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Sample records for h9c2 cell line

  1. Effect of AZT on thymidine phosphorylation in cultured H9c2, U-937, and Raji cell lines.

    PubMed

    Lynx, Matthew D; Kang, Bae-Kwang; McKee, Edward E

    2008-04-15

    3'-azido-3'-deoxythymidine (AZT) has been shown to be a potent inhibitor of thymidine kinase 2 in work from this laboratory. Inhibition results in decreased salvage of thymidine to TTP, which may lead to depletion of the TTP pool and result in the mitochondrial dysfunction and mt-DNA depletion observed with AZT toxicity. The effect of AZT on thymidine phosphorylation in growing cells expressing thymidine kinase 1 has not been shown. Three cell lines were used in these experiments: H9c2, derived from rat cardiomyoblasts; U-937, derived from human monocytes; and Raji, derived from human lymphoblasts. AZT inhibited growth in a concentration-dependent manner in U-937 cells, but not the other cell lines. The phosphorylation of [3H]-thymidine or [3H]-AZT was determined during log growth. All cell lines salvaged and phosphorylated thymidine to TTP, with TTP the major product. The U-937 cells had a much more active salvage pathway than the other cells. All cell lines phosphorylated AZT to the triphosphate, but the major product was AZTMP. The AZT inhibition of growth of the U-937 cells did not correlate with levels of the phosphorylated AZT. In contrast, pro-drug AZT was shown to inhibit thymidine phosphorylation in all lines with 50% inhibition concentrations (IC50) ranging from 4.4 to 21.9muM. Since the U-937 cells expressed higher activity of the salvage pathway than the other cell lines, the U-937 cells may rely more heavily on the salvage pathway for TTP synthesis, accounting for AZT inhibition of growth. PMID:18295188

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

    PubMed

    Zamperone, Andrea; Pietronave, Stefano; Colangelo, Donato; Antonini, Silvia; Locatelli, Monica; Travaglia, Fabiano; 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

  3. Factors released from embryonic stem cells inhibit apoptosis of H9c2 cells.

    PubMed

    Singla, Dinender K; McDonald, Debbie E

    2007-09-01

    Our recent study (Singla DK, Hacker TA, Ma L, Douglas PS, Sullivan R, Lyons GE, Kamp TJ, J Mol Cell Cardiol 40: 195-200, 2006) suggests that transplanted embryonic stem (ES) cells subsequent to myocardial infarction differentiate into the major cell types in the heart and improve cardiac function. However, the extent of regeneration is relatively meager compared with the observed functional improvement. The mechanisms underlying their improved function are completely unknown. In this report, we provide evidence using a cell culture model system for novel mechanisms that involve the release of cytoprotective, anti-apoptotic factor(s) from ES cells and inhibit H(2)O(2)-induced apoptosis in the rat cardiomyocyte-derived cell line H9c2. Conditioned medium (CM) from growing mouse ES cells treated with and without H(2)O(2) was generated. Apoptosis was induced after exposure to H(2)O(2) in H9c2 cells for 2 h followed by replacement with fresh cell culture or ES cell-CM. After 24 h, H9c2 cells treated with both ES cell-CMs demonstrated significantly decreased apoptosis, as determined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining, apoptotic ELISA, caspase-3 activity, and DNA ladder. Next, using Luminex technology, we examined the presence of antiapoptotic proteins cystatin c, osteopontin, and clusterin and anti-fibrotic, tissue inhibitor of metalloproteinase-1 (TIMP-1) in both ES cell-CMs. The levels of released factors were 2- to 170-fold higher than those in H9c2 cell-CM. Antiapoptotic effects of ES cell-CM were significantly inhibited with TIMP-1 antibody, suggesting that TIMP-1 is an important factor to inhibit apoptosis. Furthermore, we used CM from an TIMP-1-overexpressing cell line and demonstrated that H(2)O(2)-induced apoptosis in the H9c2 cells was significantly inhibited. These observations demonstrate that factors released from ES cells contain antiapoptotic factors and that the effects are mediated by TIMP-1. Moreover, these findings suggest that released factors might be useful for therapeutic applications in ischemic heart disease as well as for many other diseases. PMID:17545477

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

    PubMed

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

    2015-11-01

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

  5. 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

  6. 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

  7. 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

  8. Lipocalin-2 inhibits autophagy and induces insulin resistance in H9c2 cells.

    PubMed

    Chan, Yee Kwan; Sung, Hye Kyoung; Jahng, James Won Suk; Kim, Grace Ha Eun; Han, Meng; Sweeney, Gary

    2016-07-15

    Lipocalin-2 (Lcn2; also known as neutrophil gelatinase associated lipocalin, NGAL) levels are increased in obesity and diabetes and associate with insulin resistance. Correlations exist between Lcn2 levels and various forms or stages of heart failure. Insulin resistance and autophagy both play well-established roles in cardiomyopathy. However, little is known about the impact of Lcn2 on insulin signaling in cardiomyocytes. In this study, we treated H9c2 cells with recombinant Lcn2 for 1 h followed by dose- and time-dependent insulin treatment and found that Lcn2 attenuated insulin signaling assessed via phosphorylation of Akt and p70S6K. We used multiple assays to demonstrate that Lcn2 reduced autophagic flux. First, Lcn2 reduced pULK1 S555, increased pULK1 S757 and reduced LC3-II levels determined by Western blotting. We validated the use of DQ-BSA to assess autolysosomal protein degradation and this together with MagicRed cathepsin B assay indicated that Lcn2 reduced lysosomal degradative activity. Furthermore, we generated H9c2 cells stably expressing tandem fluorescent RFP/GFP-LC3 and this approach verified that Lcn2 decreased autophagic flux. We also created an autophagy-deficient H9c2 cell model by overexpressing a dominant-negative Atg5 mutant and found that reduced autophagy levels also induced insulin resistance. Adding rapamycin after Lcn2 could stimulate autophagy and recover insulin sensitivity. In conclusion, our study indicated that acute Lcn2 treatment caused insulin resistance and use of gain and loss of function approaches elucidated a causative link between autophagy inhibition and regulation of insulin sensitivity by Lcn2. PMID:27090568

  9. 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 μM) 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

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. Calcium as a modulator of photosensitized killing of H9c2 cardiac cells.

    PubMed

    Valenzeno, D P; Tarr, M

    2001-10-01

    Illumination of H9c2 rat heart cells in the presence of Rose Bengal resulted in dose-dependent cell killing (assessed by trypan blue staining) and modification of ionic currents flowing through the heart cell membrane. Inhibitors of voltage-gated ionic currents were shown to have little effect on cell killing. Ionic current measurements were used to assess the increase in leak conductance of these cells, which has been suggested to be a causal factor in killing of other cell types (1). Inhibitors of voltage-gated ionic currents, including the sodium channel blocker tetrodotoxin (100 microM) and the calcium channel blocker lanthanum (10 microM) were shown to have little effect on cell killing. The potassium channel inhibitor tetraethylammonium (20 mM) inhibited cell killing, but the effect is viewed as being caused by an inhibition of leak current. The time course of block of voltage-activated ionic currents during illumination, in the presence of Rose Bengal, was rapid compared with that for induction of leak current and for cell killing. These observations are consistent with a role for leak current in photosensitized killing of cardiac cells. They are interpreted with respect to calcium influx through the leak current pathway as a trigger for the cellular response. PMID:11683041

  13. 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-γ/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

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

    SciTech Connect

    Zhao Yuqin; Xue Tao; Yang Xiaochun; Zhu Hong; Ding Xiaofei; Lou Liming; 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.

  15. 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

  16. Hydrogen sulfide attenuates doxorubicin‑induced cardiotoxicity by inhibiting calreticulin expression in H9c2 cells.

    PubMed

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

    2015-10-01

    Doxorubicin (DOX) is a potent and currently available antitumor therapeutic agent; however, its clinical application is limited by the occurrence of cardiotoxicity. Preliminary evidence indicates that hydrogen sulfide (H2S) may exert protective effects against DOX cardiotoxicity. Therefore, the aim of the present study was to investigate whether calreticulin (CRT) is involved in the cardioprotection of H2S against DOX‑induced cardiotoxicity. DOX was observed to markedly induce injuries, including cytotoxicity and apoptosis, and also enhance the expression level of CRT. Notably, pretreatment of H9c2 cells with sodium hydrosulfide (a donor of H2S) significantly attenuated the decreased cell viability, the increased apoptosis rate and the increased expression level of CRT in H9c2 cells. In addition, pretreatment of H9c2 cells with N‑acetyl‑L‑cysteine, a scavenger of reactive oxygen species (ROS) prior to exposure to DOX, markedly decreased the expression of CRT. These results indicate that exogenous H2S attenuates DOX‑induced cardiotoxicity by inhibiting CRT expression in H9c2 cardiac cells. PMID:26134131

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

    PubMed

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

    2016-01-01

    Doxorubicin (DOX) is a widely used chemotherapeutic agent, which can give rise to severe cardiotoxicity, limiting its clinical use. Preliminary evidence suggests that hydrogen sulfide (H2S) may exert protective effects on DOX‑induced cardiotoxicity. Therefore, the aim of the present study was to investigate whether peroxiredoxin III is involved in the cardioprotection of H2S against DOX‑induced cardiotoxicity. The results demonstrated that DOX not only markedly induced injuries, including cytotoxicity and apoptosis, it also increased the expression levels of peroxiredoxin III. Notably, pretreatment with sodium hydrosulfide significantly attenuated the DOX‑induced decrease in cell viability and increase in apoptosis, and also reversed the increased expression levels of peroxiredoxin III in H9c2 cardiomyocytes. In addition, pretreatment of the H9c2 cells with N‑acetyl‑L‑cysteine, a scavenger of reactive oxygen species, prior to exposure to DOX markedly decreased the expression levels of peroxiredoxin III. In conclusion, the results of the present study suggested that exogenous H2S attenuates DOX‑induced cardiotoxicity by inhibiting the expression of peroxiredoxin III in H9c2 cells. In the present study, the apoptosis of H9c2 cardiomyocytes was assessed using an methyl thiazolyl tetrazolium assay and Hoechst staining. The levels of Prx III and cystathionine-γ-lyase were examined by western blotting. PMID:26573464

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

    PubMed

    Li, Bo; Kim, Do Sung; Yadav, Raj Kumar; Kim, Hyung Ryong; Chae, Han Jung

    2015-07-01

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

  19. 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

  20. 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

  1. Lysophosphatidylcholine increases Na+/Ca2+ exchanger expression via RhoB-geranylgeranylation in H9c2 cells.

    PubMed

    Maeda, Sachiko; Sakamoto, Kazuho; Matsuoka, Isao; Iwamoto, Takahiro; Kimura, Junko

    2009-04-01

    The expression levels of the Na(+)/Ca(2+) exchanger type 1 (NCX1) change under various cardiac pathophysiological conditions, but the mechanism is unknown. We previously demonstrated that lysophosphatidylcholine (LPC) increased NCX1 expression by activating RhoB in H9c2 cardiomyoblasts. Conversely, fluvastatin (Flv), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, decreased NCX1 mRNA and protein expression by inhibiting RhoB. RhoB can be isoprenylated by either geranylgeranylpyrophosphate (GGPP) or farnesylpyrophosphate (FPP). Here we investigated which of GGPP or FPP is involved in the NCX1-increasing effect of LPC. When LPC was added with GGPP to the Flv-treated H9c2 cells, NCX1 mRNA was increased to a level significantly higher than that in the control cells. Only GGPP, but not FPP, allowed LPC to increase NCX1 mRNA in the presence of Flv. Furthermore, geranylgeranyltransferase 1 inhibitor (GGTI), but not farnesyltransferase inhibitor (FTI), inhibited the LPC-induced NCX1 mRNA increase. We conclude that geranylgeranylation, but not farnesylation, of RhoB mediates LPC-induced NCX1 mRNA increase in H9c2 cells. PMID:19352075

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

    PubMed Central

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

    2007-01-01

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

  3. 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

  4. 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

  5. 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

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

    PubMed

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

    2015-06-01

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

  7. 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.

  8. Arsenic trioxide toxicity in H9c2 myoblasts--damage to cell organelles and possible amelioration with Boerhavia diffusa.

    PubMed

    Vineetha, V P; Prathapan, A; Soumya, R S; Raghu, K G

    2013-06-01

    Arsenic trioxide (ATO) has been long used as a chemotherapeutic agent because of its significant anticancer property. Unfortunately, the use of ATO is limited due to its cardiotoxic effects. The present study evaluates the protective property of ethanolic extract of Boerhavia diffusa (BDE) against ATO-induced toxicity on various cell organelles in H9c2 cardiomyocytes. The effects of different concentrations of ATO (5, 7.5 and 10 μM) on cell organelles like mitochondria, endoplasmic reticulum (ER), lysosome and actin, generation of reactive oxygen species, antioxidant enzyme status and intracellular calcium overload were evaluated. ATO significantly (P ≤ 0.05) altered mitochondrial transmembrane potential, intracellular calcium level, ER, lysosomal activity and F-actin network in addition to induction of oxidative stress. Co-treatment with BDE protected the cardiomyocytes from the adverse effects of ATO, especially at 5 μM concentration, which was evident from decreased activity of lactate dehydrogenase (5 μM ATO + 20 μg/mL BDE: 6.61 ± 1.97 μU/mL, respective control group: 16.15 ± 1.92 μU/mL), reduced oxidative stress, calcium influx and organelle damage. Results obtained from the present study allow for a better characterization of the effects of ATO on H9c2 myoblasts. In conclusion, our data suggest that cell organelles are also the targets of ATO-induced cardiotoxicity in addition to other reported targets like ion channels, and BDE has the potential to protect the cardiotoxicity induced by ATO. PMID:23161055

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

    PubMed

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

    2015-10-30

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

  10. 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

  11. 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

  12. 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

  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. Protective Effect of Boerhaavia diffusa L. against Mitochondrial Dysfunction in Angiotensin II Induced Hypertrophy in H9c2 Cardiomyoblast Cells

    PubMed Central

    Prathapan, Ayyappan; Vineetha, Vadavanath Prabhakaran; Raghu, Kozhiparambil Gopalan

    2014-01-01

    Mitochondrial dysfunction plays a critical role in the development of cardiac hypertrophy and heart failure. So mitochondria are emerging as one of the important druggable targets in the management of cardiac hypertrophy and other associated complications. In the present study, effects of ethanolic extract of Boerhaavia diffusa (BDE), a green leafy vegetable against mitochondrial dysfunction in angiotensin II (Ang II) induced hypertrophy in H9c2 cardiomyoblasts was evaluated. H9c2 cells challenged with Ang II exhibited pathological hypertrophic responses and mitochondrial dysfunction which was evident from increment in cell volume (49.09±1.13%), protein content (55.17±1.19%), LDH leakage (58.74±1.87%), increased intracellular ROS production (26.25±0.91%), mitochondrial superoxide generation (65.06±2.27%), alteration in mitochondrial transmembrane potential (ΔΨm), opening of mitochondrial permeability transition pore (mPTP) and mitochondrial swelling. In addition, activities of mitochondrial respiratory chain complexes (I-IV), aconitase, NADPH oxidase, thioredoxin reductase, oxygen consumption rate and calcium homeostasis were evaluated. Treatment with BDE significantly prevented the generation of intracellular ROS and mitochondrial superoxide radicals and protected the mitochondria by preventing dissipation of ΔΨm, opening of mPTP, mitochondrial swelling and enhanced the activities of respiratory chain complexes and oxygen consumption rate in H9c2 cells. Activities of aconitase and thioredoxin reductase which was lowered (33.77±0.68% & 45.81±0.71% respectively) due to hypertrophy, were increased in BDE treated cells (P≤0.05). Moreover, BDE also reduced the intracellular calcium overload in Ang II treated cells. Overall results revealed the protective effects of B. diffusa against mitochondrial dysfunction in hypertrophy in H9c2 cells and the present findings may shed new light on the therapeutic potential of B. diffusa in addition to its nutraceutical potentials. PMID:24788441

  16. Protective effect of Boerhaavia diffusa L. against mitochondrial dysfunction in angiotensin II induced hypertrophy in H9c2 cardiomyoblast cells.

    PubMed

    Prathapan, Ayyappan; Vineetha, Vadavanath Prabhakaran; Raghu, Kozhiparambil Gopalan

    2014-01-01

    Mitochondrial dysfunction plays a critical role in the development of cardiac hypertrophy and heart failure. So mitochondria are emerging as one of the important druggable targets in the management of cardiac hypertrophy and other associated complications. In the present study, effects of ethanolic extract of Boerhaavia diffusa (BDE), a green leafy vegetable against mitochondrial dysfunction in angiotensin II (Ang II) induced hypertrophy in H9c2 cardiomyoblasts was evaluated. H9c2 cells challenged with Ang II exhibited pathological hypertrophic responses and mitochondrial dysfunction which was evident from increment in cell volume (49.09±1.13%), protein content (55.17±1.19%), LDH leakage (58.74±1.87%), increased intracellular ROS production (26.25±0.91%), mitochondrial superoxide generation (65.06±2.27%), alteration in mitochondrial transmembrane potential (ΔΨm), opening of mitochondrial permeability transition pore (mPTP) and mitochondrial swelling. In addition, activities of mitochondrial respiratory chain complexes (I-IV), aconitase, NADPH oxidase, thioredoxin reductase, oxygen consumption rate and calcium homeostasis were evaluated. Treatment with BDE significantly prevented the generation of intracellular ROS and mitochondrial superoxide radicals and protected the mitochondria by preventing dissipation of ΔΨm, opening of mPTP, mitochondrial swelling and enhanced the activities of respiratory chain complexes and oxygen consumption rate in H9c2 cells. Activities of aconitase and thioredoxin reductase which was lowered (33.77±0.68% & 45.81±0.71% respectively) due to hypertrophy, were increased in BDE treated cells (P≤0.05). Moreover, BDE also reduced the intracellular calcium overload in Ang II treated cells. Overall results revealed the protective effects of B. diffusa against mitochondrial dysfunction in hypertrophy in H9c2 cells and the present findings may shed new light on the therapeutic potential of B. diffusa in addition to its nutraceutical potentials. PMID:24788441

  17. Docosahexaenoic Acid Attenuates Doxorubicin-induced Cytotoxicity and Inflammation by Suppressing NF-κB/iNOS/NO Signaling Pathway Activation in H9C2 Cardiac Cells.

    PubMed

    Wang, Zhi-Quan; Chen, Man-Tian; Zhang, Rui; Zhang, Yi; Li, Wei; Li, Yi-Gang

    2016-04-01

    Doxorubicin (DOX) is a widely used antineoplastic agent for a variety of carcinomas. However, it is cardiotoxic and leads to cardiomyopathy. Previous studies have indicated that omega-3 polyunsaturated acids (ω-3 PUFAs) have therapeutic effects on dilated and diabetic cardiomyopathies. However, whether ω-3 PUFAs exert therapeutic effects on DOX-induced cardiomyopathy remains unclear. In this study, we explored the effect and underlying mechanisms of docosahexaenoic acid (DHA), an important type of ω-3 PUFA, on DOX-induced cardiotoxicity and inflammation. H9C2 cardiac cells were exposed to DOX (5 μM) and interfered with by DHA (10 μM) for 4 hours. The effect of DHA on H9C2 cell viability was measured by Cell Counting Kit-8 assay. The levels of mRNA and protein expression of inflammatory cytokines were determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Reactive oxygen species and nitric oxide (NO) levels were determined by corresponding kits. The protein expression of key molecules in the nuclear factor-kappa B/inducible isoform of nitric oxide synthase/nitric oxide (NF-κB/iNOS/NO) signaling pathway was determined by western blotting. DOX-induced significant cytotoxicity and reactive oxygen species production in H9C2 cardiac cells. It also induced cardiac inflammation as evidenced by significantly increased expressions of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-1β, monocyte chemoattractant protein-1, and inducible isoform of NO synthase. However, DHA effectively attenuated DOX-induced cytotoxicity and inflammation. A further mechanistic study revealed that DHA suppressed DOX-induced activation of the NF-κB/iNOS/NO signaling pathway in H9C2 cells. Our results indicate that DHA may protect against DOX-induced cardiotoxicity by inhibiting NF-κB/iNOS/NO signaling pathway activation. PMID:26657886

  18. 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

  19. 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

  20. 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

  1. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    PubMed

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. PMID:27005940

  2. 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

  3. 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

  4. 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 300μl/ml, an exposure time of 45s, and a plasmid concentration of 15μg/ml were found to be optimal for transfection. UTMD/PEI-mediated PHD2-shRNA transfection in H9C2 cells significantly down regulated the expression of PHD2 and increased expression of HIF-1α and downstream angiogenesis factors VEGF, TGF-β and bFGF. UTMD/PEI, combined with albumin-coated microbubbles, warrants further investigation for therapeutic gene delivery. PMID:26267649

  5. 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

  6. ATP-sensitive K+ channels contribute to the protective effects of exogenous hydrogen sulfide against high glucose-induced injury in H9c2 cardiac cells.

    PubMed

    Liang, Weijie; Chen, Jingfu; Mo, Liqiu; Ke, Xiao; Zhang, Wenzhu; Zheng, Dongdan; Pan, Wanying; Wu, Shaoyun; Feng, Jianqiang; Song, Mingcai; Liao, Xinxue

    2016-03-01

    Hyperglycemia, as well as diabetes mellitus, has been shown to impair ATP-sensitive K+ (KATP) channels in human vascular smooth muscle cells. Hydrogen sulfide (H2S) is also known to be an opener of KATP channels. We previously demonstrated the cardioprotective effects exerted by H2S against high-glucose (HG, 35 mM glucose)-induced injury in H9c2 cardiac cells. As such, we hypothesized that KATP channels play a role in the cardioprotective effects of H2S against HG-induced injury. In this study, to examine this hypothesis, H9c2 cardiac cells were treated with HG for 24 h to establish a model of HG-induced insults. Our findings revealed that treatment of the cells with HG markedly decreased the expression level of KATP channels. However, the decreased expression of KATP channels was reversed by the treatment of the cells with 400 µM sodium hydrogen sulfide (NaHS, a donor of H2S) for 30 min prior to exposure to HG. Additionally, the HG-induced cardiomyocyte injuries, including cytotoxicity, apoptosis, oxidative stress and mitochondrial damage, were ameliorated by treatment with NaHS or 100 µM diazoxide (a mitochondrial KATP channel opener) or 50 µM pinacidil (a non-selective KATP channel opener) for 30 min prior to exposure to HG, as indicated by an increase in cell viability, as well as a decrease in the number of apoptotic cells, the expression of cleaved caspase-3, the generation of reactive oxygen species (ROS) and the dissipation of mitochondrial membrane potential (MMP). Notably, treatment of the H9c2 cardiac cells with 100 µM 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP channel blocker) or 1 mM glibenclamide (Gli, a non-selective KATP channel blocker) for 30 min prior to treatment with NaHS and exposure to HG significantly attenuated the above-mentioned cardioprotective effects exerted by NaHS. Notably, treatment of the cells with 500 µM N-acetyl‑L‑cysteine (NAC, a scavenger of ROS) for 60 min prior to exposure to HG markedly reduced the HG-induced inhibitory effect on the expression of KATP channels. Taken together, our results suggest that KATP channels play an important role in the cardioprotective effects of exogenous H2S against HG-induced injury. This study also provides novel data demonstraring that there is an antagonistic interaction between ROS and KATP channels in HG-exposed H9c2 cardiac cells. PMID:26820501

  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. 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. PMID:27034732

  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

    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. PMID:27034732

  10. 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

  11. 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

  12. 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

  13. 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

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

    PubMed Central

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

    2014-01-01

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

  15. Globular Adiponectin, Acting via AdipoR1/APPL1, Protects H9c2 Cells from Hypoxia/Reoxygenation-Induced Apoptosis

    PubMed Central

    Park, Min; Youn, ByungSoo; Zheng, Xi-long; Wu, Donghai; Xu, Aimin; Sweeney, Gary

    2011-01-01

    Cardiomyocyte apoptosis is an important remodeling event contributing to heart failure and adiponectin may mediate cardioprotective effects at least in part via attenuating apoptosis. Here we used hypoxia-reoxygenation (H/R) induced apoptosis in H9c2 cells to examine the effect of adiponectin and cellular mechanisms of action. We first used TUNEL labeling in combination with laser scanning cytometry to demonstrate that adiponectin prevented H/R-induced DNA fragmentation. The anti-apoptotic effect of adiponectin was also verified via attenuation of H/R-induced phosphatidylserine exposure using annexin V binding. H/R-induced apoptosis via the mitochondrial-mediated intrinsic pathway of apoptosis as assessed by cytochrome c release into cytosol and caspase-3 activation, both of which were attenuated by adiponectin. Mechanistically, we demonstrated that adiponectin enhanced anti-oxidative potential in these cells which led to attenuation of the increase in intracellular reactive oxygen species (ROS) caused by H/R. To further address the mechanism of adiponctins anti-apoptotic effects we used siRNA to efficiently knockdown adiponectin receptor (AdipoR1) expression and found that this attenuated the protective effects of adiponectin on ROS production and caspase 3 activity. Knockdown of APPL1, an important intracellular binding partner for AdipoR, also significantly reduced the ability of adiponectin to prevent H/R-induced ROS generation and caspase 3 activity. In summary, H/R-induced ROS generation and activation of the intrinsic apoptotic pathway was prevented by adiponectin via AdipoR1/APPL1 signaling and increased anti-oxidant potential. PMID:21552570

  16. 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. PMID:26465230

  17. 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

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-02-01

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

  20. 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

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

    PubMed

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

    2015-08-01

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

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

    PubMed

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

    2015-01-01

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

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

    PubMed

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

    2015-12-15

    Diabetic cardiomyopathy is the major cause of death in type 2 diabetic patients. Berberine is an isoquinoline alkaloid extract from traditional chinese herbs and its hypoglycemic and hypolipidemic effects make it a promising drug for treatment of type 2 diabetes. We examined if berberine improved cardiac function and attenuated cardiac hypertrophy and fibrosis in high fat diet and streptozotocin induced-type 2 diabetic rats in vivo and reduced expression of hypertrophy markers in palmitate-induced hypertrophic H9c2 cells in vitro. Treatment of diabetic animals with berberine partially improved cardiac function and restored fasting blood insulin, fasting blood glucose, total cholesterol, and triglyceride levels to that of control. In addition, berberine treatment of diabetic animals increased cardiac 5'-adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) activation and reduced glycogen synthase kinase 3 beta (GSK3β) activation compared to control. Palmitate incubation of H9c2 cells resulted in cellular hypertrophy and decreased expression of alpha-myosin heavy chain (α-MHC) and increased expression of beta-myosin heavy chain (β-MHC) compared to controls. Berberine treatment of palmitate-incubated H9c2 cells reduced hypertrophy, increased α-MHC expression and decreased β-MHC expression. In addition, berberine treatment of palmitate-incubated H9c2 cells increased AMPK and AKT activation and reduced GSK3β activation. The presence of the AMPK inhibitor Compound C attenuated the effects of berberine. The results strongly indicate that berberine treatment may be protective against the development of diabetic cardiomyopathy. PMID:26522928

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

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

    PubMed

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

    2015-01-01

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

  10. 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

  11. Kobophenol A inhibits sodium nitroprusside-induced cardiac H9c2 cell death through suppressing activation of JNK and preserving mitochondrial anti-apoptotic Bcl-2 and Mcl-1.

    PubMed

    Lee, Sung Ryul; Kwak, Jong Hwan; Noh, Su Jin; Pronto, Julius Ryan; Ko, Kyung Soo; Rhee, Byoung Doo; Xu, Zhelong; Kim, Nari; Han, Jin

    2014-01-01

    Sodium nitroprusside (SNP) releases nitric oxide (NO), a powerful vasodilator, and thus widely used in intensive care unit for treating hypertension emergency. However, cardiac toxicity after SNP administration is a clinical problem. For finding a natural compound that suppressing SNP-induced cardiac toxicity, we tested the protective potential of kobophenol A (Kob A), purified from the root of Caragana sinica, against the toxic effects of SNP. The severe cardiac H9c2 cell death was induced by SNP (2 mM) treatment. Kob A ameliorated SNP-induced cardiac H9c2 cell death, and this protective effect of Kob A may be related to the inhibition of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase activation following SNP administration. In addition, the downregulation of cellular Bcl-2 and Mcl-1 levels by SNP exposure was strongly abrogated in the presence of Kob A. These biological properties of Kob A might provide insights into developing new cardioprotectant against SNP-induced cardiac cell death. PMID:24759620

  12. Boerhaavia diffusa L. attenuates angiotensin II-induced hypertrophy in H9c2 cardiac myoblast cells via modulating oxidative stress and down-regulating NF-κβ and transforming growth factor β1.

    PubMed

    Prathapan, A; Vineetha, V P; Abhilash, P A; Raghu, K G

    2013-10-01

    The present study evaluated the antihypertrophic potential of the ethanolic extract of Boerhaavia diffusa (BDE), a well-known edible cardiotonic plant reported in Ayurveda against angiotensin II-induced hypertrophy in H9c2 cardiac myoblast cells. Markers of hypertrophy such as cell size, protein content and the concentrations of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were analysed for the confirmation of hypertrophy induction. Angiotensin II (100 nM) caused an increase in cell volume (69·26 (SD 1·21)%),protein content (48·48 (SD 1·64)%), ANP (81·90 (SD 1·22)%) and BNP (108·57 (SD 1·47)%). BDE treatment significantly reduced cell volume, protein content and the concentrations of ANP and BNP (P#0·05) in H9c2 cells. The activity of various antioxidant enzymes and the concentration of reduced glutathione, which was lowered due to hypertrophy, were increased in BDE-treated cells. The BDE treatment also reduced intracellular reactive oxygen species generation, lipid peroxidation and protein carbonyls in cells. In addition,the expression patterns of NF-kb and transforming growth factor b1 were found to be increased during hypertrophy, and their expressions were reduced on BDE treatment. In vitro chemical assays showed that BDE inhibits angiotensin-converting enzyme and xanthine oxidase in a dose-dependent manner with an estimated 50% effective concentration (EC50) value of 166·12 (SD 2·42) and 60·05 (SD 1·54) mg/ml,respectively. The overall results clearly indicate the therapeutic potential of B. diffusa against cardiac hypertrophy, in addition to its nutritional qualities. PMID:23591029

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

    PubMed

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

    2015-08-01

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

  14. 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

  15. 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

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

    PubMed Central

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

    2016-01-01

    Background Mitochondrial dysfunction would ultimately lead to myocardial cell apoptosis and death during ischemia-reperfusion injuries. Autophagy could ameliorate mitochondrial dysfunction by autophagosome forming, which is a catabolic process to preserve the mitochondrial’s structural and functional integrity. HO-1 induction and expression are important protective mechanisms. This study in order to investigate the role of HO-1 during mitochondrial damage and its mechanism. Methods and Results The H9c2 cardiomyocyte cell line were incubated by hypoxic and then reoxygenated for the indicated time (2, 6, 12, 18, and 24 h). Cell viability was tested with CCK-8 kit. The expression of endogenous HO-1(RT-PCR and Western blot) increased with the duration of reoxygenation and reached maximum levels after 2 hours of H/R; thereafter, the expression gradually decreased to a stable level. Mitochondrial dysfunction (Flow cytometry quantified the ROS generation and JC-1 staining) and autophagy (The Confocal microscopy measured the autophagy. RFP-GFP-LC3 double-labeled adenovirus was used for testing.) were induced after 6 hours of H/R. Then, genetic engineering technology was employed to construct an Lv-HO1-H9c2 cell line. When HO-1 was overexpressed, the LC3II levels were significantly increased after reoxygenation, p62 protein expression was significantly decreased, the level of autophagy was unchanged, the mitochondrial membrane potential was significantly increased, and the mitochondrial ROS level was significantly decreased. Furthermore, when the HO-1 inhibitor ZnPP was applied the level of autophagy after reoxygenation was significantly inhibited, and no significant improvement in mitochondrial dysfunction was observed. Conclusions During myocardial hypoxia-reoxygenation injury, HO-1 overexpression induces autophagy to protect the stability of the mitochondrial membrane and reduce the amount of mitochondrial oxidation products, thereby exerting a protective effect. PMID:27138700

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

    PubMed

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

    2016-01-01

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

  18. 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

  19. 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

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

    PubMed

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

    2015-06-01

    Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as α-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5 kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for β1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis. PMID:25836924

  1. 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

  2. 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 ω-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 ω-3 fatty acyl chains, indicating a ω-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

  3. 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

  4. 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

  5. Parthenolide-Induced Cytotoxicity in H9c2 Cardiomyoblasts Involves Oxidative Stress

    PubMed Central

    Tsai, Tien-Yao; Chan, Paul; Gong, Chi-Li; Wong, Kar-Lok; Su, Tzu-Hui; Shen, Pei-Chen; Leung, Yuk-Man; Liu, Zhong-Min

    2015-01-01

    Background Cardiac cellular injury as a consequence of ischemia and reperfusion involves nuclear factor-κB (NF-κ B), amongst other factors, and NF-κ B inhibitors could substantially reduce myocardial infarct size. Parthenolide, a sesquiterpene lactone compound which could inhibit NF-κ B, has been shown to ameliorate myocardial reperfusion injury but may also produce toxic effects in cardiomyocytes at high concentrations. The aim of this study was to examine the cytotoxic effects of this drug on H9c2 cardiomyoblasts, which are precursor cells of cardiomyocytes. Methods Cell viability and apoptosis were examined by MTT and TUNEL assay, respectively, and protein expression was analyzed by western blot. Reactive oxygen species (ROS) production was measured using DCFH-DA as dye. Cytosolic Ca2+ concentration and mitochondrial membrane potential were measured microfluorimetrically using, respectively, fura 2 and rhodamine 123 as dyes. Results Parthenolide caused apoptosis at 30 μ M, as judged by TUNEL assay and Bax and cytochrome c translocation. It also caused collapse of mitochondrial membrane potential and endoplasmic reticulum stress. Parthenolide triggered ROS formation, and vitamin C (antioxidant) partially alleviated parthenolide-induced cell death. Conclusions The results suggested that parthenolide at high concentrations caused cytotoxicity in cardiomyoblasts in part by inducing oxidative stress, and demonstrated the imperative for cautious and appropriate use of this agent in cardioprotection. PMID:27122844

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

    PubMed

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

    2009-09-01

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

  7. Cytosolic renin is targeted to mitochondria and inducesapoptosis in H9c2 rat cardiomyoblasts

    PubMed Central

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

    2009-01-01

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

  8. 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

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

    PubMed

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

    2016-07-01

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

  10. 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

  11. 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

  12. 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

  13. Cerium oxide nanoparticles inhibit oxidative stress and nuclear factor-κB activation in H9c2 cardiomyocytes exposed to cigarette smoke extract.

    PubMed

    Niu, Jianli; Wang, Kangkai; Kolattukudy, Pappachan E

    2011-07-01

    Cigarette smoke contains and generates a large amount of reactive oxygen species (ROS) that affect normal cellular function and have pathogenic consequences in the cardiovascular system. Increased oxidative stress and inflammation are considered to be an important mechanism of cardiovascular injury induced by cigarette smoke. Antioxidants may serve as effective therapeutic agents against smoke-related cardiovascular disease. Because of the presence of oxygen vacancies on its surface and self-regenerative cycle of its dual oxidation states, Ce(3+) and Ce(4+), cerium oxide (CeO(2)) nanoparticles offer a potential to quench ROS in biological systems. In this study, we determined the ability of CeO(2) nanoparticles to protect against cigarette smoke extract (CSE)-induced oxidative stress and inflammation in cultured rat H9c2 cardiomyocytes. CeO(2) nanoparticles pretreatment of H9c2 cells resulted in significant inhibition of CSE-induced ROS production and cell death. Pretreatment of H9c2 cells with CeO(2) nanoparticles suppressed CSE-induced phosphorylation of IκBα, nuclear translocation of p65 subunit of nuclear factor-κB (NF-κB), and NF-κB reporter activity in H9c2 cells. CeO(2) nanoparticles pretreatment also resulted in a significant down-regulation of NF-κB-regulated inflammatory genes tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric-oxide synthase and further inhibited CSE-induced depletion of antioxidant enzymes, such as copper zinc superoxide dismutase, manganese superoxide dismutase, and intracellular glutathione content. These results indicate that CeO(2) nanoparticles can inhibit CSE-induced cell damage via inhibition of ROS generation, NF-κB activation, inflammatory gene expression, and antioxidant depletion and may have a great potential for treatment of smoking-related diseases. PMID:21464334

  14. 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

  15. 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

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

    PubMed

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

    2016-01-01

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

  17. Chemical chaperone 4-phenylbutyric acid protects H9c2 cardiomyocytes from ischemia/reperfusion injury by attenuating endoplasmic reticulum stress-induced apoptosis.

    PubMed

    Jian, Lian; Lu, Yuan; Lu, Shan; Lu, Chengzhi

    2016-05-01

    Myocardial ischemia/reperfusion (I/R) is a potential contributor to high rates of mortality in several cardiovascular diseases. I/R initiates the unfolded protein response and endoplasmic reticulum (ER) stress, which may lead to apoptotic pathways and exaggerate I/R injury. 4‑phenylbutyric acid (4‑PBA), a low molecular weight, terminal aromatic substituted fatty acid, has been reported to function as an ER chaperone. The aim of the present study was to investigate whether 4‑PBA is able to reduce ER stress‑induced apoptosis and prevent cardiomyocyte damage during the process of I/R in vitro. Accordingly, the rat cardiomyocyte line, H9c2, was treated with hypoxia/reoxygenation as an I/R model in vitro. Myocardium apoptosis was determined with TUNEL staining. The expression of ER stress‑related proteins were examined by western blotting. The resulting data showed that I/R activates the ER stress proteins, glucose‑regulated protein 78, activating transcription factor 6 and protein kinase RNA‑like endoplasmic reticulum kinase, which were all reduced by pretreatment with 4‑PBA. In addition, pretreatment with 4‑PBA significantly inhibited the expression levels of pro‑apoptotic proteins, C/EBP homologous protein, B cell lymphoma (Bcl‑2)‑associated X protein and phosphorylated c‑Jun N‑terminal kinase, and enhanced the expression of the anti‑apoptotic protein Bcl‑2 (n=3; P<0.05). The data demonstrated that I/R initiates ER stress‑associated apoptotic pathways, and 4‑PBA pretreatment protected the cardiomyocytes from I/R‑induced cell death. To the best of our knowledge, the present study is the first to report on the cell repair mechanism of 4‑PBA against I/R damage in cardiomyocytes based on ER stress‑associated apoptotic pathways. PMID:27035223

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

    PubMed

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

    2016-03-01

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

  19. 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

  20. An embryonic heart cell line is susceptible to dengue virus infection.

    PubMed

    Angel-Ambrocio, Antonio H; Soto-Acosta, Rubén; Tammineni, Eshwar R; Carrillo, Elba D; Bautista-Carbajal, Patricia; Hernández, Ascención; Sánchez, Jorge A; del Angel, Rosa M

    2015-02-16

    Dengue virus (DENV) is the causative agent of dengue fever. In recent years, patients with more severe form of the disease with acute heart failure or progression to cardiogenic shock and death have been reported. However, the pathogenesis of myocardial lesions and susceptibility of cardiomyocytes to DENV infection have not been evaluated. Under this perspective, the susceptibility of the myoblast cell line H9c2, obtained from embryonic rat heart, to DENV infection was analyzed. Our findings indicate that H9c2 cells are susceptible to the infection with the four DENV serotypes. Moreover, virus translation/replication and viral production in this cell line is as efficient as in other susceptible cell lines, supporting the idea that DENV may target heart cells as evidenced by infection of H9c2 cells. This cell line may thus represent an excellent model for the study and characterization of cardiac physiopathology in DENV infection. PMID:25598317

  1. 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

  2. The Protective Role of the TOPK/PBK Pathway in Myocardial Ischemia/Reperfusion and H2O2-Induced Injury in H9C2 Cardiomyocytes

    PubMed Central

    Sun, Guozhe; Ye, Ning; Dai, Dongxue; Chen, Yintao; Li, Chao; Sun, Yingxian

    2016-01-01

    T-LAK-cell-originated protein kinase (TOPK) is a PDZ-binding kinase (PBK) that was recently identified as a novel member of the mitogen-activated protein kinase (MAPK) family. It has been shown to play an important role in many cellular functions. However, its role in cardiac function remains unclear. Thus, we have herein explored the biological function of TOPK in myocardial ischemia/reperfusion (I/R) and oxidative stress injury in H9C2 cardiomyocytes. I/R and ischemic preconditioning (IPC) were induced in rats by 3-hour reperfusion after 30-min occlusion of the left anterior descending coronary artery and by 3 cycles of 5-min I/R. Hydrogen peroxide (H2O2) was used to induce oxidative stress in H9C2 cardiomyocytes. TOPK expression was analyzed by western blotting, RT-PCR, immunohistochemical staining, and immunofluorescence imaging studies. The effects of TOPK gene overexpression and its inhibition via its inhibitor HI-TOPK-032 on cell viability and Bcl-2, Bax, ERK1/2, and p-ERK1/2 protein expression were analyzed by MTS assay and western blotting, respectively. The results showed that IPC alleviated myocardial I/R injury and induced TOPK activation. Furthermore, H2O2 induced TOPK phosphorylation in a time-dependent manner. Interestingly, TOPK inhibition aggravated the H2O2-induced oxidative stress injury in myocardiocytes, whereas overexpression relieved it. In addition, the ERK pathway was positively regulated by TOPK signaling. In conclusion, our results indicate that TOPK might mediate a novel survival signal in myocardial I/R, and that its effect on anti-oxidative stress involves the ERK signaling pathway. PMID:26907268

  3. 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.

  4. 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

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

    PubMed Central

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

    2016-01-01

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

  6. 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

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

    PubMed

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

    2016-02-01

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

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

    PubMed 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

  9. 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

  10. Cardioprotective effect of breviscapine: inhibition of apoptosis in H9c2 cardiomyocytes via the PI3K/Akt/eNOS pathway following simulated ischemia/reperfusion injury.

    PubMed

    Wang, Jun; Ji, Shu-Yun; Liu, Si-Zhu; Jing, Rui; Lou, Wei-Juan

    2015-09-01

    Breviscapine (BE) is a standardized Chinese herbal medicine extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely used to treat cardiovascular and cerebrovascular diseases. However, there are no reports on the protective effects and underlying molecular mechanisms of BE action on myocardial ischemia/reperfusion (MI/R)-induced cardiomyocyte apoptosis. In the present study, we aimed to confirm the cardioprotective effect of BE from MI/R injury in vivo, and investigate the potential molecular mechanisms against simulated ischemia/reperfusion (SI/R)-induced cardiomyocyte apoptosis in vitro. The rat model of MI/R injury was induced by 30 min of transient vessel occlusion followed by 3 h of reperfusion. BE significantly reduced the myocardium infarct size and production of cardiac troponin (cTnl) in serum. In an in vitro experiment, H9c2 cardiomyocytes were incubated with vehicle or ischemic buffer during hypoxia; then, they were reoxygenated with or without BE. BE markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. We confirmed the anti-apoptotic effect of BE with the Hoechst 33258 staining assay, and this effect was associated with an increase in Bcl-2 and a decrease in active caspase-3 expression. Western blot analysis also showed that BE increased the phosphorylation of Akt and eNOS in H9c2 cells, and the protective effects of BE were partially inhibited by the phosphatidylinositol 3'-kinase (PI3K) specific inhibitor LY294002. Our results suggested that BE could provide significant cardioprotection against MI/R injury, and the potential mechanisms might involve suppression of cardiomyocyte apoptosis through activating the PI3K/Akt/eNOS signaling pathway. PMID:26492644

  11. 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

  12. Expression of HSP72 after ELF-EMF exposure in three cell lines.

    PubMed

    Gottwald, Eric; Sontag, Werner; Lahni, Brigitte; Weibezahn, Karl-Friedrich

    2007-10-01

    It has been reported that magnetic fields with flux densities ranging from microT to mT are able to induce heat shock factor, HSP72 mRNA or heat shock proteins in various cells. In this study we investigated changes in the HSP72 mRNA transcription level in three cell lines (HL-60, H9c2, and Girardi heart cells) and in the intracellular HSP72 protein content in two cell lines (HL-60 and Girardi heart cells) after treatment schemes using electromagnetic fields with a flux density of 2 microT to 4 mT, a frequency of 50 Hz and exposure times from 15 to 30 min. None of the treatments or modalities showed any significant effect on the HSP72 protein level, although HSP72 mRNA could be induced, at least to some extent, with one of the parameter combinations in all cell lines tested. Obviously, HSP72 mRNA transcription and translation are not necessarily coupled in certain cells. This leads to the conclusion that electromagnetic field effects on HSP72 mRNA levels are not indicative for downstream effects unless increased mRNA levels can be correlated with increased HSP72 protein levels as well. PMID:17508393

  13. 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

  14. 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.

  15. CLO: The cell line ontology

    PubMed Central

    2014-01-01

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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. High Glucose Induces Down-Regulated GRIM-19 Expression to Activate STAT3 Signaling and Promote Cell Proliferation in Cell Culture

    PubMed Central

    Li, Yong-Guang; Han, Bei-Bei; Li, Feng; Yu, Jian-Wu; Dong, Zhi-Feng; Niu, Geng-Ming; Qing, Yan-Wei; Li, Jing-Bo; Wei, Meng; Zhu, Wei

    2016-01-01

    Recent studies indicated that Gene Associated with Retinoid-IFN-Induced Mortality 19 (GRIM-19), a newly discovered mitochondria-related protein, can regulate mitochondrial function and modulate cell viability possibly via interacting with STAT3 signal. In the present study we sought to test: 1) whether GRIM-19 is involved in high glucose (HG) induced altered cell metabolism in both cancer and cardiac cells, 2) whether GRIM-19/STAT3 signaling pathway plays a role in HG induced biological effects, especially whether AMPK activity could be involved. Our data showed that HG enhanced cell proliferation of both HeLa and H9C2 cells, which was closely associated with down-regulated GRIM-19 expression and increased phosphorylated STAT3 level. We showed that GRIM-19 knock-down alone in normal glucose cultured cells can also result in an increase in phosphorylated STAT3 level and enhanced proliferation capability, whereas GRIM-19 over-expression can abolished HG induced STAT3 activation and enhanced cell proliferation. Importantly, both down-regulated or over-expression of GRIM-19 increased lactate production in both HeLa and H9C2 cells. The activated STAT3 was responsible for increased cell proliferation as either AG-490, an inhibitor of JAK2, or siRNA targeting STAT3 can attenuate cell proliferation increased by HG. In addition, HG increased lactate acid levels in HeLa cells, which was also observed when GRIM-19 was genetically manipulated. However, HG did not affect the lactate levels in H9C2 cells. Of note, over-expression of GRIM-19 and silencing of STAT3 both increased lactate production in H9C2 cells. As expected, HG resulted in significant decreases in phosphorylated AMPKα levels in H9C2 cells, but not in HeLa cells. Interestingy, activation of AMPKα by metformin was associated with a reversal of the suppressed GRIM-19 expression in H9C2 cells, the fold of changes in GRIM-19 expression by metformin were much less in HeLa cells. Metformin did not affect the phosphorylated STAT3 lelvels, however, decreased its levels in H9C2, especially in the setting of HG culture. Not like HG alone which resulted in no changes in lactate acid in H9C2 cells, metformin can increase lactate acid levels in H9C2 cells. Increased lactate induced by metformin was also observed in HeLa cells. PMID:27101310

  2. 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.

  3. Cell-host, LINE and environment

    PubMed Central

    Del Re, Brunella; Giorgi, Gianfranco

    2013-01-01

    Long interspersed nuclear elements -1 (LINEs, L1s) are retroelements occupying almost 17% of the human genome. L1 retrotransposition can cause deleterious effects on the host-cell and it is generally inhibited by suppressive mechanisms, but it can occur in some specific cells during early development as well as in some tumor cells and in the presence of several environmental factors. In a recent publication we reported that extremely low frequency pulsed magnetic field can affect L1 retrotransposition in neuroblastoma cells. In this commentary we discuss the interaction between environment and L1 activity in the light of the new emerging paradigm of host-LINE relationship. PMID:23734298

  4. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

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

  5. Differential SELEX in Human Glioma Cell Lines

    PubMed Central

    Cerchia, Laura; Esposito, Carla Lucia; Jacobs, Andreas H.; Tavitian, Bertrand; de Franciscis, Vittorio

    2009-01-01

    The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers. PMID:19956692

  6. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

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

    1995-07-30

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

  7. 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....

  8. 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.

  9. 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

  10. Characterization of swine testicular cell line as immature porcine Sertoli cell line.

    PubMed

    Ma, Changping; Song, Huibin; Guan, Kaifeng; Zhou, Jiawei; Xia, Xuanyan; Li, Fenge

    2016-04-01

    Swine testicular (ST) cell line is isolated from swine fetal testes and has been widely used in biomedical research fields related to pig virus infection. However, the potential benefit and utilization of ST cells in boar reproductive studies has not been fully explored. As swine fetal testes mainly contain multiple types of cells such as Leydig cells, Sertoli cells, gonocytes, and peritubular myoid cells, it is necessary to clarify the cell type of ST cell line. In this study, we identified ST cell line was a collection of Sertoli cells by analyzing the unique morphological characteristic with satellite karyosomes and determining the protein expression of two markers (androgen-binding protein, ABP; Fas ligand, FASL) of Sertoli cells. Then ST cells were further confirmed to be immature Sertoli cells by examining the expression of three markers (anti-Mullerian hormone, AMH; keratin 18, KRT18; follicle-stimulating hormone receptor, FSHR). In conclusion, ST cells are a collection of immature Sertoli cells which can be good experimental materials for the researches involved in Sertoli cell functions and maturation, or even in boar reproductions. PMID:26744029

  11. Stem cell factor suppresses apoptosis in neuroblastoma cell lines.

    PubMed

    Timeus, F; Crescenzio, N; Valle, P; Pistamiglio, P; Piglione, M; Garelli, E; Ricotti, E; Rocchi, P; Strippoli, P; Cordero di Montezemolo, L; Madon, E; Ramenghi, U; Basso, G

    1997-11-01

    Stem cell factor (SCF) is a glycoprotein growth factor produced by marrow stromal cells that acts after binding to its specific surface receptor, which is the protein encoded by the protooncogene c-kit. SCF synergizes with specific lineage factors in promoting the proliferation of primitive hematopoietic progenitors, and has been administered to expand the pool of these progenitors in cancer patients treated with high-dose chemotherapy. SCF and its c-kit receptor are expressed by some tumor cells, including myeloid leukemia, breast carcinoma, small cell lung carcinoma, melanoma, gynecological tumors, and testicular germ cell tumors. Previous studies of SCF in neuroblastoma have produced conflicting conclusions. To explore the role of SCF in neuroblastoma, we studied five neuroblastoma lines (IMR-5, SK-N-SH, SK-N-BE, AF8, and SJ-N-KP) and the neuroepithelioma line CHP-100. All lines expressed mRNA for c-kit and c-kit protein at low intensity as measured by flow cytometry, and secreted SCF in medium culture as shown by ELISA. Exogenous SCF did not modify 3H thymidine uptake in the neuroblastoma and neuroepithelioma cell lines. After 6 days' culture in the presence of anti-c-kit, the number of viable neuroblastoma cells was significantly lower than the control, and terminal deoxynucleotidyl transferase assay showed a substantial increase of apoptotic cells: The percentage of positive cells was 1-3% in the control lines, whereas in the presence of anti c-kit it varied from 29% of SK-N-BE to 92% of CHP-100. After 9 days' culture in the presence of anti-c-kit, no viable cells were detectable. These data indicate that SCF is produced by some neuroblastoma cell lines via an autocrine loop to protect them from apoptosis. PMID:9357969

  12. Spontaneous Cell Competition in Immortalized Mammalian Cell Lines

    PubMed Central

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

    2015-01-01

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

  13. Functional features of cancer stem cells in melanoma cell lines

    PubMed Central

    2013-01-01

    Background Recent evidence suggests a subset of cells within a tumor with "stem-like" characteristics. These cells are able to transplant tumors in immunodeficient hosts. Distinct from non-malignant stem cells, cancer stem cells (CSC) show low proliferative rates, high self-renewing capacity, propensity to differentiate into actively proliferating tumor cells, and resistance to chemotherapy or radiation. They are often characterized by elevated expression of stem cell surface markers, in particular CD133, and sets of differentially expressed stem cell-associated genes. CSC are usually rare in clinical specimens and hardly amenable to functional studies and gene expression profiling. In this study, a panel of heterogenous melanoma cell lines was screened for typical CSC features. Methods Nine heterogeneous metastatic melanoma cell lines including D10 and WM115 were studied. Cell lines were phenotyped using flow cytometry and clonogenic assays were performed by limiting dilution analysis on magnetically sorted cells. Spheroidal growth was investigated in pretreated flasks. Gene expression profiles were assessed by using real-time rt-PCR and DNA microarrays. Magnetically sorted tumor cells were subcutaneously injected into the flanks of immunodeficient mice. Comparative immunohistochemistry was performed on xenografts and primary human melanoma sections. Results D10 cells expressed CD133 with a significantly higher clonogenic capacity as compared to CD133- cells. Na8, D10, and HBL cells formed spheroids on poly-HEMA-coated flasks. D10, Me39, RE, and WM115 cells expressed at least 2 of the 3 regulatory core transcription factors SOX2, NANOG, and OCT4 involved in the maintenance of stemness in mesenchymal stem cells. Gene expression profiling on CD133+ and CD133- D10 cells revealed 68 up- and 47 downregulated genes (+/-1.3 fold). Two genes, MGP and PROM1 (CD133), were outstandingly upregulated. CD133+ D10 cells formed tumors in NSG mice contrary to CD133- cells and CD133 expression was detected in xenografts and primary human melanoma sections using immunohistochemistry. Conclusions Established melanoma cell lines exhibit, to variable extents, the typical features of CSCs. The tumorigenic cell line D10, expressing CD133 and growing in spheroids and might qualify as a potential model of melanoma CSCs. PMID:23915418

  14. Lactoferrin binding by leukemia cell lines

    SciTech Connect

    Yamada, Y.; Amagasaki, T.; Jacobsen, D.W.; Green, R.

    1987-07-01

    Monocytes and macrophages have receptors for the iron-binding protein lactoferrin. Lactoferrin acts as a potent inhibitor of granulocyte-macrophage colony stimulating factor production when it binds to these cells. Using a rosette assay and immunofluorescence, we have shown that cultured leukemia cells, including the human erythroid leukemia cell line K562, also have lactoferrin binding sites. The number of binding sites on K562 cells was estimated using soluble /sup 59/Fe-lactoferrin. Inhibition studies demonstrate that lactoferrin binding sites are distinct and unrelated to receptors for transferrin or the Fc portion of IgG, which are present on K562 cells. However, electrostatic forces may be important for lactoferrin binding, since other polycationic proteins (eg, protamine) inhibit lactoferrin binding. Prior treatment of K562 cells with trypsin nearly abolishes lactoferrin binding. However, these cells recover their ability to bind lactoferrin when trypsin is removed. Unlike transferrin receptors, the expression of lactoferrin binding sites is not regulated by cellular iron status. Cytosine arabinoside arrests the proliferation of K562 cells and simultaneously leads to a reduction in lactoferrin surface binding, suggesting that lactoferrin binding may be dependent on cell proliferation.

  15. Cytosine methylation profiling of cancer cell lines.

    PubMed

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

    2008-03-25

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

  16. 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

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

    PubMed Central

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

    2016-01-01

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

  18. A bioinformatics analysis of the cell line nomenclature

    PubMed Central

    Sarntivijai, Sirarat; Ade, Alexander S.; Athey, Brian D.; States, David J.

    2008-01-01

    Motivation: Cell lines are used extensively in biomedical research, but the nomenclature describing cell lines has not been standardized. The problems are both linguistic and experimental. Many ambiguous cell line names appear in the published literature. Users of the same cell line may refer to it in different ways, and cell lines may mutate or become contaminated without the knowledge of the user. As a first step towards rationalizing this nomenclature, we created a cell line knowledgebase (CLKB) with a well-structured collection of names and descriptive data for cell lines cultured in vitro. The objectives of this work are: (i) to assist users in extracting useful information from biomedical text and (ii) to highlight the importance of standardizing cell line names in biomedical research. This CLKB contains a broad collection of cell line names compiled from ATCC, Hyper CLDB and MeSH. In addition to names, the knowledgebase specifies relationships between cell lines. We analyze the use of cell line names in biomedical text. Issues include ambiguous names, polymorphisms in the use of names and the fact that some cell line names are also common English words. Linguistic patterns associated with the occurrence of cell line names are analyzed. Applying these patterns to find additional cell line names in the literature identifies only a small number of additional names. Annotation of microarray gene expression studies is used as a test case. The CLKB facilitates data exploration and comparison of different cell lines in support of clinical and experimental research. Availability: The web ontology file for this cell line collection can be downloaded at http://www.stateslab.org/data/celllineOntology/cellline.zip. Contact: dstates@umich.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:18849319

  19. 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

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

    PubMed Central

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

    2013-01-01

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

  1. 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

  2. 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.

  3. Translational research on esophageal adenocarcinoma: from cell line to clinic.

    PubMed

    Boonstra, J J; Tilanus, H W; Dinjens, W N M

    2015-01-01

    Human esophageal adenocarcinoma (EAC) cell lines have made a substantial contribution to elucidating mechanisms of carcinogenesis and drug discovery. Model research on EAC relies almost entirely on a relatively small set of established tumor cell lines because appropriate animal models are lacking. Nowadays, more than 20% of all fundamental translational research studies regarding EAC are partially or entirely based on these cell lines. The ready availability of these cell lines to investigators worldwide have resulted in more than 250 publications, including many examples of important biomedical discoveries. The high genomic similarities (but certainly not completely identical) between the EAC cell lines and their original tumors provide rational for their use. Recently, in a collaborative effort all available EAC cell lines have been verified resulting in the establishment of a reliable panel of 10 EAC cell lines. It could be expected that the value of these cell lines increases as unlimited source of tumor material because new biomedical techniques require more tumor cells and the supply of viable tumor cells is diminishing because of neoadjuvant chemo(radio)therapy of patients with EAC. Here, we review the history of the EAC cell lines and their utility in translational research and biomedical discovery. PMID:23795680

  4. 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

  5. Lymphoid cell line identification and the detection of cross contamination.

    PubMed

    Conner, B R; Ferrone, S; Pellegrino, M A; Glaser, R

    1980-05-01

    Four lymphoid cell lines, presumably recent derivatives from non-neoplastic human tonsils, were identified in actuality as established Burkitt lymphoma and lymphoblastoid cell lines (LCL) when tested by chromosome banding and typing for HLA antigens. Additionally, the morphology, growth characteristics, tumorigenicity, expression of Epstein-Barr virus antigens and surface membrane immunoglobulins (SmIg) of the four lymphoid cell lines confirmed the correct identifications. In determining the possibility of cross contamination, the most reliable criteria for the identification of lymphoid cell lines were found to be HLA antigenic profile, karyotype, cellular morphology, and growth characteristics. PMID:6248453

  6. 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.

  7. 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.

  8. 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...

  9. 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

  10. AB241. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P

    PubMed Central

    Huang, Bin; Wang, Dao-Hu; Chen, Jun-Xing; Qiu, Shao-Peng

    2016-01-01

    Background Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Methods We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Results Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells among five cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Conclusions Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

  11. 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

  12. 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.

  13. 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.

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

    SciTech Connect

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

    1987-11-01

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

  15. 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

  16. Replicative Capacity of MERS Coronavirus in Livestock Cell Lines

    PubMed Central

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

    2014-01-01

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

  17. 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.

  18. 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

  19. 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

  20. Establishment of a Human Thymic Myoid Cell Line

    PubMed Central

    Wakkach, Abdel; Poea, Sandrine; Chastre, Eric; Gespach, Christian; Lecerf, Florence; De la Porte, Sabine; Tzartos, Socrates; Coulombe, Alain; Berrih-Aknin, Sonia

    1999-01-01

    The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. α-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells. PMID:10514405

  1. Conventional and novel methods for embryonic stem cell line derivation.

    PubMed

    Kiatpongsan, Sorapop; Tannirandorn, Yuen; Numchaisrikhabsc, Pranee; Rungsiwiwut, Ruttachuk

    2006-06-01

    Cell therapy is the promising therapeutic tool for the next decade. "Regenerative Medicine" based on cell and tissue replacement therapy is proposed as a revolutionary approach to various chronic and incurable conditions. The first key step for successful cell therapy is the establishment of clinical grade human Embryonic Stem Cell (hESC) lines. This article provides a concise summary on conventional and novel methods for hESC line derivation. There is also discussion on progression, future direction and problems in hESC line development. In Thailand, more advance knowledge, skill, and technology are required to develop the first human embryonic stem cell line and step forward to make cell therapy a reality. PMID:16850695

  2. 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

  3. 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

  4. Cell line models for differentiation: preadipocytes and adipocytes.

    PubMed

    Poulos, Sylvia P; Dodson, Michael V; Hausman, Gary J

    2010-10-01

    In vitro models have been invaluable in determining the mechanisms involved in adipocyte proliferation, differentiation, adipokine secretion and gene/protein expression. The cells presently available for research purposes all have unique advantages and disadvantages that one should be aware of when selecting cells. Established cell lines, such as 3T3-L1 cells, are easier and less costly to use than freshly isolated cells, even though freshly isolated cells allow for various comparisons such as the in vitro evaluation of different in vivo conditions that may not be possible using cell lines. Moreover, stem cells, transdifferentiated cells or dedifferentiated cells are relatively new cell models being evaluated for the study of adipocyte regulation and physiology. The focus of this brief review is to highlight similarities and differences in adipocyte models to aid in appropriate model selection and data interpretation for successful advancement of our understanding of adipocyte biology. PMID:20864461

  5. 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

  6. 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

  7. Generation and characterization of human insulin-releasing cell lines

    PubMed Central

    Labriola, Leticia; Peters, Maria G; Krogh, Karin; Stigliano, Iván; Terra, Letícia F; Buchanan, Cecilia; Machado, Marcel CC; Joffé, Elisa Bal de Kier; Puricelli, Lydia; Sogayar, Mari C

    2009-01-01

    Background The in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines. Results We generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate. Conclusion We have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction. PMID:19545371

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

    PubMed Central

    LUVIANO, AXEL; AGUIÑIGA-SÁNCHEZ, ITZEN; DEMARE, PATRICIA; TIBURCIO, REYNALDO; LEDESMA-MARTÍNEZ, EDGAR; SANTIAGO-OSORIO, EDELMIRO; REGLA, IGNACIO

    2014-01-01

    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

  9. Characterization of three new serous epithelial ovarian cancer cell lines

    PubMed Central

    Ouellet, Véronique; Zietarska, Magdalena; Portelance, Lise; Lafontaine, Julie; Madore, Jason; Puiffe, Marie-Line; Arcand, Suzanna L; Shen, Zhen; Hébert, Josée; Tonin, Patricia N; Provencher, Diane M; Mes-Masson, Anne-Marie

    2008-01-01

    Background Cell lines constitute a powerful model to study cancer, and here we describe three new epithelial ovarian cancer (EOC) cell lines derived from poorly differentiated serous solid tumors (TOV-1946, and TOV-2223G), as well as the matched ascites for one case (OV-1946). Methods In addition to growth parameters, the cell lines were characterized for anchorage independent growth, migration and invasion potential, ability to form spheroids and xenografts in SCID mice. Results While all cell lines were capable of anchorage independent growth, only the TOV-1946 and OV-1946 cell lines were able to form spheroid and produce tumors. Profiling of keratins, p53 and Her2 protein expression was assessed by immunohistochemistry and western blot analyses. Somatic TP53 mutations were found in all cell lines, with TOV-1946 and OV-1946 harboring the same mutation, and none harbored the commonly observed somatic mutations in BRAF, KRAS or germline BRCA1/2 mutations found to recur in the French Canadian population. Conventional cytogenetics and spectral karyotype (SKY) analyses revealed complex karyotypes often observed in ovarian disease. Conclusion This is the first report of the establishment of matched EOC cell lines derived from both solid tumor and ascites of the same patient. PMID:18507860

  10. 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

  11. 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

  12. Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    PubMed Central

    Fausther, Michel; Goree, Jessica R.; Lavoie, Élise G.; Graham, Alicia L.; Sévigny, 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

  13. Establishment and characterization of a chicken mononuclear cell line.

    PubMed

    Qureshi, M A; Miller, L; Lillehoj, H S; Ficken, M D

    1990-11-01

    A new chicken mononuclear cell line (MQ-NCSU) has been established. The starting material used to initiate this cell line was a transformed spleen from a female Dekalb XL chicken which had been experimentally challenged with the JM/102W strain of the Marek's disease virus. After homogenization, a single cell suspension of splenic cells was cultured using L.M. Hahn medium supplemented with 10 microM 2-mercaptoethanol. Under these culture conditions, a rapidly proliferating cell was observed and then expanded after performing limiting dilution cultures. These cells were moderately adherent and phagocytic for sheep red blood cells and Salmonella typhimurium. When tested against a panel of monoclonal antibodies (mAb) using the flow cytometry, MQ-NCSU cells stained readily with anti-chicken monocyte specific (K-1) mAb but did not stain with mAb detecting T-helper, T-cytotoxic/suppressor, and NK cells. MQ-NCSU cells expressed very high levels of Ia antigens and transferrin receptors. In addition, cell-free supernatant obtained from MQ-NCSU culture contained a factor which exhibited cytolytic activity against tumor cell targets. Based on their cultural, morphological, and functional characteristics and mAb reactivity profile, we conclude that MQ-NCSU cell line represents a malignantly-transformed cell which shares features characteristic of cells of the mononuclear phagocyte lineage. PMID:2176014

  14. 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...

  15. Permanent cell lines from erythrophoromas in goldfish (Carassius auratus).

    PubMed

    Matsumoto, J; Ishikawa, T; Masahito, P; Takayama, S

    1980-04-01

    Attempts to establish permanent cell lines from spontaneous erythrophoromas (tumors derived from red pigment cells or erythrophores) of goldfish were made with the use of biopsy specimens from 12 tumors in 10 fish. Three cell lines were established that grew in vitro in synthetic medium (L-15 or Dulbecco's modified Eagle's minimum essential medium) supplemented with 20% fetal bovine serum for more than 8 months. One of these lines (GEM-81) with high proliferative activity was cultured for over 200 generations without an obvious change in growth rate. From this cell line, clonal cultures were obtained that formed clones with relatively intense yellow pigmentation. Descendants of these cell lines and clones contained low but detectable amounts of pteridine pigments (such as 7-hydroxybiopterin, biopterin, xanthopterin, and isoxanthopterin) and numerous cytoplasmic organelles analogous to pterinosomes. Both these characteristics are phenotypic markers of normal erythrophores and their neoplastic counterparts. After numerous subcultivations, these long-term cultures differed from those of the initial explants in having much lower contents of total pteridines and relatively lowered contents of 7-hydroxybiopterin. As manifestations of their neoplastic origins, all cell lines examined showed disoriented growth with dense focal mounding in monolayer cultures. The population doubling time of the uncloned GEM-81 cell line was 31 hours at 35 degrees C over a feeder cell layer and 3 days at 25 degrees C without feeder cells. Injection of cultured cells (1.5 x 10(7) cells/fish) into normal goldfish that had not been immunosuppressed did not give rise to tumors within 5 months. PMID:6928999

  16. Surface charge characteristics of cells from malignant cell lines and normal cell lines of the human hematopoietic system.

    PubMed

    Marikovsky, Y; Ben-Bassat, H; Leibovich, S J; Cividalli, L; Fischler, H; Danon, D

    1979-02-01

    Cells from malignant and normal lines of human hematopoietic origin were studied for their surface charge characteristics with the use of the following criteria: 1) the electron microscopic appearance of cell membranes after labeling with cationized ferritin (CF) either before or after glutaraldehyde fixation, 2) electrophoretic mobility, 3) total sialic acid content, and 4) agglutinability with poly-L-lysine (PLL). CF induced a time-dependent redistribution of surface receptors in unfixed malignant cells but not in unfixed normal cells. After 10 seconds of labeling with CF, both normal and malignant unfixed cells showed a uniform and even labeling pattern. After 5 minutes of labeling, malignant cells exhibited a highly pronounced pattern of clusters and patches, as distinct from a random and even pattern exhibited by normal cells. Both normal and malignant cells after fixation exhibited an equivalent random and even labeling pattern with CF, independent of the duration of labeling. The malignant cells studied possessed less sialic acid, had a lower electric mobility, and were agglutinated more readily with PLL than were the normal cells. PMID:310907

  17. 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

  18. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

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

    1990-02-01

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

  19. 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.

  20. 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

  1. 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

  2. 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

  3. 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.

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

    PubMed Central

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

    2012-01-01

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

  5. 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

  6. Xenotropic retrovirus Bxv1 in human pancreatic β cell lines.

    PubMed

    Kirkegaard, Jeannette S; Ravassard, Philippe; Ingvarsen, Signe; Diedisheim, Marc; Bricout-Neveu, Emilie; Grønborg, Mads; Frogne, Thomas; Scharfmann, Raphael; Madsen, Ole D; Rescan, Claude; Albagli, Olivier

    2016-03-01

    It has been reported that endogenous retroviruses can contaminate human cell lines that have been passaged as xenotransplants in immunocompromised mice. We previously developed and described 2 human pancreatic β cell lines (EndoC-βH1 and EndoC-βH2) that were generated in this way. Here, we have shown that B10 xenotropic virus 1 (Bxv1), a xenotropic endogenous murine leukemia virus (MuLV), is present in these 2 recently described cell lines. We determined that Bxv1 was also present in SCID mice that were used for in vivo propagation of EndoC-βH1/2 cells, suggesting that contamination occurred during xenotransplantation. EndoC-βH1/2 cells released Bxv1 particles that propagated to human 293T and Mus dunni cells. Mobilization assays demonstrated that Bxv1 transcomplements defective MuLV-based retrovectors. In contrast, common rodent β cell lines, rat INS-1E and RIN-5F cells and mouse MIN6 and βTC3 cells, displayed either no or extremely weak xenotropic helper activity toward MuLV-based retrovectors, although xenotropic retrovirus sequences and transcripts were detected in both mouse cell lines. Bxv1 propagation from EndoC-βH1/2 to 293T cells occurred only under optimized conditions and was overall poorly efficient. Thus, although our data imply that MuLV-based retrovectors should be cautiously used in EndoC-βH1/2 cells, our results indicate that an involuntary propagation of Bxv1 from these cells can be easily avoided with good laboratory practices. PMID:26901817

  7. Hamster cell line suitable for transfection assay of transforming genes.

    PubMed Central

    Higashi, T; Sasai, H; Suzuki, F; Miyoshi, J; Ohuchi, T; Takai, S; Mori, T; Kakunaga, T

    1990-01-01

    We established a subclone, SHOK, from the GHE-L cell line, an immortal line derived from a primary culture of Syrian hamster embryo cells, as a recipient cell line useful for the detection of oncogenes by transfection. SHOK cells were almost as susceptible as NIH 3T3 cells to focus formation by many oncogenes, including v-raf, v-Ha-ras, v-Ki-ras, or activated c-Ha-ras. The susceptibility of SHOK to focus formation was higher than that of NIH 3T3 for v-mos but was lower for v-fps, v-fgr, v-src, v-sis, and v-abl. When DNAs extracted from 27 human and murine tumors were tested for focus formation, 5 DNAs were positive in NIH 3T3 cells, whereas 9 were positive in SHOK cells at the primary transfection. Using SHOK cells as recipients of tumor cellular DNA, we isolated another oncogene and a c-Ki-ras2 gene mutated at codon 146 that were difficult to detect in NIH 3T3 cells. SHOK cells have a low rate of spontaneous transformation, produce easily distinguishable foci, and maintain a stable karyotype in transformed cells. In addition to being useful for the screening of human tumor DNAs, SHOK cells will be useful for the isolation of oncogenes from murine tumors because of their hamster origin. Images PMID:2181436

  8. 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

  9. Experimental Adaptation of Rotaviruses to Tumor Cell Lines.

    PubMed

    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

  10. Tumorigenicity of human hematopoietic cell lines in athymic nude mice.

    PubMed

    Nilsson, K; Giovanella, B C; Stehlin, J S; Klein, G

    1977-03-15

    Human hematopoietic cell lines, which had been classified on the basis of studies on clonality, and morphological, chromosomal and functional parameters as lymphoblastoid cell lines (LCL) of presumed non-neoplastic origin, and lymphoma, myeloma and leukemia lines of proven malignant origin, were tested for tumorigenic potential on subcutaneous transplantation to nude mice and for capacity to grow in semi-solid medium in vitro. Recently established LCL failed to grow both in nude mice and in agarose. In contrast, some of the LCL which had developed secondary chromosomal alterations during continuous cultivation for periods exceeding several years were tumorigenic and/or had the capacity to form colonies in agarose. Most lymphoma lines formed colonies in agarose and tumors in the mice. One of the two myeloma lines formed subcutaneous tumor which, however, showed no progressive growth. The other myeloma line failed to grow. Both myeloma lines, however, formed colonies in agarose. The myeloid leukemia line was tumorigenic while two of the three tested lymphocytic leukemia lines failed to grow in the mice. All leukemia lines formed colonies in agarose. We conclude from this study that: (1) Of the two types of Epstein-Barr virus containing cell lines [LCL and Burkitt lymphoma (BL) lines], only BL lines were shown to form tumors when inoculated subcutaneously in nude mice and had the capacity to grow in agarose in vitro. This shows that EBV transformation per se does not necessarily render lymphocytes tumorigenic in nude mice. The capacity to form colonies in agarose is not acquired either. (2) Changes of the karyotype and several phenotypic characteristics which occur in the originally diploid LCL during prolonged cultivation in vitro may be accompanied by the acquisition of the potential to grow subcutaneously in nude mice and in agarose in vitro. (3) The inconsistency with regard to the capacity of come of the neoplastic cell lines to grow in nude mice or in agarose seems to underline that neither of the two tests is a reliable criterion for malignancy of human lymphoma, leukemia and myeloma cell lines. PMID:14896

  11. 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.

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

    PubMed

    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

  13. 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

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

    PubMed Central

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

    1991-01-01

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

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. 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 ...

  1. 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.

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

    PubMed

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

    2014-10-01

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

  3. 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

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

    PubMed Central

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

    2014-01-01

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

  5. 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

  6. 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

  7. Female Sex Bias in Human Embryonic Stem Cell Lines

    PubMed Central

    Ben-Yosef, Dalit; Amit, Ami; Malcov, Mira; Frumkin, Tsvia; Ben-Yehudah, Ahmi; Eldar, Ido; Mey-Raz, Nava; Azem, Foad; Altarescu, Gheona; Renbaum, Paul; Beeri, Rachel; Varshaver, Irit; Eldar-Geva, Talia; Epsztejn-Litman, Silvina; Levy-Lahad, Ephrat

    2012-01-01

    The factors limiting the rather inefficient derivation of human embryonic stem cells (HESCs) are not fully understood. The aim of this study was to analyze the sex ratio in our 42 preimplantation genetic diagnosis (PGD)-HESC lines, in an attempt to verify its affect on the establishment of HESC lines. The ratio between male and female PGD-derived cell lines was compared. We found a significant increase in female cell lines (76%). This finding was further confirmed by a meta-analysis for combining the results of all PGD-derived HESC lines published to date (148) and all normal karyotyped HESC lines derived from spare in vitro fertilization embryos worldwide (397). Further, gender determination of embryos demonstrated that this difference originates from the actual derivation process rather than from unequal representation of male and female embryos. It can therefore be concluded that the clear-cut tendency for female preponderance is attributed to suboptimal culture conditions rather than from a true gender imbalance in embryos used for derivation of HESC lines. We propose a mechanism in which aberrant X chromosome inactivation and/or overexpression of critical metabolic X-linked genes might explain this sex dimorphism. PMID:21585244

  8. Reconstruction of Endometrium from Human Endometrial Side Population Cell Lines

    PubMed Central

    Cervelló, Irene; Mas, Aymara; Gil-Sanchis, Claudia; Peris, Laura; Faus, Amparo; Saunders, Philippa T. K.; Critchley, Hilary O. D.; Simón, Carlos

    2011-01-01

    Endometrial regeneration is mediated, at least in part, by the existence of a specialized somatic stem cell (SSC) population recently identified by several groups using the side population (SP) technique. We previously demonstrated that endometrial SP displays genotypic, phenotypic and the functional capability to develop human endometrium after subcutaneous injection in NOD-SCID mice. We have now established seven human endometrial SP (hESP) cell lines (ICE 1–7): four from the epithelial and three from the stromal fraction, respectively. SP cell lines were generated under hypoxic conditions based on their cloning efficiency ability, cultured for 12–15 passages (20 weeks) and cryopreserved. Cell lines displayed normal 46XX karyotype, intermediate telomerase activity pattern and expressed mRNAs encoding proteins that are considered characteristic of undifferentiated cells (Oct-4, GDF3, DNMT3B, Nanog, GABR3) and those of mesodermal origin (WT1, Cardiac Actin, Enolase, Globin, REN). Phenotype analysis corroborated their epithelial (CD9+) or stromal (vimentin+) cell origin and mesenchymal (CD90+, CD73+ and CD45−) attributes. Markers considered characteristic of ectoderm or endoderm were not detected. Cells did not express either estrogen receptor alpha (ERα) or progesterone receptor (PR). The hESP cell lines were able to differentiate in vitro into adipocytes and osteocytes, which confirmed their mesenchymal origin. Finally, we demonstrated their ability to generate human endometrium when transplanted beneath the renal capsule of NOD-SCID mice. These findings confirm that SP cells exhibit key features of human endometrial SSC and open up new possibilities for the understanding of gynecological disorders such as endometriosis or Asherman syndrome. Our cell lines can be a valuable model to investigate new targets for endometrium proliferation in endometriosis. PMID:21712999

  9. 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

  10. Definitive Molecular Cytogenetic Characterization of 15 Colorectal Cancer Cell Lines

    PubMed Central

    Knutsen, Turid; Padilla-Nash, Hesed M.; Wangsa, Danny; Barenboim-Stapleton, Linda; Camps, Jordi; McNeil, Nicole; Difilippantonio, Michael J.; Ried, Thomas

    2009-01-01

    In defining the genetic profiles in cancer, cytogenetically aberrant cell lines derived from primary tumors are important tools for the study of carcinogenesis. We here present the results of a comprehensive investigation of 15 established colorectal cancer cell lines utilizing spectral karyotyping (SKY), fluorescence in situ hybridization, and comparative genomic hybridization (CGH). Detailed karyotypic analysis by SKY on five of the lines (P53HCT116, T84, NCI-H508, NCI-H716, and SK-CO-1) are described here for the first time. The five lines with karyotypes in the diploid range and that are characterized by defects in DNA mismatch repair had a mean of 4.8 chromosomal abnormalities per line, whereas the 10 aneuploid lines exhibited complex karyotypes and a mean of 30 chromosomal abnormalities. Of the 150 clonal translocations, only eight were balanced and none were recurrent among the lines. We also reviewed the karyotypes of 345 cases of adenocarcinoma of the large intestine listed in the Mitelman Database of Chromosome Aberrations in Cancer. The types of abnormalities observed in the cell lines reflected those seen in primary tumors: there were no recurrent translocations in either tumors or cell lines, isochromosomes were the most common recurrent abnormalities, and breakpoints occurred most frequently at the centromeric/pericentromeric and telomere regions. Of the genomic imbalances detected by array CGH, 87% correlated with chromosome aberrations observed in the SKY studies. The fact that chromosome abnormalities result predominantly in copy number changes rather than specific chromosome or gene fusions, suggests this may be the major mechanism leading to carcinogenesis in colorectal cancer. PMID:19927377

  11. 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

  12. 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

  13. 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

  14. Skin Biopsy and Patient-Specific Stem Cell Lines

    PubMed Central

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

    2016-01-01

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

  15. 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.

  16. 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

  17. 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

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

    PubMed Central

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

    2010-01-01

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

  19. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  1. 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

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

    PubMed Central

    2010-01-01

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

  3. Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines

    PubMed Central

    Coram, Marc A.; Reddy, Anupama; Deng, Glenn; Telli, Melinda L.; Advani, Ranjana H.; Carlson, Robert W.; Mollick, Joseph A.; Sheth, Shruti; Kurian, Allison W.; Ford, James M.; Stockdale, Frank E.; Quake, Stephen R.; Pease, R. Fabian; Mindrinos, Michael N.; Bhanot, Gyan; Dairkee, Shanaz H.; Davis, Ronald W.; Jeffrey, Stefanie S.

    2012-01-01

    Background To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery. Methodology/Principal Findings We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy. Conclusions/Significance For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of ‘liquid biopsies’ to better model drug discovery. PMID:22586443

  4. 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

  5. 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

  6. 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

  7. Amniotic membrane-derived cells inhibit proliferation of cancer cell lines by inducing cell cycle arrest

    PubMed Central

    Magatti, Marta; Munari, Silvia; Vertua, Elsa; Parolini, Ornella

    2012-01-01

    Cells derived from the amniotic foetal membrane of human term placenta have drawn particular attention mainly for their plasticity and immunological properties, which render them interesting for stem-cell research and cell-based therapeutic applications. In particular, we have previously demonstrated that amniotic mesenchymal tissue cells (AMTC) inhibit lymphocyte proliferation in vitro and suppress the generation and maturation of monocyte-derived dendritic cells. Here, we show that AMTC also significantly reduce the proliferation of cancer cell lines of haematopoietic and non-haematopoietic origin, in both cell–cell contact and transwell co-cultures, therefore suggesting the involvement of yet-unknown inhibitory soluble factor(s) in this ‘cell growth restraint’. Importantly, we provide evidence that the anti-proliferative effect of AMTC is associated with induction of cell cycle arrest in G0/G1 phase. Gene expression analyses demonstrate that AMTC can down-regulate cancer cells' mRNA expression of genes associated with cell cycle progression, such as cyclins (cyclin D2, cyclin E1, cyclin H) and cyclin-dependent kinase (CDK4, CDK6 and CDK2), whilst they up-regulate cell cycle negative regulator such as p15 and p21, consistent with a block in G0/G1 phase with no progression to S phase. Taken together, these findings warrant further studies to investigate the applicability of these cells for controlling cancer cell proliferation in vivo. PMID:22260183

  8. 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

  9. 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

  10. 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

  11. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

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

    2005-07-15

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

  12. 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

  13. Characterisation of seven newly established head and neck squamous cell carcinoma cell lines.

    PubMed

    Görögh, Tibor; Quabius, Elgar Susanne; Meyer, Patrick; Hoffmann, Markus

    2015-05-01

    Seven squamous cell carcinoma cell lines were disintegrated from biopsies of patients with head and neck cancer. Genotyping tests verified the authenticity and the human origin of all seven lines. The cell lines designated as University of Kiel, Head and Neck (UKHN) -1 to -3 and UKHN-6 to -9 were subjected to flow cytometry and indirect immunofluorescence to assess aberrant DNA content. To confirm the squamous epithelial origin of the cells, the cytokeratin profile was immunocytologically analysed. The cell lines showed individual differences in mitotic frequency. UKHN-1, -6, -7 and -9 grew as monolayers, whereas UKHN-2, -3, and -8 tend to multilayer stratification. Overexpression of LOXL4 and Pim-1 proteins as distinctive features of head and neck carcinomas were shown in all seven cell lines. Inoculating SCID mice with these cell lines resulted in tumour formation, hence corroborating the tumourigenicity of all seven cell lines. The cell lines were also tested for high-risk HPV types using different DNA-based assays and found to be negative. PMID:24792014

  14. Establishment of a human neonatal hepatocyte cell line.

    PubMed

    Reid, Yvonne; Gaddipati, Jaya P; Yadav, Deepmala; Kantor, Judy

    2009-10-01

    Hepatocytes are routinely used to generate and identify drug metabolites and hepatic toxicity. Primary cultures of human hepatocytes are the model cell of choice for most of these pharmacological and toxicological studies. However, major problems are encountered with primary liver cell cultures: the dwindling availability of viable livers, hepatocytes having a limited life span, the loss of liver-specific functions in culture, and the donor to donor variability. These limitations have created a significant need for an in vitro hepatocyte system, which has both the potential for use in toxicological and pharmaceutical studies as well as clinical applications. Ectopic expression of human telomerase reverse transcriptase (hTERT) is one of the major strategies used to develop immortalized cells. Immortalization of primary cells using hTERT allows retention of the original cellular characteristics and functions and avoids some of the genetic and phenotypic instabilities associated with using known oncogenes. In the present study, we developed a cell line from human neonatal hepatocytes by transduction with a recombinant retrovirus expressing the hTERT gene. Induction of stable expression of hTERT in the neonatal cells led to immortalization of these cells. The cell line was cultured continuously for more than 25 passages, equivalent to >25 population doublings, whereas the parental cells senesced within five passages. Analysis of telomerase activity as measured by telomeric repeat amplification protocol assay indicated elevated levels of telomerase activity in immortalized cells compared to the parental cells. These immortalized human hepatocytes cells maintained a normal diploid karyotype as well as the gene expression profile similar to that of human normal neonatal hepatocytes. The data suggest that these immortalized cells preserved some of the biological characteristics of hepatic progenitor cells and might be useful as an in vitro model for pharmacological and toxicity studies. PMID:19565302

  15. Evolution and ultrastructure of the bovine spermatogonia precursor cell line.

    PubMed

    Wrobel, K H; Bickel, D; Kujat, R; Schimmel, M

    1995-08-01

    The spermatogonial stem cell line in prepubertal and adult bovine testis was studied by electron microscopy and protein gene product 9.5 immunohistochemistry. Three successive spermatogonia precursor cell configurations were observed. Small basal stem cells were found to possess a spherical shape and nuclei with two to three nucleoli. They were observed in prepubertal testes (25 and 30 weeks) and in low numbers during all the stages of the seminiferous epithelial cycle in the adult. Aggregated spermatogonia precursor cells are the dominating germ cell type in the 25-week-old and 30-week-old calf. In the adult seminiferous epithelium, they cause expansion of the basal tubular compartment as they form dense groups containing up to 15 cells. These groups are observed concomitantly with cycling A-spermatogonia and preleptotenes at the beginning of spermatocytogenesis. At the end of A-spermatogonia propagation, the aggregated spermatogonia precursor cells separate and intermingle with cycling A-spermatogonia. The spermatogonia precursor cells can later be found together with I-spermatogonia as members of an interconnected cellular network of medium-sized cells. When the I-spermatogonia divide to form the smaller B-spermatogonia, the precursor cells, which stay connected with the cycling spermatogonial population, pass through a growth phase. They can now be considered as committed spermatogonia precursor cells and are continuously being transformed into A1-spermatogonia to start a new round of spermatocytogenesis. Ultrastructurally, all members of the precursor cell line are similar. However, a number of features have been found to show a quantitative increase (endoplasmic reticulum, mitochondria) or to exhibit a rising degree of complexity (nucleolus) during the progression from basal stem cells to committed spermatogonia precursor cells. PMID:7648620

  16. Aphid-symbiotic bacteria cultured in insect cell lines.

    PubMed

    Darby, A C; Chandler, S M; Welburn, S C; Douglas, A E

    2005-08-01

    The cells and tissues of many aphids contain bacteria known as "secondary symbionts," which under specific environmental circumstances may be beneficial to the host insect. Such symbiotic bacteria are traditionally described as intractable to cultivation in vitro. Here we show that two types of aphid secondary symbionts, known informally as T type and U type, can be cultured and maintained in three insect cell lines. The identities of the cultured bacteria were confirmed by PCR with sequencing of 16S rRNA gene fragments and fluorescence in situ hybridization. In cell lines infected with bacteria derived from aphids harboring both T type and U type, the U type persisted, while the T type was lost. We suggest that the two bacteria persist in aphids because competition between them is limited by differences in tropism for insect tissues or cell types. The culture of these bacteria in insect cell lines provides a new and unique research opportunity, offering a source of unibacterial material for genomic studies and a model system to investigate the interactions between animal cells and bacteria. We propose the provisional taxon names "Candidatus Consessoris aphidicola" for T type and "Candidatus Adiaceo aphidicola" for U type. PMID:16085881

  17. [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

  18. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

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

  19. USING NEUROBLASTOMA CELL LINES TO EXAMINE ORGANOPHOSPHATE NEUROTOXICITY

    EPA Science Inventory

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

  20. 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...

  1. 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

  2. DIFFERENCES IN ARACHIDONIC ACID METABOLISM BY HUMAN MYELOMONCYTIC CELL LINES

    EPA Science Inventory

    The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines were determined after incubation with interferongamma (IFNg; 500 U/ml) or vehicle for 4 days. ells were prelabeled with tritiated arachidonic acid for 4 hours, and media supernata...

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

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

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

  4. 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...

  5. 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.

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

    PubMed

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

    2011-09-01

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

  7. 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.

  8. 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.

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

    PubMed

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

    2015-12-01

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

  10. 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

  11. 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

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

    PubMed Central

    2014-01-01

    Background Euterpe oleracea Mart., a plant from the Amazon region, is commonly known as açaí or juçara; it has high nutritional value and elevated levels of lipids, proteins, and minerals. Açaí is an abundant and much consumed fruit by the Amazon local population, and studies have demonstrated that it is rich in phytochemicals with antioxidant, anti-inflammatory, and anticancer activities. Therefore, the aim of this study was to test this plant for anticancer activity in different human malignant cell lines. Methods Cell lines derived from breast and colorectal adenocarcinomas were treated with 10, 20, and 40 μg/mL of bark, seed, and total açaí fruit hydroalcoholic extracts for 24 and 48 h. After treatment, cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, and cell morphological features were observed by light and transmission electron microscopy. The type of cell death was also evaluated. The data were analyzed statistically by one-way analysis of variance (ANOVA), followed by Dunnett’s or Tukey’s post hoc tests, as appropriate. Results We observed that of all the cell lines tested, MCF-7 was the only line that responded to açaí treatment. The extracts caused significant reduction (p < 0.01) in cell viability and altered cell morphological features by inducing the appearance of autophagic vacuoles, as observed by transmission electron microscopy. Furthermore, increased expression of LC3BII, a protein marker of autophagosome formation, was observed by western blotting. Caspase Glo™ assays and morphologic observations by DAPI nuclear staining and transmission electron microscopy did not indicate any apoptotic events. Conclusions The present study demonstrated that açaí possesses antitumorigenic potential in the MCF-7 cell line. Further studies are needed to identify the compound (s) responsible for this cytotoxic activity and the molecular target in the cell. This discovery of the anticancer potential of açaí may help in the development of chemopreventive drugs and may have therapeutic effects in the treatment of breast cancer. PMID:24886139

  13. 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

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

    PubMed

    Kowarz, Eric; Löscher, 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

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

    PubMed

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

    2006-07-01

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

  16. 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

  17. 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

  18. Characterization of experimental rat nephroblastoma and its cell line.

    PubMed

    Nagashima, Y; Miyagi, Y; Sumino, K; Ohaki, Y; Umeda, M; Oshimura, M; Misugi, K

    1992-10-01

    Rat nephroblastoma (Wilms' tumor) was induced by transplacental administration of N-ethyl-nitrosourea (ENU). The induced renal tumors were histologically compatible with human nephroblastoma. A cultured cell line (ENU-T-1) established from a xenotransplant, showed similar morphological and biological features to cultured embryonal kidney cells. Introduction of normal human chromosome #11 (#11) bearing Wilms' tumor suppressor gene(s) (WT) suppressed colony-forming ability on soft agar plates (CFA) but tumorigenicity of ENU-T-1 was not affected. Whereas tumorigenicity of human nephroblastoma cell line, SK-NEP-1 was completely suppressed, CFA was unchanged. These facts indicated that pathogenetic mechanism is different between human and experimental rat nephroblastomas. PMID:1339105

  19. 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.

  20. Ellipticine cytotoxicity to cancer cell lines - a comparative study.

    PubMed

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

    2011-06-01

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

  1. 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

  2. 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

  3. 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

  4. The potassium channel opener NS1619 modulates calcium homeostasis in muscle cells by inhibiting SERCA.

    PubMed

    Wrzosek, Antoni

    2014-07-01

    NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca(2+)-activated K(+) (BKCa) channel opener. It was previously reported that activation of BKCa channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca(2+) homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca(2+) homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca(2+) uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca(2+) from internal stores in a concentration-dependent manner. Ca(2+) accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca(2+) concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered. PMID:24813114

  5. 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

  6. 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,...

  7. Cytolytic replication of echoviruses in colon cancer cell lines

    PubMed Central

    2011-01-01

    Background Colorectal cancer is one of the most common cancers in the world, killing nearly 50% of patients afflicted. Though progress is being made within surgery and other complementary treatments, there is still need for new and more effective treatments. Oncolytic virotherapy, meaning that a cancer is cured by viral infection, is a promising field for finding new and improved treatments. We have investigated the oncolytic potential of several low-pathogenic echoviruses with rare clinical occurrence. Echoviruses are members of the enterovirus genus within the family Picornaviridae. Methods Six colon cancer cell lines (CaCo-2, HT29, LoVo, SW480, SW620 and T84) were infected by the human enterovirus B species echovirus 12, 15, 17, 26 and 29, and cytopathic effects as well as viral replication efficacy were investigated. Infectivity was also tested in spheroids grown from HT29 cells. Results Echovirus 12, 17, 26 and 29 replicated efficiently in almost all cell lines and were considered highly cytolytic. The infectivity of these four viruses was further evaluated in artificial tumors (spheroids), where it was found that echovirus 12, 17 and 26 easily infected the spheroids. Conclusions We have found that echovirus 12, 17 and 26 have potential as oncolytic agents against colon cancer, by comparing the cytolytic capacity of five low-pathogenic echoviruses in six colon cancer cell lines and in artificial tumors. PMID:21999585

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

    PubMed

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

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering. PMID:25842107

  9. Processing of proSAAS in neuroendocrine cell lines.

    PubMed Central

    Mzhavia, Nino; Qian, Yimei; Feng, Yun; Che, Fa-Yun; Devi, Lakshmi A; Fricker, Lloyd D

    2002-01-01

    ProSAAS, a recently discovered granin-like protein, potently inhibits prohormone convertase (PC)1, and might also perform additional functions. In the present study, the processing of proSAAS was compared in two neuroendocrine cell lines overexpressing this protein: the AtT-20 mouse pituitary corticotrophic line and the PC12 rat adrenal phaeochromocytoma line. The processing of proSAAS was examined by pulse-chase analysis using [(3)H]leucine, by MS, and by chromatography and radioimmunoassay. Various smaller forms of proSAAS were detected, including peptides designated as little SAAS, PEN and big LEN. Because the PC-12 cells used in the present study do not express either PC1 or PC2, the finding that these cells efficiently cleave proSAAS indicates that these cleavages do not require either enzyme. Two of the peptides identified in AtT-20 media represent novel C-terminally truncated forms of PEN. In both cell lines, the secretion of the small proSAAS-derived peptides is stimulated by secretagogues. However, long-term treatment of wild-type AtT-20 cells with two different secretagogues (8-bromo-cAMP and a phorbol ester) does not affect levels of proSAAS mRNA; this treatment significantly increases PC1 mRNA by approx. 60-80%. The lack of co-regulation of proSAAS and PC1 mRNA implies that enzyme activity can be induced without an accompanying increase in the inhibitor. In addition, the finding that the peptides are secreted via the regulated pathway is consistent with the proposal that they may function as neuropeptides. PMID:11742530

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. 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

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

    PubMed Central

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

    2016-01-01

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

  15. 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

  16. 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

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

    PubMed Central

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

    2015-01-01

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

  18. 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.

  19. Cell line profiling to improve monoclonal antibody production.

    PubMed

    Kang, Sohye; Ren, Da; Xiao, Gang; Daris, Kristi; Buck, Lynette; Enyenihi, Atim A; Zubarev, Roman; Bondarenko, Pavel V; Deshpande, Rohini

    2014-04-01

    Mammalian cell culture performance is influenced by both intrinsic (genetic) and extrinsic (media and process) factors. In this study, intrinsic capacity of various monoclonal antibody-producing Chinese Hamster Ovary (CHO) cell lines was compared by exposing them to the same culture condition. Microarray-based transcriptomics and LC-MS/MS shotgun proteomics technologies were utilized to obtain expression landscape of different cell lines. Specific transcripts and proteins correlating with productivity, growth rate and cell size have been identified. The proteomics analysis results showed a strong correlation between the intracellular protein expression levels of the recombinant DHFR and productivity. In contrast, neither the light chain nor the heavy chain of the recombinant monoclonal antibody showed correlation to productivity. Other top ranked proteins which demonstrated positive correlation to productivity included the adaptor protein complex subunits AP3D1and AP2B2, DNA repair protein DDB1 and the ER translocation complex component, SRPR. The subunits of molecular chaperone T-complex protein 1 and the regulator of mitochondrial one-carbon metabolism MTHFD2 showed negative correlation to productivity. The transcriptomics analysis has identified the regulators of calcium signaling, Tmem20 and Rcan1, as the top ranked genes displaying positive and negative correlation to productivity, respectively. For the second part of the study, the principal component analysis (PCA) was generated to view the underlying global structure of the expression data. A clear division and expression polarity was observed between the two distinct clusters of cell lines, independent of link to productivity or any other traits examined. The primary component of the PCA generated from either transcriptomics or proteomics data displayed a strong correlation to cell size and doubling time, while none of the main principal components showed correlation to productivity. Our findings suggest that productivity is rather a minor feature in the context of global transcriptional or protein expression space. PMID:24249214

  20. Vitamin K2-induced cell growth inhibition via autophagy formation in cholangiocellular carcinoma cell lines.

    PubMed

    Enomoto, Masanobu; Tsuchida, Akihiko; Miyazawa, Keisuke; Yokoyama, Tomohisa; Kawakita, Hideaki; Tokita, Hiromi; Naito, Munekazu; Itoh, Masahiro; Ohyashiki, Kazuma; Aoki, Tatsuya

    2007-12-01

    Vitamin K2 (MK4) has antitumor effects on various types of cancer cell lines in vitro, and its efficacy has also been reported in clinical applications for patients with leukemia, myelodysplastic syndrome, and hepatocellular carcinoma (HCC). However, details of the mechanism of the antitumor effects of MK4 remain unclear. In the present study, we examined the antitumor effects of MK4 on cholangiocellular carcinoma (CCC) cell lines and its mechanism of action using the HL-60 leukemia cell line that exerts MK4-induced cell growth inhibition via apoptosis induction and cell cycle arrest as a control. MK4 exerted dose-dependent antitumor effects on all three types of CCC cell lines. However, apoptosis occurred in a smaller percentage of cells and there was less cell cycle arrest compared with other cancer cell lines studied previously, which suggested slight MK4-induced cell growth inhibition via apoptosis induction and cell cycle arrest. On the contrary, histopathological fidings showed a large number of cells containing vacuoles in their cytoplasm, and electron microscopic findings showed a large number of cytoplasmic autophagosomes and autolysosomes. These findings suggested evidence of autophagy-related cell death. Fluorescence microscopy following acridine orange staining revealed an increase in the number of cytoplasmic acidic vesicular organelles characteristic of autophagy. Moreover, there were few cells forming autophagic vesicles in the control group, while the percentage of cells containing vacuoles in the MK4-treated group increased with the duration of culture. These results suggested that, unlike in leukemia, gastric cancer, HCC, and other cancer cells, the antitumor effects of MK4 on CCC cells are induced via autophagy formation. PMID:17982686

  1. 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.

  2. 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

  3. 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

  4. A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells.

    PubMed

    Greenwood-Goodwin, Midori; Yang, Jiwei; Hassanipour, Mohammad; Larocca, David

    2016-01-01

    Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy. PMID:27109637

  5. A novel lineage restricted, pericyte-like cell line isolated from human embryonic stem cells

    PubMed Central

    Greenwood-Goodwin, Midori; Yang, Jiwei; Hassanipour, Mohammad; Larocca, David

    2016-01-01

    Pericytes (PCs) are endothelium-associated cells that play an important role in normal vascular function and maintenance. We developed a method comparable to GMP quality protocols for deriving self-renewing perivascular progenitors from the human embryonic stem cell (hESC), line ESI-017. We identified a highly scalable, perivascular progenitor cell line that we termed PC-A, which expressed surface markers common to mesenchymal stromal cells. PC-A cells were not osteogenic or adipogenic under standard differentiation conditions and showed minimal angiogenic support function in vitro. PC-A cells were capable of further differentiation to perivascular progenitors with limited differentiation capacity, having osteogenic potential (PC-O) or angiogenic support function (PC-M), while lacking adipogenic potential. Importantly, PC-M cells expressed surface markers associated with pericytes. Moreover, PC-M cells had pericyte-like functionality being capable of co-localizing with human umbilical vein endothelial cells (HUVECs) and enhancing tube stability up to 6 days in vitro. We have thus identified a self-renewing perivascular progenitor cell line that lacks osteogenic, adipogenic and angiogenic potential but is capable of differentiation toward progenitor cell lines with either osteogenic potential or pericyte-like angiogenic function. The hESC-derived perivascular progenitors described here have potential applications in vascular research, drug development and cell therapy. PMID:27109637

  6. Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines.

    PubMed

    Musk, S R; Hatton, D H; Bouffler, S D; Margison, G P; Johnson, R T

    1989-07-01

    The responses of two Indian muntjac cell lines to two monofunctional alkylating agents were investigated. An SV40-transformed line (SVM) had an increased sensitivity to cell killing when compared to the other, euploid line (DM) after exposure both to methyl nitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibited higher frequencies of sister chromatid exchanges (SCEs) following alkylation. The hypersensitivity of SVM to DMS correlates with the defective repair of single-strand breaks that results in the generation of long-lived breaks in the DNA following exposure, leading eventually to the formation of chromosome aberrations. In contrast no difference is seen in the formation of long-lived breaks in the DNA of SVM and DM after treatment with biologically relevant doses of MNU; in this case hypersensitivity may be due to the loss of O6-alkylguanine-DNA-alkyltransferase activity. The conclusion that the hypersensitivites of SVM to MNU and to DMS have different molecular bases is supported by transfection of SVM with plasmids containing the protein coding region of the Escherichia coli ada+ gene; subsequent expression within the cell corrects its hypersensitivity to the cytotoxic and SCE-inducing effects of MNU but has very little influence upon the lethality, SCE induction or the repair of long-lived DNA strand breaks after exposure to DMS. PMID:2544312

  7. Cancer cell lines for drug discovery and development.

    PubMed

    Wilding, Jennifer L; Bodmer, Walter F

    2014-05-01

    Despite the millions of dollars spent on target validation and drug optimization in preclinical models, most therapies still fail in phase III clinical trials. Our current model systems, or the way we interpret data from them, clearly do not have sufficient clinical predictive power. Current opinion suggests that this is because the cell lines and xenografts that are commonly used are inadequate models that do not effectively mimic and predict human responses. This has become such a widespread belief that it approaches dogma in the field of drug discovery and optimization and has spurred a surge in studies devoted to the development of more sophisticated animal models such as orthotopic patient-derived xenografts in an attempt to obtain more accurate estimates of whether particular cancers will respond to given treatments. Here, we explore the evidence that has led to the move away from the use of in vitro cell lines and toward various forms of xenograft models for drug screening and development. We review some of the pros and cons of each model and give an overview of ways in which the use of cell lines could be modified to improve the predictive capacity of this well-defined model. PMID:24717177

  8. Effects of cylindrospermopsin on a common carp leucocyte cell line.

    PubMed

    Sieroslawska, Anna; Rymuszka, Anna

    2015-01-01

    Cylindrospermopsin (CYN) is a cytotoxin produced by different cyanobacterial species, increasingly detected in water reservoirs worldwide. There is very little information available concerning the effects of the toxin on fish immune cells. The aim of the study was to elucidate the potential impact of cylindrospermopsin on the selected parameters of a common carp (Cyprinus carpio L.) leucocyte cell line (CLC). The cells were incubated with the cyanotoxin at concentrations of 10, 1 or 0.1 µg ml(-1) for up to 48 h. Cell viability and proliferation, apoptosis/necrosis induction, cell morphology and phagocytic activity were determined. The two higher toxin concentrations occurred to be evidently cytotoxic in a time-dependent manner and influenced all studied parameters. The lowest used concentration had no effects on cell viability and cell number; however, a strong reduction of bacteria uptake after 24-h exposure was detected. The obtained results indicate that cylindrospermopsin may interfere with the basic functions of fish phagocytic cells and as a consequence influence the fish immunity. PMID:24477983

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

    PubMed

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

    2009-12-01

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

  10. Antimanic drug sensitizes breast cancer cell line to ionizing radiation.

    PubMed

    Rouhani, Maryam; Goliaei, Bahram; Khodagholi, Fariba; Nikoofar, Alireza

    2014-01-01

    Breast cancer is one of the most prevalent types of cancer among women. Lithium chloride (LiCl) is an FDA-approved drug for bipolar disorder. Breast cancer is reported to occur with higher rate in women with bipolar disorder. The effect of LiCl on the response of breast cancer cells to ionizing radiation has not been studied. We studied the effect of LiCl on the radiosensitivity of radioresistant T47D breast cancer cell line. Treatment of T47D cells with 20 mM LiCl for 24 hours decreased the radioresistance of these cells indicated by clonogenic survival assay. Comet assay demonstrated decreased DNA repair in LiCl-treated cells. LiCl treatment also decreased the meiotic recombination 11 (Mre11) mRNA level. Mre11 is an essential protein for DNA repair whose transcription is regulated by β-catenin protein. Western blot analysis indicated that the β-catenin protein level was decreased in LiCl-treated cells. LiCl increased glycogen synthase kinase-3β (GSK-3β) protein that is involved in β-catenin degradation. The results demonstrated that LiCl could radiosensitize T47D cells by decreasing DNA repair, partially through Mre11 repression. GSK-3β/β-catenin/Mre11 pathway might be the connection between LiCl treatment and the decreased DNA repair in T47D cells. PMID:24448371

  11. Cytogenetic instability of dental pulp stem cell lines.

    PubMed

    Duailibi, Monica Talarico; Kulikowski, Leslie Domenici; Duailibi, Silvio Eduardo; Lipay, Monica Vannucci Nunes; Melaragno, Maria Isabel; Ferreira, Lydia Masako; Vacanti, Joseph Phillip; Yelick, Pamela Crotty

    2012-02-01

    Human adult stem cells (hASCs) offer a potentially renewable source of cell types that are easily isolated and rapidly expanded for use in regenerative medicine and cell therapies without the complicating ethical problems that are associated with embryonic stem cells. However, the eventual therapeutic use of hASCs requires that these cells and their derivatives maintain their genomic stability. There is currently a lack of systematic studies that are aimed at characterising aberrant chromosomal changes in cultured ASCs over time. However, the presence of mosaicism and accumulation of karyotypic abnormalities within cultured cell subpopulations have been reported. To investigate cytogenetic integrity of cultured human dental stem cell (hDSC) lines, we analysed four expanded hDSC cultures using classical G banding and fluorescent in situ hybridisation (FISH) with X chromosome specific probe. Our preliminary results revealed that about 70% of the cells exhibited karyotypic abnormalities including polyploidy, aneuploidy and ring chromosomes. The heterogeneous spectrum of abnormalities indicates a high frequency of chromosomal mutations that continuously arise upon extended culture. These findings emphasise the need for the careful analysis of the cytogenetic stability of cultured hDSCs before they can be used in clinical therapies. PMID:22109772

  12. 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

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

    PubMed Central

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

    2016-01-01

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

  14. Investigation of native fluorescence spectral difference among prostate cancer cell lines with different risk levels

    NASA Astrophysics Data System (ADS)

    Pu, Yang; Xue, Jianpeng; Xu, Baogang; Wang, Wubao; Gu, Yueqing; Tang, Rui; Achilefu, S.; Ackerstaff, Ellen; Koutcher, Jason A.; Alfano, R. R.

    2013-03-01

    The alteration of native fluorophores among different types of cancer cell lines was investigated by the fluorescence spectroscopy. Different types of cancer cell lines with different risk levels, such as moderate metastatic (DU-145) and advanced metastatic (PC-3) cell lines as well as normal cell line (Fibroblast), were excited by the selective excitation wavelength of 300 nm to explore changes of the relative contents of tryptophan and NADH using principal component analysis (PCA). The higher relative content of tryptophan was observed in the advanced metastatic cancer cell lines in comparison with the moderate metastatic and non aggressive cell lines.

  15. 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.

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

    PubMed Central

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

    2014-01-01

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

  17. 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

  18. 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.

  19. Interactions of Streptococcus iniae with phagocytic cell line.

    PubMed

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

    2015-04-01

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

  20. 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.

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

    PubMed

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

    2016-04-01

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

  2. 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

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

    PubMed

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

    2015-07-01

    Melatonin is a small indole produced by the pineal gland and other tissues, and has numerous functions that aid in the maintenance of the whole body homeostasis, ranging from the regulation of circadian rhythms and sleep to protection from oxidative stress. Melatonin has also been reported to counteract cell growth and chemoresistance in different types of cancer. In the present study, we investigated the effects of exogenous melatonin administration on different human cell lines and primary mouse tumor cultures of rhabdomyosarcoma (RMS), the most frequent soft tissue sarcoma affecting childhood. The results showed that melatonin significantly affected the behavior of RMS cells, leading to inhibition of cell proliferation and impairment of myogenic differentiation followed by increased apoptotic cell death, as observed by immunoblotting analysis of apoptosis-related markers including Bax, Bcl-2 and caspase-3. Similar findings were observed using a combination of microscopy techniques, including scanning/transmission electron and confocal microscopy. Furthermore, melatonin in combination with doxorubicin or cisplatin, two compounds commonly used for the treatment of solid tumors, increased the sensitivity of RMS cells to apoptosis. These data indicated that melatonin may be effective in counteracting RMS tumor growth and chemoresistance. PMID:25998836

  4. 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

  5. Iron oxide nanoparticle-mediated development of cellular gap junction crosstalk to improve mesenchymal stem cells' therapeutic efficacy for myocardial infarction.

    PubMed

    Han, Jin; Kim, Bokyoung; Shin, Jung-Youn; Ryu, Seungmi; Noh, Myungkyung; Woo, Jongsu; Park, Jin-Sil; Lee, Youjin; Lee, Nohyun; Hyeon, Taeghwan; Choi, Donghoon; Kim, Byung-Soo

    2015-03-24

    Electrophysiological phenotype development and paracrine action of mesenchymal stem cells (MSCs) are the critical factors that determine the therapeutic efficacy of MSCs for myocardial infarction (MI). In such respect, coculture of MSCs with cardiac cells has windowed a platform for cardiac priming of MSCs. Particularly, active gap junctional crosstalk of MSCs with cardiac cells in coculture has been known to play a major role in the MSC modification through coculture. Here, we report that iron oxide nanoparticles (IONPs) significantly augment the expression of connexin 43 (Cx43), a gap junction protein, of cardiomyoblasts (H9C2), which would be critical for gap junctional communication with MSCs in coculture for the generation of therapeutic potential-improved MSCs. MSCs cocultured with IONP-harboring H9C2 (cocultured MSCs: cMSCs) showed active cellular crosstalk with H9C2 and displayed significantly higher levels of electrophysiological cardiac biomarkers and a cardiac repair-favorable paracrine profile, both of which are responsible for MI repair. Accordingly, significantly improved animal survival and heart function were observed upon cMSC injection into rat MI models compared with the injection of unmodified MSCs. The present study highlights an application of IONPs in developing gap junctional crosstalk among the cells and generating cMSCs that exceeds the reparative potentials of conventional MSCs. On the basis of our finding, the potential application of IONPs can be extended in cell biology and stem cell-based therapies. PMID:25688594

  6. 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

  7. 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

  8. 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.

  9. 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

  10. Internalization of cystatin C in human cell lines.

    PubMed

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

    2008-09-01

    Altered protease activity is considered important for tumour invasion and metastasis, processes in which the cysteine proteases cathepsin B and L are involved. Their natural inhibitor cystatin C is a secreted protein, suggesting that it functions to control extracellular protease activity. Because cystatins added to cell cultures can inhibit polio, herpes simplex and coronavirus replication, which are intracellular processes, the internalization and intracellular regulation of cysteine proteases by cystatin C should be considered. The extension, mechanism and biological importance of this hypothetical process are unknown. We investigated whether internalization of cystatin C occurs in a set of human cell lines. Demonstrated by flow cytometry and confocal microscopy, A-431, MCF-7, MDA-MB-453, MDA-MB-468 and Capan-1 cells internalized fluorophore-conjugated cystatin C when exposed to physiological concentrations (1 microm). During cystatin C incubation, intracellular cystatin C increased after 5 min and accumulated for at least 6 h, reaching four to six times the baseline level. Western blotting showed that the internalized inhibitor was not degraded. It was functionally intact and extracts of cells exposed to cystatin C showed a higher capacity to inhibit papain and cathepsin B than control cells (decrease in enzyme activity of 34% and 37%, respectively). The uptake of labelled cystatin C was inhibited by unlabelled inhibitor, suggesting a specific pathway for the internalization. We conclude that the cysteine protease inhibitor cystatin C is internalized in significant quantities in various cancer cell lines. This is a potentially important physiological phenomenon not previously described for this group of inhibitors. PMID:18699780

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. Establishment of immortalized murine mesothelial cells and a novel mesothelioma cell line.

    PubMed

    Blum, Walter; Pecze, László; Felley-Bosco, Emanuela; Worthmüller-Rodriguez, Janine; Wu, Licun; Vrugt, Bart; de Perrot, Marc; Schwaller, Beat

    2015-08-01

    Mesothelial cells are susceptible to asbestos fiber-induced cytotoxicity and on longer time scales to transformation; the resulting mesothelioma is a highly aggressive neoplasm that is considered as incurable at the present time Zucali et al. (Cancer Treatment Reviews 37:543-558, 2011). Only few murine cell culture models of immortalized mesothelial cells and mesothelioma cell lines exist to date. We generated SV40-immortalized cell lines derived from wild-type (WT) and neurofibromatosis 2 (merlin) heterozygote (Nf2+/-) mice, both on a commonly used genetic background, C57Bl/6J. All immortalized mesothelial clones consistently grow in DMEM supplemented with fetal bovine serum. Cells can be passaged for more than 40 times without any signs of morphological changes or a decrease in proliferation rate. The tumor suppressor gene NF2 is one of the most frequently mutated genes in human mesothelioma, but its detailed function is still unknown. Thus, these genotypically distinct cell lines likely relevant for malignant mesothelioma formation are expected to serve as useful in vitro models, in particular to compare with in vivo studies in mice of the same genotype. Furthermore, we generated a novel murine mesothelioma cell line RN5 originating from an Nf2+/- mouse subjected to repeated crocidolite exposure. RN5 cells are highly tumorigenic. PMID:25877069

  17. 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

  18. 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

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

    PubMed Central

    Mehta, Rajvi H.

    2014-01-01

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

  20. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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.

  6. Bcl-2 transfection protects Hut78 cell line from different types of cytotoxic effector cells.

    PubMed

    Saxena, R K; Collins, G D; Chrest, J F; Adler, W H

    1996-09-01

    The human T lymphoma cell line Hut78 was transfected with Bel-2 gene or with control G418 Neomycin resistance-conferring plasmid. Bcl-2 transfected and Neomycin selected Hut78 cells expressed about ten-fold greater Bcl-2 protein than the neo-transfected controls. Susceptibility of Bcl-2-transfected Hut78 cells to human NK cells, human mixed lymphocyte reaction-generated cytotoxic effector cells, and mouse cytotoxic spleen cells generated by immunization with Hut78 cells was examined. In all systems tested, Bcl-2-transfected Hut78 target cells were significantly less susceptible to lysis than the neo-transfected control target cells. These results suggest that in Hut78 cells, Bcl-2 gene product may confer partial resistance to different types of cytotoxic effector mechanisms. PMID:8905402

  7. Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function?

    PubMed Central

    Herrmann, Jens; Gressner, Axel M; Weiskirchen, Ralf

    2007-01-01

    Abstract At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts. PMID:17760834

  8. 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.

  9. Establishment and characterization of a cell line from human circulating colon cancer cells.

    PubMed

    Cayrefourcq, Laure; Mazard, Thibault; Joosse, Simon; Solassol, Jérôme; Ramos, Jeanne; Assenat, Eric; Schumacher, Udo; Costes, Valérie; Maudelonde, Thierry; Pantel, Klaus; Alix-Panabières, Catherine

    2015-03-01

    Circulating tumor cells (CTC) in blood are promising new biomarkers potentially useful for prognostic prediction and monitoring of therapies in patients with solid tumors including colon cancer. Moreover, CTC research opens a new avenue for understanding the biology of metastasis in patients with cancer. However, an in-depth investigation of CTCs is hampered by the very low number of these cells, especially in the blood of patients with colorectal cancer. Thus, the establishment of cell cultures and permanent cell lines from CTCs has become the most challenging task over the past year. Here, we describe, for the first time, the establishment of cell cultures and a permanent cell line from CTCs of one patient with colon cancer. The cell line designated CTC-MCC-41 has been cultured for more than one year, and the cells have been characterized at the genome, transcriptome, proteome, and secretome levels. This thorough analysis showed that CTC-MCC-41 cells resemble characteristics of the original tumor cells in the patient with colon cancer and display a stable phenotype characterized by an intermediate epithelial/mesenchymal phenotype, stem cell-like properties, and an osteomimetic signature, indicating a bone marrow origin. Functional studies showed that CTC-MCC-41 cells induced rapidly in vitro endothelial cell tube formation and in vivo tumors after xenografting in immunodeficient mice. The establishment of this first colon cancer CTC line allows now a wealth of functional studies on the biology of CTCs as well as in vitro and in vivo drug testing. PMID:25592149

  10. 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

  11. 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.

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

    PubMed

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

    2014-02-28

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

  13. Expression of survivin in HTLV-I-infected T-cell lines and primary ATL cells.

    PubMed

    Mori, N; Yamada, Y; Hata, T; Ikeda, S; Yamasaki, Y; Tomonaga, M; Yamamoto, N

    2001-04-20

    Human T-cell leukemia virus type I (HTLV-I) is the etiological agent of adult T-cell leukemia (ATL), although the precise mechanisms involved in the transformation process have not yet been defined. Deregulated inhibition of apoptosis may facilitate the insurgence of leukemia. Survivin is an inhibitor of apoptosis and considered to play a role in oncogenesis. We investigated the expression of survivin in HTLV-I-infected T-cell lines and primary ATL cells. Northern blot analysis showed expression of survivin transcript in all 9 HTLV-I-positive T-cell lines. High survivin expression levels were detected in primary cells of patients with acute-type ATL, but no such expression was noted in the cells of chronic-type ATL or normal peripheral blood mononuclear cells. Treatment of an HTLV-I-infected T-cell line, MT-1, with survivin specific antisense oligonucleotide resulted in reduced cell growth. Our results suggest that expression of survivin may play an important role in the development and pathogenesis of ATL. PMID:11302729

  14. 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.

  15. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  16. 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...

  17. 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.

  18. Characterisation of a new cell line (CJ18) from a patient with 'hairy' cell leukaemia.

    PubMed

    Skinnider, L F; Ha, T; Bergen, S; Wang, H C

    1985-10-01

    A cell line (CJ18) has been established from the peripheral blood of a patient with hairy cell leukaemia (HCL) in a leukaemic phase. They grow slowly in large clumps with a doubling time of 3-4 d. 8% show positivity for surface immunoglobulin G and a small percentage (5%) are positive for intracytoplasmic immunoglobulin. They are B1,Ia1 positive and CALL, TdT and OKM1 negative. Although they are Epstein Barr Virus Nuclear Antigen (EBNA) positive they have several features not found in other EBNA positive B lymphoblastoid cell lines which suggest they may be derived from the patient's leukaemic hairy cells. They are strongly positive for tartrate resistant acid phosphatase, esterase positive, contain numerous lysosomes and are able to phagocytose sheep red blood cells after exposure to tetradecanoyl-12, 13-phorbol acetate (TPA). Following exposure to retinoic acid and TPA they adhere to plastic with numerous slender processes, a feature seen in HCL cells. PMID:4081642

  19. 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

  20. 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

  1. 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

  2. Labeling of multiple cell lines using a new iron oxide agent for cell tracking by MRI.

    PubMed

    McFadden, C; Mallett, C L; Foster, P J

    2011-01-01

    Stem cells, cancer cells and immune cells were labeled by co-incubation with a new ultra-small iron oxide nanoparticle called Molday ION Rhodamine-B (MIRB). Iron staining, fluorescence imaging, transmission electron microscopy and flow cytometry were used to assess cell viability, function and labeling efficiency. This study has shown that MIRB can be used to label both adherent and nonadherent cell lines, with high viability and loading levels sufficient for their detection in vivo by MRI at 3 T. PMID:22144030

  3. 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

  4. 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

  5. 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

  6. 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

  7. Enrichment of Oct3/4-positive cells from a human bronchial epithelial cell line.

    PubMed

    Li, Xin; Jia, Lanling; Jia, Xinshan; Shi, Mumu; Li, Xiaolei; Ye, Xulv; Wang, Ruiyue; Xiong, Yanlei; Wang, Enhua; Li, Fang

    2013-07-01

    Most adult stem cells are in the G0 phase of the cell cycle, accounting for only a small percentage of the cells in the tissue. Thus, isolation of stem cells from tissues for further study represents a major challenge. The anti-tumor drug 5-fluorouracil (5-FU) selectively kills proliferating cells, sparing cells in the G0 phase. Thus, the objective of this study was to determine whether 5-FU can be used to enrich stem cells in a human bronchial epithelial (HBE) cell population in vitro. Side population (SP) cells were isolated from untreated HBE cells or HBE cells treated with 5-FU, and the resulting cells were subjected to colony formation assays, culturing of cell spheres, and tumorigenicity assays. Expression of Oct3/4, Sox2, PCK, and β-catenin were examined by Western blot analysis and immunofluorescence. Treatment with 5-FU increased the percentage of SP cells from 0.3% to 1.5%, and the clonogenic ability of 5-FU-treated cells was more than twofold higher than that of HBE cells. Cells that survived after 5-FU treatment exhibited a higher capacity for sphere formation. Furthermore, spheres formed from 5-FU-treated cells possessed the capacity to generate differentiated progenies. Cells treated with 5-FU also exhibited tumorigenic potential, based on tumor formation assays in nude mice, and Oct3/4-positive cell aggregates were identified in the resulting tumors. In this study, we have shown that 5-FU treatment enriched the population of cells expressing the putative embryonic markers Oct3/4 and Sox2 and exhibiting nuclear accumulation of β-catenin. Furthermore, 5-FU-treated cells expressed low levels of the epithelial differentiation marker PCK. Analysis of epigenetic modifications suggested that Oct3/4-positive cells possessed characteristics of stem cells. These results demonstrate that treatment with 5-FU can enrich the stem cell population present in a human bronchial epithelial cell line, and implicate combined treatment with 5-FU and serum-free medium as a new method for isolation of stem-like cells from the HBE cell line. PMID:23216104

  8. 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

  9. 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

  10. 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

  11. Gene expression profiling analysis of osteosarcoma cell lines

    PubMed Central

    SUN, LU; LI, JIE; YAN, BING

    2015-01-01

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

  12. Gene expression profiling analysis of osteosarcoma cell lines.

    PubMed

    Sun, Lu; Li, Jie; Yan, Bing

    2015-09-01

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

  13. 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.

  14. 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

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

    PubMed

    Talbot, Neil C; Wang, Ling; Garrett, Wesley M; Caperna, Thomas J; Tang, Young

    2016-03-01

    The establishment and initial characterization of bovine fetal liver cell lines are described. Bovine fetal hepatocytes were cultured from the liver of a 34-d bovine fetus by physical disruption of the liver tissue. Released liver cells and clumps of cells were plated on STO (SIMS mouse strain, thioguanine- and ouabain-resistant) feeder layers and were cultured in a medium supplemented with 10% fetal bovine serum. After 2-3 wk, primary colonies of hepatocytes were observed by phase-contrast microscopic observation. Individual hepatocyte colonies were colony-cloned into independent bovine fetal liver (BFL) cell lines. Two cell lines, BFL-6 and BFL-9, grew the best of several isolates, and they were further characterized for growth potential and for hepatocyte morphology and function. The two cell lines were found to grow markedly better in the presence of the transforming growth factor (TGF)-beta inhibitor, SB431542 (1 μM). Their continuous culture also depended on a particular medium height-for T12.5 flasks, 3 ml total medium produced optimum growth. Higher or lower amounts of medium caused less cell growth or cessation of growth. The cell lines were propagated for over a year at split ratios of 1:2 or 1:3 at each passage until reaching senescence at approximately 30 passages. The cells were laterally polarized with well-developed canalicular spaces occurring between adjacent BFL cells. Treatment of the cultures with cyclic adenosine monophosphate (cAMP)-stimulating chemicals or peptides (e.g., forskolin or glucagon) caused physical expansion of the canaliculi between the cells within 15 min. The cells secreted a spectrum of serum proteins, were positive for the expression of several hepatocyte-specific genes, and converted ammonia to urea, although at a relatively low rate. The culture system provides an in vitro model of fetal bovine hepatocytes and is the first demonstration of the continuous culture of normal bovine hepatocytes as cell lines. PMID:26659396

  16. Glioblastoma cell secretome: analysis of three glioblastoma cell lines reveal 148 non-redundant proteins.

    PubMed

    Polisetty, Ravindra V; Gupta, Manoj Kumar; Nair, Sudha C; Ramamoorthy, Kalidoss; Tiwary, Shivani; Shiras, Anjali; Chandak, Giriraj R; Sirdeshmukh, Ravi

    2011-09-01

    Glioblastoma multiforme (GBM) is the most aggressive among human gliomas with poor prognosis. Study of tumor cell secretome - proteins secreted by cancer cell lines, is a powerful approach to discover potential diagnostic or prognostic biomarkers. Here we report, for the first time, proteins secreted by three GBM cell lines, HNGC2, LN229 and U87MG. Analysis of the conditioned media of these cell lines by LC-MS/MS using ESI-IT mass spectrometer (LTQ) resulted in the confident identification of 102, 119 and 64 proteins, respectively. Integration of the results from all the three cell lines lead to a dataset of 148 non-redundant proteins. Subcellular classification using Genome Ontology indicated that 42% of the proteins identified belonged to extracellular or membrane proteins, viz. Vinculin, Tenascin XB, SERPIN F1 and TIMP-1. 52 proteins matched with the secretomes of 11 major cancer types reported earlier whereas remaining 96 are unique to our study. 25 protein identifications from the dataset represent proteins related to GBM or other cancer tissues as per Human Protein Atlas; at least 22 are detectable in plasma, 11 of them being reported even in cerebrospinal fluid. Our study thus provides a valuable resource of GBM cell secretome with potential for further investigation as GBM biomarkers. PMID:21601021

  17. Clonal heterogeneity in physiological properties of melanized cells induced from goldfish erythrophoroma cell lines.

    PubMed

    Matsumoto, J; Ishikawa, T; Masahito, P; Takayama, S; Taylor, J D; Tchen, T T

    1984-01-01

    Cells of the uncloned goldfish erythrophoroma lines, GEM-81 and GEM-218, have been induced to melanize by cultivation in autologous serum. The melanized cells continue to proliferate and exhibit clonal heterogeneity in terms of morphology, growth rates, contact behavior and pigment content, and distribution and translocation in response to hormones. Based on these characteristics and those of their normal counterparts, the melanized tumor cells have been categorized as type-I and type-II melanocytomas, and melanophoromas. The melanophoroma cells are capable of pigment translocation in response to epinephrine, melatonin, and/or MSH, whereas melanocytoma cells are not. The distinguishing characteristics of each type are apparent at the first appearance of melanized cells and appear to be stable except in some type-II melanocytoma clones which contain cells capable of differentiating into melanophoroma cells in long-term cultures. It appears that the parent erythrophoroma lines contain stem cells, melanoblastomas, which are capable of melanogenesis. These stem cells may themselves be a heterogeneous population with respect to the characteristics of the melanized cells to which they give rise. PMID:6468803

  18. 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

  19. 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 0.05°C to 0.54°C within the range from 28°C to 38°C in HeLa cell extracts. Using our optimized protocol, this cell-permeable FPT spontaneously diffused into HeLa cells within 10 min of incubation and exhibited minimal toxicity over several hours of observation. FLIM analysis confirmed a temperature difference between the nucleus and the cytoplasm and heat production near the mitochondria, which were also detected previously using the microinjected FPT. We also showed that this cell-permeable FPT protocol can be applied to other mammalian cell lines, COS7 and NIH/3T3 cells. Thus, this cell-permeable FPT represents a promising tool to study cellular states and functions with respect to temperature. PMID:25692871

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

    PubMed Central

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

    2015-01-01

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

  1. 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...

  2. 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

  3. 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

  4. 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.

  5. 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...

  6. Cell death induced by Bothrops asper snake venom metalloproteinase on endothelial and other cell lines.

    PubMed

    Brenes, Oscar; Muñóz, Eduardo; Roldán-Rodríguez, Raquel; Díaz, 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

  7. 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

  8. Identification of tumor-initiating cells in a canine hepatocellular carcinoma cell line.

    PubMed

    Michishita, Masaki; Ezaki, Shiori; Ogihara, Kikumi; Naya, Yuko; Azakami, Daigo; Nakagawa, Takayuki; Sasaki, Nobuo; Arai, Toshiro; Shida, Takuo; Takahashi, Kimimasa

    2014-04-01

    Tumor-initiating cells (TICs) or cancer stem cells (CSCs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and have the ability to self-renew and differentiate tumors at a high frequency. We established a canine HCC cell line, HCC930599, and analyzed it for stem and progenitor cell marker expression using flow cytometry. HCC930599 showed high CD44 and CD29, moderate CD90, and low CD133, CD34, CD24, CD117, and CD13 expression. CD90(+)CD44(+) and CD90(-)CD44(+) cells were characterized using the in vitro sphere assay and an in vivo transplant model. CD90(+)CD44(+) cells acquired enhanced self-renewal capacity, proliferative activity and tumourigenicity compared with CD90(-)CD44(+) cells, suggesting that TICs exist in the HCC930599 cell line and that CD90 is a marker for enriched TICs. Understanding TIC characteristics may help elucidate hepatic carcinogenesis and HCC therapy development. PMID:24534130

  9. 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.

  10. 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

  11. [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

  12. Lymphoblastoid cell lines in pharmacogenomic discovery and clinical translation

    PubMed Central

    Wheeler, Heather E; Dolan, M Eileen

    2012-01-01

    The ability to predict how an individual patient will respond to a particular treatment is the ambitious goal of personalized medicine. The genetic make up of an individual has been shown to play a role in drug response. For pharmacogenomic studies, human lymphoblastoid cell lines (LCLs) comprise a useful model system for identifying genetic variants associated with pharmacologic phenotypes. The availability of extensive genotype data for many panels of LCLs derived from individuals of diverse ancestry allows for the study of genetic variants contributing to interethnic and interindividual variation in susceptibility to drugs. Many genome-wide association studies for drug-induced phenotypes have been performed in LCLs, often incorporating gene-expression data. LCLs are also being used in follow-up studies to clinical findings to determine how an associated variant functions to affect phenotype. This review describes the most recent pharmacogenomic findings made in LCLs, including the translation of some findings to clinical cohorts. PMID:22176622

  13. 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

  14. Rejection by syngeneic mice of cell variants obtained by mutagenesis of a malignant teratocarcinoma cell line.

    PubMed Central

    Boon, T; Kellermann, O

    1977-01-01

    Cells from the malignant teratocarcinoma line PCC4.azal were treated with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine. Fifty-five clones were isolated from the surviving cells. Twelve clones are unable to form tumors in the syngeneic 129/Sv mice. However, these "tum-" clones form tumors as readily as the original cells when they are injected into irradiated mice. Moreover, they stimulate the production of immune memory cells, which protect the injected animals and confer resistance by adoptive transfer. The tum- clones are therefore unable to generate tumors in syngeneic mice because they elicit an immune rejection response. PMID:264681

  15. 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

  16. Chemokines induce migrational responses in human breast carcinoma cell lines.

    PubMed

    Youngs, S J; Ali, S A; Taub, D D; Rees, R C

    1997-04-10

    Chemokines have been shown to chemoattract and activate different leukocyte populations. Here we report the in vitro effect of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, regulated on activation, normal T-cells, expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interferon inducible protein-10 (IP-10), neutrophil-activating peptide-2 (NAP-2), growth-related protein (GRO)-alpha and GRO-gamma, on the migration of 3 human breast carcinoma cell lines, MCF-7, T47D and ZR-75-1, using a microchemotaxis chamber to assess migration across fibronectin-coated polycarbonate membranes. MCF-7 cells responded chemotactically to all chemokines tested in a pattern which was dose and time dependent, although responses to the different chemokines were variable. ZR-75-1 responded to MIP-1beta and GRO-alpha, giving maximum migration indices of 3.7 and 5.3, respectively, and exhibited a migratory response to MIP-1alpha, IL-8 and MCP-1 although to a lower degree. T47D was unresponsive to the chemokines tested, but both MCF-7 and T47D cells bound radiolabelled ligands with binding constants (Kd) ranging from 0.6 to 2.2 nM and 0.6 to 2.1 nM, respectively. The specificity of the chemotactic response of MCF-7 to MIP-1alpha and MIP-1beta was confirmed using chemokine-specific neutralising antibodies and heat denaturation, and was demonstrated to involve G protein and cyclic AMP signalling pathways. MIP-1beta and MIP-1alpha were shown to induce changes in the organisation of the actin cytoskeleton and the level of F-actin in MCF-7 cells, as determined using flow cytometric analysis and confocal microscopy. Our results show that breast carcinoma cells can respond to chemokines, and suggests a potential role for these molecules in the process of tumour cell migration, invasion and metastasis. PMID:9139852

  17. Primary cell lines: false representation or model system? a comparison of four human colorectal tumors and their coordinately established cell lines

    PubMed Central

    Pastor, Danielle M; Poritz, Lisa S; Olson, Thomas L; Kline, Christina L; Harris, Leonard R; Koltun, Walter A; Chinchilli, Vernon M; Irby, Rosalyn B

    2010-01-01

    Cultured cell lines have played an integral role in the study of tumor biology since the early 1900's. The purpose of this study is to evaluate colorectal cancer (CRC) cell lines with respect to progenitor tumors and assess whether these cells accurately and reliably represent the cancers from which they are derived. Primary cancer cell lines were derived from fresh CRC tissue. Tumorigenicity of cell lines was assessed by subcutaneous injection of cells into athymic mice and calculation of tumor volume after 3 weeks. DNA ploidy was evaluated by flow cytometry. Invasive ability of the lines was tested with the MATRIGEL™ invasion assay at 24 or 48 hours. Cells were assessed for the presence of Kirsten-Ras (K-Ras), p-53, deleted in colon cancer (DCC), and Src. Protein profiling of cells and tissue was performed by surface enhanced laser desorption/ionization-time of flight/mass spectroscopy. microRNA expression in cell and tumor tissue samples was evaluated by FlexmiR™ MicroRNA Assays. Four cell lines were generated from tumor tissue from patients with CRC and confirmed to be tumorigenic (mean tumor volume 158.46 mm3). Two cell lines were noted to be diploid; two were aneuploid. All cell lines invaded the MATRIGEL™ starting as early as 24 hours. K-Ras, p53, DCC, and Src expression were markedly different between cell lines and corresponding tissue. Protein profiling yielded weak-to-moderate correlations between cell samples and respective tissues of origin. Weak-to-moderate tau correlations for levels of expression of human microRNAs were found between cells and respective tissue samples for each of the four pairings. Although our primary CRC cell lines vary in their expression of several tumor markers and known microRNAs from their respective progenitor tumor tissue, they do not statistically differ in overall profiles. Characteristics are retained that deem these cell lines appropriate models of disease; however, data acquired through the use of cell culture may not always be a completely reliable representation of tumor activity in vivo. PMID:20369042

  18. Genetic inheritance of gene expression in human cell lines.

    PubMed

    Monks, S A; Leonardson, A; Zhu, H; Cundiff, P; Pietrusiak, P; Edwards, S; Phillips, J W; Sachs, A; Schadt, E E

    2004-12-01

    Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value <.000005. Of these, 13 genes possessed a QTL within 5 Mb of their physical location. Hierarchical clustering was performed on the basis of both Pearson correlation of gene expression and genetic correlation. Both reflected biologically relevant activity taking place in the lymphoblastoid cell lines, with greater coherency represented in Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways than in Gene Ontology database pathways. However, more pathway coherence was observed in KEGG pathways when clustering was based on genetic correlation than when clustering was based on Pearson correlation. As more expression data in segregating populations are generated, viewing clusters or networks based on genetic correlation measures and shared QTLs will offer potentially novel insights into the relationship among genes that may underlie complex traits. PMID:15514893

  19. Flow cytometric analysis of human uterine sarcomas and cell lines.

    PubMed

    Nelson, K G; Haskill, J S; Sloan, S; Siegfried, J M; Siegal, G P; Walton, L; Kaufman, D G

    1987-06-01

    Flow cytometric techniques were used to characterize multiple human uterine sarcomas and cell lines derived from some of these tumors. Analysis of DNA content showed that 9 of the 11 uterine sarcomas investigated were composed of at least one aneuploid population as well as a distinct diploid population. These data indicate that aneuploidy, as measured by flow cytometry, is a characteristic more common to uterine sarcomas than that previously reported for uterine adenocarcinomas. Unlike the original tumors, the cell lines established from three of the sarcomas contained predominantly diploid populations with only minor aneuploid populations. Treatment of one of the sarcoma cultures with tumor promoters did not result in an increase in the aneuploid populations. Tumors which arose in nude mice upon transplantation of two of the sarcomas did not contain the same distribution of tumor subpopulations as found in the original sarcomas. Apparently, the in vitro culture and and in vivo nude mouse conditions were not appropriate for maintaining the original equilibrium between the aneuploid and diploid subpopulations but instead provided a selective environment that resulted in the preferential growth of only certain tumor populations. Dual-parameter analysis of DNA content and alkaline phosphatase levels of one of the sarcomas were useful for distinguishing the aneuploid from the diploid population coexisting in this tumor. Our data suggest that flow cytometry is a valuable tool to analyze the characteristics of the tumor populations residing in primary uterine sarcomas as well as to determine which of these tumor subpopulations survive in culture and transplantation to nude mice. PMID:3567904

  20. 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...

  1. 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

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

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

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

  3. 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

  4. Characterization of cell-mediated cytolytic mechanisms against human transitional cell carcinoma line, 253J.

    PubMed Central

    Catalona, W J; Ratliff, T L; McCool, R E

    1980-01-01

    We examined the nature of effector cells that mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and spontaneous cell-mediated cytotoxicity (SCMC) against the 253J human bladder transitional cell carcinoma cell line. Fractionation of peripheral blood lymphocytes (PBL) showed that ADCC and SCMC against the 253J targets were mediated by non-adherent, surface immunoglobulin-negative lymphocytes lacking or having only weak affinity for sheep erythrocytes. Macrophages were not cytotoxic against 253J cells in 18 h culture, as removal of macrophages by glass adherence or treatment with silica particles did not reduce ADCC or SCMC, and macrophage-enriched effector cell preparations were only weakly cytotoxic. PMID:7380462

  5. 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

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

    PubMed

    Patil, Shankargouda; Rao, Roopa S; Raj, A Thirumal

    2015-11-01

    Oral cell lines have provided valuable insights into the various molecular pathways in oral carcinogenesis. Several landmark studies in oral oncology have utilized commercially available normal, dysplastic and cancer cell lines to decode the genetic alterations leading to the development of oral cancer. Most of these studies have shown a significant degree of variation in their mutation landscapes. These variations were thought to represent the heterogeneity of oral cancer.(1) But in a recent study, Dickman et al have shown that normal and dysplastic cell lines carry specific genetic alterations within the parent cell line, thus questioning the authenticity of several published mutation profiles. These genetic alterations in the commercial cell lines have been attributed to several factors, the most common being immortalization. Normal and dysplastic cell lines unlike cancer cell lines attain senescence following limited number of replication. Immortalization of the normal and dysplastic cell lines would aid the researcher in maintaining a viable population of cells for further studies. Ideally, the immortalized cell line must possess potential for indefinite replication and must retain the genetic makeup of its parent cell line.(2). PMID:26718303

  7. Insulin synthesis in a clonal cell line of simian virus 40-transformed hamster pancreatic beta cells.

    PubMed Central

    Santerre, R F; Cook, R A; Crisel, R M; Sharp, J D; Schmidt, R J; Williams, D C; Wilson, C P

    1981-01-01

    A clonal hamster beta cell line (HIT) was established by simian virus 40 transformation of Syrian hamster pancreatic islet cells. Cytoplasmic insulin was detected in all cells by indirect fluorescent antibody staining, and membrane-bound secretory granules were observed ultrastructurally. Acidified-ethanol extracts of HIT cell cultures contained hamster insulin as determined by radioimmunoassay, radioreceptor assay, and bioassay. One subclone at passage 39 contained 2.6 micrograms of insulin per mg of cell protein. [3H]Leucine-labeled HIT insulin and proinsulin were identical to islet-derived proteins when compared by NaDodSO4/polyacrylamide gel electrophoresis of immunoprecipitates. HIT cell insulin secretion was stimulated by glucose, glucagon, and 3-isobutyl-1-methylxanthine. Insulin secretion at optimal glucose concentration (7.5 mM) was 2.4 milliunits per 10(6) cells per hr. Somatostatin and dexamethasone markedly inhibited HIT insulin secretion. The HIT cell line represents a unique in vitro system for studying beta cell metabolism and insulin biosynthesis. Images PMID:6270673

  8. Isolation and characterization of cancer stem cells from a human glioblastoma cell line U87.

    PubMed

    Yu, Shi-Cang; Ping, Yi-Fang; Yi, Liang; Zhou, Zhi-Hua; Chen, Jian-Hong; Yao, Xiao-Hong; Gao, Lei; Wang, Ji Ming; Bian, Xiu-Wu

    2008-06-28

    A variety of malignant cancers have been found to contain a subpopulation of stem cell-like tumor cells, or cancer stem cells (CSCs). However, the existence of CSCs in U87, a most commonly used glioma cell line, is still controversial. In this study, we demonstrate that U87 cell line contained a fraction of tumor cells that could form tumor spheres and were enriched by progressively increasing the concentration of serum-free neural stem cell medium with or without low dose vincristine. These cells possessed the ability of self-renewal and multipotency, the defined characteristics of CSCs. Moreover, the tumors formed by the secondary spheres displayed typical histological features of human glioblastoma, including cellular pleomorphism, pseudopalisades surrounding necrosis, hyperchromatic nuclei, high density of microvessels and invasion to the brain parenchyma. These results indicate that gradually increasing the concentration of serum-free neural stem cell culture medium with or without vincristine is a simple and effective method for isolation of CSCs to study the initiation and progression of human glioblastoma. PMID:18343028

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. 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

  18. 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

  19. Ectopic expression of Flt3 kinase inhibits proliferation and promotes cell death in different human cancer cell lines.

    PubMed

    Oveland, Eystein; Wergeland, Line; Hovland, Randi; Lorens, James B; Gjertsen, Bjørn Tore; Fladmark, Kari E

    2012-08-01

    Stable ectopic expression of Flt3 receptor tyrosine kinase is usually performed in interleukin 3 (IL-3)-dependent murine cell lines like Ba/F3, resulting in loss of IL-3 dependence. Such high-level Flt3 expression has to date not been reported in human acute myeloid leukemia (AML) cell lines, despite the fact that oncogenic Flt3 aberrancies are frequent in AML patients. We show here that ectopic Flt3 expression in different human cancer cell lines might reduce proliferation and induce apoptotic cell death, involving Bax/Bcl2 modulation. Selective depletion of Flt3-expressing cells occurred in human AML cell lines transduced with retroviral Flt3 constructs, shown here using the HL-60 leukemic cell line. Flt3 expression was investigated in two cellular model systems, the SAOS-2 osteosarcoma cell line and the human embryonic kidney HEK293 cell line, and proliferation was reduced in both systems. HEK293 cells underwent apoptosis upon ectopic Flt3 expression and cell death could be rescued by overexpression of Bcl-2. Furthermore, we observed that the Flt3-induced inhibition of proliferation in HL-60 cells appeared to be Bax-dependent. Our results thus suggest that excessive Flt3 expression has growth-suppressive properties in several human cancer cell lines. PMID:22422053

  20. 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

  1. CD133 Is Not Suitable Marker for Isolating Melanoma Stem Cells from D10 Cell Line

    PubMed Central

    Rajabi Fomeshi, Motahareh; Ebrahimi, Marzieh; Mowla, Seyed Javad; Firouzi, Javad; Khosravani, Pardis

    2016-01-01

    Objective Cutaneous melanoma is the most hazardous malignancy of skin cancer with a high mortality rate. It has been reported that cancer stem cells (CSCs) are responsible for malignancy in most of cancers including melanoma. The aim of this study is to compare two common methods for melanoma stem cell enriching; isolating based on the CD133 cell surface marker and spheroid cell culture. Materials and Methods In this experimental study, melanoma stem cells were enriched by fluorescence activated cell sorting (FACS) based on the CD133 protein expression and spheroid culture of D10 melanoma cell line,. To determine stemness features, the mRNA expression analysis of ABCG2, c-MYC, NESTIN, OCT4-A and -B genes as well as colony and spheroid formation assays were utilized in unsorted CD133+, CD133- and spheroid cells. Significant differences of the two experimental groups were compared using student’s t tests and a two-tailed value of P<0.05 was statistically considered as a significant threshold. Results Our results demonstrated that spheroid cells had more colony and spheroid forming ability, rather than CD133+ cells and the other groups. Moreover, melanospheres expressed higher mRNA expression level of ABCG2, c-MYC, NESTIN and OCT4-A com- pared to other groups (P<0.05). Conclusion Although CD133+ derived melanoma cells represented stemness fea- tures, our findings demonstrated that spheroid culture could be more effective meth- od to enrich melanoma stem cells. PMID:27054115

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

    SciTech Connect

    Kawata, Shigehisa; Suzuki, Jun; Maruoka, Masahiro; Mizutamari, Megumi; Ishida-Kitagawa, Norihiro; Yogo, Keiichiro; Jat, Parmjit S.; Shishido, Tomoyuki . E-mail: shishid@bs.naist.jp

    2006-11-10

    Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 deg. C, but stopped growing at the non-permissive temperature of 39 deg. C. In the presence of receptor activator of NF{kappa}B ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 deg. C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.

  3. Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822).

    PubMed

    Chaudhary, Dharmendra K; Sood, Neeraj; Swaminathan, T Raja; Rathore, Gaurav; Pradhan, P K; Agarwal, N K; Jena, J K

    2013-01-10

    A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28°C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish. PMID:23026220

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

    SciTech Connect

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

    2013-11-15

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

  5. 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

  6. 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

  7. Rapid Selection and Proliferation of CD133(+) Cells from Cancer Cell Lines: Chemotherapeutic Implications

    PubMed Central

    Kelly, Sarah E.; Di Benedetto, Altomare; Greco, Adelaide; Howard, Candace M.; Sollars, Vincent E.; Primerano, Donald A.; Valluri, Jagan V.; Claudio, Pier Paolo

    2010-01-01

    Cancer stem cells (CSCs) are considered a subset of the bulk tumor responsible for initiating and maintaining the disease. Several surface cellular markers have been recently used to identify CSCs. Among those is CD133, which is expressed by hematopoietic progenitor cells as well as embryonic stem cells and various cancers. We have recently isolated and cultured CD133 positive [CD133(+)] cells from various cancer cell lines using a NASA developed Hydrodynamic Focusing Bioreactor (HFB) (Celdyne, Houston, TX). For comparison, another bioreactor, the rotary cell culture system (RCCS) manufactured by Synthecon (Houston, TX) was used. Both the HFB and the RCCS bioreactors simulate aspects of hypogravity. In our study, the HFB increased CD133(+) cell growth from various cell lines compared to the RCCS vessel and to normal gravity control. We observed a (+)15-fold proliferation of the CD133(+) cellular fraction with cancer cells that were cultured for 7-days at optimized conditions. The RCCS vessel instead yielded a (−)4.8-fold decrease in the CD133(+)cellular fraction respect to the HFB after 7-days of culture. Interestingly, we also found that the hypogravity environment of the HFB greatly sensitized the CD133(+) cancer cells, which are normally resistant to chemo treatment, to become susceptible to various chemotherapeutic agents, paving the way to less toxic and more effective chemotherapeutic treatment in patients. To be able to test the efficacy of cytotoxic agents in vitro prior to their use in clinical setting on cancer cells as well as on cancer stem cells may pave the way to more effective chemotherapeutic strategies in patients. This could be an important advancement in the therapeutic options of oncologic patients, allowing for more targeted and personalized chemotherapy regimens as well as for higher response rates. PMID:20386701

  8. Nuclear motility in glioma cells reveals a cell-line dependent role of various cytoskeletal components.

    PubMed

    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

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

    SciTech Connect

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

    1995-01-01

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

  10. 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.

  11. Oxidative stress-induced DNA damage and cell cycle regulation in B65 dopaminergic cell line.

    PubMed

    Pizarro, Javier G; Folch, Jaume; Vazquez De la Torre, Aurelio; Verdaguer, Ester; Junyent, Felix; Jordán, Joaquín; Pallàs, Mercè; Camins, Antoni

    2009-10-01

    Reactive oxygen species and oxidative stress are associated with neuronal cell death in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are still unclear. This study used the B65 rat neuroblastoma cell line as a model to study the molecular events that occur after H(2)O(2) treatment. Treatment of B65 cells with H(2)O(2) rapidly up-regulated the DNA damage pathway involved in double-strand breakage. Subsequently, proteins involved in p53 regulation, such as sirtuin 1 and STAT1, were modified. In addition, H(2)O(2) treatment altered the pattern of cell cycle protein expression. Specifically, a decrease was found in the expression of cyclin D1, cdk4 and surprisingly the levels of cyclin A and the retinoblastoma protein phosphorylated at ser780 were increased. Furthermore, this study shows that pre-treatment of B65 cells with 50 microM trolox confers almost total protection against apoptotic cell death and restores the cell cycle. Likewise, the increase in retinoblastoma phosphorylation was attenuated by KU-55993, a selective ATM inhibitor, and also by trolox. These observations indicate that DNA damage and oxidative stress are responsible for cell cycle regulation. In summary, this study describes the molecular mechanisms involved in cell cycle alterations induced by oxidative stress in B65 cells. These findings highlight the relevance of ATM in the regulation of cell cycle after oxidative stress. PMID:19657808

  12. 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

  13. 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. ...

  14. 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 ...

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

    PubMed

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

    2010-04-01

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

  16. 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

  17. 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

  18. Enrichment and characterization of cancer stem cells from a human non-small cell lung cancer cell line.

    PubMed

    Zhao, Changhong; Setrerrahmane, Sarra; Xu, Hanmei

    2015-10-01

    Tumor cells from the same origin comprise different cell populations. Among them, cancer stem cells (CSCs) have higher tumorigenicity. It is necessary to enrich CSCs to determine an effective way to suppress and eliminate them. In the present study, using the non-adhesive culture system, tumor spheres were successfully generated from human A549 non-small cell lung cancer (NSCLC) cell line within 2 weeks. Compared to A549 adherent cells, sphere cells had a higher self-renewal ability and increased resistance to cytotoxic drugs. Sphere cells were more invasive and expressed stem cell markers including octamer‑binding transcription factor 4 (Oct4) and sex-determining region Y-box 2 (Sox2) at high levels. CD133, a disputed marker of lung CSCs, was also upregulated. Tumor sphere cells showed higher tumorigenic ability in vivo, indicating that more CSCs were enriched in the sphere cells. More blood vessels were formed in the tumor generated by sphere cells suggesting the interaction between CSCs and blood vessel. A reliable model of enriching CSCs from the human A549 NSCLC cell line was established that was simple and cost-effective compared to other methods. PMID:26239272

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

    SciTech Connect

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

    2010-05-04

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

  20. Unit-length line-1 transcripts in human teratocarcinoma cells.

    PubMed Central

    Skowronski, J; Fanning, T G; Singer, M F

    1988-01-01

    We have characterized the approximately 6.5-kilobase cytoplasmic poly(A)+ Line-1 (L1) RNA present in a human teratocarcinoma cell line, NTera2D1, by primer extension and by analysis of cloned cDNAs. The bulk of the RNA begins (5' end) at the residue previously identified as the 5' terminus of the longest known primate genomic L1 elements, presumed to represent "unit" length. Several of the cDNA clones are close to 6 kilobase pairs, that is, close to full length. The partial sequences of 18 cDNA clones and full sequence of one (5,975 base pairs) indicate that many different genomic L1 elements contribute transcripts to the 6.5-kilobase cytoplasmic poly(A)+ RNA in NTera2D1 cells because no 2 of the 19 cDNAs analyzed had identical sequences. The transcribed elements appear to represent a subset of the total genomic L1s, a subset that has a characteristic consensus sequence in the 3' noncoding region and a high degree of sequence conservation throughout. Two open reading frames (ORFs) of 1,122 (ORF1) and 3,852 (ORF2) bases, flanked by about 800 and 200 bases of sequence at the 5' and 3' ends, respectively, can be identified in the cDNAs. Both ORFs are in the same frame, and they are separated by 33 bases bracketed by two conserved in-frame stop codons. ORF 2 is interrupted by at least one randomly positioned stop codon in the majority of the cDNAs. The data support proposals suggesting that the human L1 family includes one or more functional genes as well as an extraordinarily large number of pseudogenes whose ORFs are broken by stop codons. The cDNA structures suggest that both genes and pseudogenes are transcribed. At least one of the cDNAs (cD11), which was sequenced in its entirety, could, in principle, represent an mRNA for production of the ORF1 polypeptide. The similarity of mammalian L1s to several recently described invertebrate movable elements defines a new widely distributed class of elements which we term class II retrotransposons. Images PMID:2454389

  1. 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 viabilit