B lymphocytes. The leukemic cell lines were variable and contained different percentages of lymphoid cells with the idiotype-specific membrane Ig and, in addition, different percentages of plasma cells with intracellular Ig of the same specificity. Specific Ig synthesis was also demonstrated by hemagglutination-inhibition analysis of cell line supernatants. Aside from Ig specificity, no differences have been found between the leukemic cell lines and those derived from normal cells. One of the leukemic cell lines was cloned in soft agarose. All the clones were shown to be of leukemic origin. Images PMID:82971

  1. Functional characterization of gefitinib uptake in non-small cell lung cancer cell lines.

    PubMed

    Galetti, Maricla; Alfieri, Roberta R; Cavazzoni, Andrea; La Monica, Silvia; Bonelli, Mara; Fumarola, Claudia; Mozzoni, Paola; De Palma, Giuseppe; Andreoli, Roberta; Mutti, Antonio; Mor, Marco; Tiseo, Marcello; Ardizzoni, Andrea; Petronini, Pier Giorgio

    2010-07-15

    Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC). In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2. After 15min of treatment at 1microM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment. Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation. PMID:20363215

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

    SciTech Connect

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

    2013-11-15

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

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

    PubMed Central

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

    2015-01-01

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

  4. Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

    PubMed Central

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

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

  5. Characterization of three cell lines derived from fine needle biopsy of choroidal melanoma with metastatic outcome

    PubMed Central

    Burgess, Barry L.; Rao, Nagesh P.; Eskin, Ascia; Nelson, Stanley F.

    2011-01-01

    Purpose To report three low-passage cell lines from primary choroidal melanoma with metastatic outcome, which were stable for cytogenetic patterns and expression profiles of the primary melanoma. Methods In patients with choroidal melanoma, transscleral fine needle aspiration biopsy (FNAB) was performed immediately before plaque placement for 125iodine brachytherapy or immediately after enucleation. Cells were examined for cytopathology, evaluated by fluorescence in-situ hybridization (FISH) for the centromere of chromosome 3, analyzed by 250K whole genome Mapping Array and U133 plus 2.0 Expression Array, and placed in cell culture. At passage 3, the cell lines were analyzed by Mapping Array and Expression Array. Results Three cell lines were propagated from primary choroidal melanomas in three patients who subsequently developed metastasis. Two cell lines were stable for the entire chromosomal aberration pattern of the respective primary tumor. In the third, necrotic material from the biopsy prevented further analysis, yet resulted in a stable cell line. Each cell line had chromosome 3 loss, 6q loss, 8p loss, multiple 8q gain, and 16q loss. Additionally, two cell lines had chromosome 6p gain. Two cell lines had RNA expression profiles similar to the respective primary tumors; the third cell line had a similar RNA expression profile relative to the other two cell lines. Conclusions FNAB of primary choroidal melanomas resulted in highly characterized, low-passage cell lines, which were stable for the cytogenetic patterns and expression profiles found in the primary tumor. These cell lines represent novel tools for the study of metastatic choroidal melanoma biology. PMID:21386926

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

    PubMed

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

    2000-02-01

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

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

    SciTech Connect

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

    2010-06-04

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

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2000-04-14

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

  11. Akabane Virus Utilizes Alternative Endocytic Pathways to Entry into Mammalian Cell Lines

    PubMed Central

    BANGPHOOMI, Norasuthi; TAKENAKA-UEMA, Akiko; SUGI, Tatsuki; KATO, Kentaro; AKASHI, Hiroomi; HORIMOTO, Taisuke

    2014-01-01

    ABSTRACT The entry mechanisms of Akabane virus (AKAV), Bunyaviridae family, have not yet been determined. In this study, chemical inhibitors were used to analyze endocytic mechanisms during AKAV infection of mammalian cell lines. The analyses using drug treatments followed by quantitative measurement of viral RNA and N protein revealed that AKAV enters non-bovine-derived cell lines (Vero, HmLu-1 and BHK cells) in a manner indicative of clathrin endocytosis. By contrast, AKAV infection in bovine-derived cell lines (LB9.K and MDBK cells) is independent of this pathway. Further analyses indicated that AKAV entry into bovine cell lines involves a non-clathrin, non-caveolae endocytic pathway that is dependent on dynamin. We conclude that although both cell types require a low pH for AKAV penetration, AKAV utilizes alternative entry pathways into mammalian cell lines. PMID:25056673

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

    PubMed Central

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

    1983-01-01

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

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

    PubMed Central

    2011-01-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Comparison of initial feasibility of host cell lines for viral vaccine production.

    PubMed

    Vlecken, Danielle H W; Pelgrim, Ralf P M; Ruminski, Slawomir; Bakker, Wilfried A M; van der Pol, Leo A

    2013-10-01

    In order to reduce the time required for the development and production of viral vaccines, host cell lines should be available as expression systems for production of viral vaccines against groups of viral pathogens. A selection of cell lines was compared for their initial feasibility as expression system for the replication of polioviruses, influenza A viruses and respiratory syncytial virus (wild type strain A2). Six adherent cell lines (Vero, HEK-293, MRC-5, CHO-K1, BHK-21 c13, MDCK) and six single cell suspension cell lines (CAP, AGE1.CR.HS, sCHO-K1, BHK-21 c13 2p, MDCK SFS) were studied for their ability to propagate viruses. First, maximum cell densities were determined. Second, virus receptor expression and polarization of the cell lines regarding receptor distribution of eight different viruses were monitored using flow cytometry and immunocytochemistry. Organization of the actin cytoskeleton was studied by transfection of the cells with Lifeact, a construct coding for actin-EGFP. Finally, the ability to produce virus progeny of the viruses studied was assayed for each cell line. The results suggest that single cell suspension cell lines grown on serum free medium are the best candidates to serve as host cell lines for virus replication. PMID:23684847

  16. 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 ...

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

    SciTech Connect

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

    2006-11-01

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

  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. PMID:26131123

  19. Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

    PubMed Central

    Roh, Cecilia; Roche, Michael; Guo, Zhiru; Photopoulos, Christos; Tao, Qingfeng; Lyle, Stephen

    2009-01-01

    We previously demonstrated that keratin-15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin-15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 months of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland and hair follicle lineages. Intact ?-catenin dependent signaling which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation. PMID:18568376

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

    SciTech Connect

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

    2010-05-04

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

  1. The Influence of Clinical and Demographic Risk Factors on the Establishment of Head and Neck Squamous Cell Carcinoma Cell Lines

    PubMed Central

    White, Jason S.; Weissfeld, Joel L.; Ragin, Camille C. R.; Rossie, Karen M.; Martin, Christa Lese; Shuster, Michele; Ishwad, Chandramohan S.; Law, John C.; Myers, Eugene N.; Johnson, Jonas T.; Gollin, Susanne M.

    2007-01-01

    The purpose of this study was to generate stable cell cultures from head and neck squamous cell carcinomas (HNSCC), and retrospectively analyze the factors associated with successful cell line establishment. Fifty-two HNSCC cell lines were isolated from a series of 199 tumors collected between 1992 and 1997 at the University of Pittsburgh Medical Center. Cell lines were characterized at the molecular and cellular level to determine the features associated with cell line formation. Successful cell line formation was dependent on multiple factors, including gene amplification involving chromosomal band 11q13, local and/or regional involvement of lymph nodes, and alcohol usage. The establishment of HNSCC cell lines enriches the resources available for cancer research. Our findings indicate that generation of stable cell lines from HNSCC is biased towards tumors with a poor prognosis. Our 52 stable lines comprise one of the largest series of HNSCC cell lines in the literature, with complete demographic, histopathologic, clinical, and survival data. PMID:17112776

  2. 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

  3. 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

  4. Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line.

    PubMed

    Lee, Suk Kyoo; Lee, Gyun Min

    2003-06-30

    Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins. PMID:12701155

  5. Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

    SciTech Connect

    Ageberg, Malin; Rydstroem, Karin; Linden, Ola; Linderoth, Johan; Jerkeman, Mats; Drott, Kristina

    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.

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

    PubMed Central

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

    2015-01-01

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

  7. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude

  8. Glioma cells on the run the migratory transcriptome of 10 human glioma cell lines

    PubMed Central

    Demuth, Tim; Rennert, Jessica L; Hoelzinger, Dominique B; Reavie, Linsey B; Nakada, Mitsutoshi; Beaudry, Christian; Nakada, Satoko; Anderson, Eric M; Henrichs, Amanda N; McDonough, Wendy S; Holz, David; Joy, Anna; Lin, Richard; Pan, Kuang H; Lih, Chih J; Cohen, Stan N; Berens, Michael E

    2008-01-01

    Background Glioblastoma multiforme (GBM) is the most common primary intracranial tumor and despite recent advances in treatment regimens, prognosis for affected patients remains poor. Active cell migration and invasion of GBM cells ultimately lead to ubiquitous tumor recurrence and patient death. To further understand the genetic mechanisms underlying the ability of glioma cells to migrate, we compared the matched transcriptional profiles of migratory and stationary populations of human glioma cells. Using a monolayer radial migration assay, motile and stationary cell populations from seven human long term glioma cell lines and three primary GBM cultures were isolated and prepared for expression analysis. Results Gene expression signatures of stationary and migratory populations across all cell lines were identified using a pattern recognition approach that integrates a priori knowledge with expression data. Principal component analysis (PCA) revealed two discriminating patterns between migrating and stationary glioma cells: i) global down-regulation and ii) global up-regulation profiles that were used in a proband-based rule function implemented in GABRIEL to find subsets of genes having similar expression patterns. Genes with up-regulation pattern in migrating glioma cells were found to be overexpressed in 75% of human GBM biopsy specimens compared to normal brain. A 22 gene signature capable of classifying glioma cultures based on their migration rate was developed. Fidelity of this discovery algorithm was assessed by validation of the invasion candidate gene, connective tissue growth factor (CTGF). siRNA mediated knockdown yielded reduced in vitro migration and ex vivo invasion; immunohistochemistry on glioma invasion tissue microarray confirmed up-regulation of CTGF in invasive glioma cells. Conclusion Gene expression profiling of migratory glioma cells induced to disperse in vitro affords discovery of genomic signatures; selected candidates were validated clinically at the transcriptional and translational levels as well as through functional assays thereby underscoring the fidelity of the discovery algorithm. PMID:18230158

  9. 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.

  10. Differential cell surface antigen expression on metastatic variant lymphoma cell lines.

    PubMed

    Joshi, S S; Brunson, K W

    1990-01-01

    In the present investigation we have studied the cell surface antigenicity of a syngeneic murine metastatic lymphoma model system. This system is comprised of a highly malignant and metastatic RAW117-H10 subline and the less malignant/metastatic parental RAW117-P cell line. Using rabbit antisera raised against whole tumor cells we have been able to identify two major glycoprotein antigens which are differentially expressed on the cells. Although these antigens have a similar molecular weight (70 kD), they are antigenically distinct as determined by in vitro cytotoxicity assays. The levels of glycosylation on these glycoproteins were also found to be different. Increased expression of one of the antigenic components on the metastatic RAW117-H10 cells appeared to be associated with metastasis in this tumor model system. PMID:1694982

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

    PubMed

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

    2015-01-01

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

  12. Characterization of a porcine intestinal epithelial cell line for influenza virus production.

    PubMed

    Sun, Zhi; Huber, Victor C; McCormick, Kara; Kaushik, Radhey S; Boon, Adrianus C M; Zhu, Longchao; Hause, Ben; Webby, Richard J; Fang, Ying

    2012-09-01

    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 log(10) TCID(50) 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 log(10) TCID(50) 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