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1

Protein acylation in the cardiac muscle like cell line, H9c2  

Microsoft Academic Search

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with 3H-palmitate

Danny M. Hasselbaink; Theo H. M. Roemen; Ger J. van der Vusse

2002-01-01

2

Stable transfection of fatty acid translocase (CD36) in a rat heart muscle cell line (H9c2)  

Microsoft Academic Search

Fatty acid translocase (FAT\\/CD36) is a mem- brane protein putatively involved in the transmembrane transport of long-chain fatty acids. We tested the hypothesis that expression of this protein in H9c2, a rat heart cell line normally not expressing FAT, would increase cellular palmi- tate uptake. We were able to stably transfect H9c2 cells with FAT, yielding 15 cell lines showing

Frans A. Van Nieuwenhoven; Joost J. F. P. Luiken; Yvonne F. De Jong; Paul A. Grimaldi; Ger J. Van der Vusse; Jan F. C. Glatz

3

The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro  

Microsoft Academic Search

Cardiac hypertrophy is a major risk factor for heart failure and associated patient morbidity and mortality. Research investigating\\u000a the aberrant molecular processes that occur during cardiac hypertrophy uses primary cardiomyocytes from neonatal rat hearts\\u000a as the standard experimental in vitro system. In addition, some studies make use of the H9C2 rat cardiomyoblast cell line,\\u000a which has the advantage of being

Sarah J. Watkins; Gillian M. Borthwick; Helen M. Arthur

2011-01-01

4

Ultrarapid Delayed Rectifier K+ Current in H9c2 Rat Ventricular Cell Line: Biophysical Property and Molecular Identity  

Microsoft Academic Search

Ultrarapid delayed rectifier K+ currents (IKurs) contribute importantly to cardiac repolarization. However, understanding of IKurs has been hampered by the difficulty of dissecting them from overlapping currents in primary cells. We found with whole-cell patch-clamp recordings that H9c2 cells, a rat ventricular cell line, under 50% confluence (single myoblasts) express exclusively IKur-like current which activates rapidly upon depolarization and partially

Hong Shi; Huizhen Wang; Hong Han; Donghui Xu; Baofeng Yang; Zhiguo Wang

2002-01-01

5

Isoproterenol Cytotoxicity is Dependent on the Differentiation State of the Cardiomyoblast H9c2 Cell Line  

Microsoft Academic Search

H9c2 cells are used as a surrogate for cardiac cells in several toxicological studies, which are usually performed with cells\\u000a in their undifferentiated state, raising questions on the applicability of the results to adult cardiomyocytes. Since H9c2\\u000a myoblasts have the capacity to differentiate into skeletal and cardiac muscle cells under different conditions, the hypothesis\\u000a of the present work was that

Ana F. Branco; Sandro L. Pereira; Ana C. Moreira; Jon Holy; Vilma A. Sardão; Paulo J. Oliveira

2011-01-01

6

Cytoprotective potential of anti-ischemic drugs against chemotherapy-induced cardiotoxicity in H9c2 myoblast cell line.  

PubMed

To investigate potential prevention or attenuation of anti- cancer drug induced cardiotoxicity using anti-ischemic drugs, a rat myoblast (H9c2) cell line was used as our in vitro cardiac model. Irinotecan and doxorubicin were found to be cytotoxic for the H9c2 cell line with IC50 of 30.69 ± 6.20 and 20.94 ± 6.05 mmol L-1, respectively. 5-Flurouracil and cladribine were not cytotoxic and thus IC50 could not be calculated. When 100 mmol L-1 doxorubicin was incubated for 72 hours with 50 mmol L-1 diltiazem, 100 mmol L-1 dexrazoxane and 100 mmol L-1 losartan, respectively, there was a 58.7 ± 10.2, 52.2 ± 11.7 and 44.7 ± 5.4 % reduction in cell death. When 200 mmol L-1 irinotecan was incubated for 72 hours with 100 mmol L-1 dexrazoxane, losartan and diltiazem, respectively, a 27.7 ± 6.9, 25.6 ± 5.1, and 19.1 ± 2.3 % reduction in cell death was observed. Our data suggests that losartan and diltiazem were as effective as dexrazoxane in protecting the cells against irinotecan- and doxorubicin-induced cell toxicity. These findings offer potential uses of anti- -ischemic drugs for ablation of cytotoxicity in response to mitochondrial injury, thereby improving patient outcomes and reducing health-care costs. PMID:24451074

Feridooni, Tiam; Mac Donald, Chris; Shao, Di; Yeung, Pollen; Agu, Remigius U

2013-12-01

7

Translocation of HSP27 to Cytoskeleton by Repetitive Hypoxia-Reoxygenation in the Rat Myoblast Cell Line, H9c2  

Microsoft Academic Search

We investigated the possible changes in the distribution of HSP27 after a brief hypoxia-reoxygenation stress in the rat myoblast cell line, H9c2, as a model of ischemic preconditioning. Cells were exposed to 4 cycles of 5 min. of hypoxia and 5 min. of reoxygenation. In the normoxic condition, HSP27 was exclusively found in the cytosolic fraction. After the hypoxia-reoxygenation cycle,

Kenji Sakamoto; Tetsuro Urushidani; Taku Nagao

1998-01-01

8

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

Microsoft Academic Search

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

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

2008-01-01

9

Cytochrome P450 epoxygenase metabolite, 14,15-EET, protects against isoproterenol-induced cellular hypertrophy in H9c2 rat cell line.  

PubMed

We have previously shown that isoproterenol-induced cardiac hypertrophy causes significant changes to cytochromes P450 (CYPs) and soluble epoxide hydrolase (sEH) gene expression. Therefore, in this study, we examined the effect of isoproterenol in H9c2 cells, and the protective effects of 14,15-EET against isoproterenol-induced cellular hypertrophy. Isoproterenol was incubated with H9c2 cells for 24 and 48 h. To determine the protective effects of 14,15-EET, H9c2 cells were incubated with isoproterenol in the absence and presence of 14,15-EET. Thereafter, the expression of hypertrophic markers and different CYP genes were determined by real time-PCR. Our results demonstrated that isoproterenol significantly increased the expression of hypertrophic marker, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), parallel to a significant increase in cell surface area. Also, isoproterenol increased the mRNA expression of CYP1A1, CYP1B1, CYP2J3, CYP4F4 and CYP4F5, as well as the gene encoding sEH, EPHX2. On other hand, 14,15-EET significantly attenuated the isoproterenol-mediated induction of ANP, BNP, CYP1A1, CYP2J3, CYP4F4, CYP4F5 and EPHX2. Moreover 14,15-EET prevented the isoproterenol-mediated increase in cell surface area. Interestingly, 20-hydroxyeicosatetraenoic acid (20-HETE) treatment caused similar effects to that of isoproterenol treatment and induced cellular hypertrophy in H9c2 cells. In conclusion, isoproterenol induces cellular hypertrophy and modulates the expression of CYPs and EPHX2 in H9c2 cells. Furthermore, 14,15-EET exerts a protective effect against isoproterenol-induced cellular hypertrophy whereas, 20-HETE induced cellular hypertrophy in H9c2 cells. PMID:23466634

Tse, Mandy M Y; Aboutabl, Mona E; Althurwi, Hassan N; Elshenawy, Osama H; Abdelhamid, Ghada; El-Kadi, Ayman O S

2013-01-01

10

Phosphoinositide 3-kinase accelerates autophagic cell death during glucose deprivation in the rat cardiomyocyte-derived cell line H9c2  

Microsoft Academic Search

We investigated cell death during glucose deprivation in rat cardiomyocyte-derived H9c2 cells. Electron microscopic analysis revealed accumulation of autophagic vacuoles during glucose deprivation. The addition of 3-methyladenine or LY294002, which are known to inhibit autophagosome formation, reduced cell death while Z-VAD-FMK, a caspase inhibitor, slightly affected cell death. Thus, cell death during glucose deprivation is not type I programmed cell

Toshihiko Aki; Kazuhito Yamaguchi; Tatsuya Fujimiya; Yoichi Mizukami

2003-01-01

11

Activation of Rac1 Increases c-Jun NH 2Terminal Kinase Activity and DNA Fragmentation in a Calcium-Dependent Manner in Rat Myoblast Cell Line H9c2  

Microsoft Academic Search

We examined the role of intracellular Ca2+ in c-Jun NH2-terminal kinase (JNK) activation and DNA fragmentation in the rat myoblast cell line H9c2 using small GTP-binding protein Rac1. A constitutively active mutant of Rac1 (V12-Rac1) increased JNK-responsive gene expression 6-fold, although this increase was attenuated by the intracellular Ca2+ chelator BAPTA-AM. V12-Rac1 also increased the number of DNA fragmentated cells.

Motohiro Nishida; Taku Nagao; Hitoshi Kurose

1999-01-01

12

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

Microsoft Academic Search

Leptin has been hypothesized to be a pathophysiologic link between obesity and cardiovascular diseases. Because the adenylate cyclase (AC) system is a main effector of ?-adrenergic receptors and leptin has been shown to modulate AC activity in other cell lines, a leptin impact on cardiac AC activity was hypothesized. Therefore, acute and chronic effects of leptin on a rat cardiac

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

2002-01-01

13

H9c2 cell line is a valuable in vitro model to study the drug metabolizing enzymes in the heart  

Microsoft Academic Search

IntroductionRecent studies demonstrated that cultured primary cardiomyocytes are a valuable tool for studying the metabolic capacity of the heart. However, a major limitation for isolated cardiomyocytes is that they are rather fragile and difficult to isolate. Therefore, there is an urgent need for an in vitro cell line model.

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

2007-01-01

14

Differential effects of chloroquine on cardiolipin biosynthesis in hepatocytes and H9c2 cardiac cells  

Microsoft Academic Search

Chloroquine is a potent lysomotropic therapeutic agent used in the treatment of malaria. The mechanism of the chloroquine-mediated modulation of new cardiolipin biosynthesis in isolated rat liver hepatocytes and H9c2 cardiac myoblast cells was addressed in this study. Hepatocytes or H9c2 cells were incubated with [1,3-3H]glycerol in the absence or presence of chloroquine and cardiolipin biosynthesis was examined. The presence

Timothy K. Ross; Fred Y. Xu; William A. Taylor; Grant Hatch

2000-01-01

15

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

PubMed

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

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

2014-01-01

16

Nardosinone protects H9c2 cardiac cells from angiotensin II-induced hypertrophy.  

PubMed

Pathological cardiac hypertrophy induced by angiotensin II (AngII) can subsequently give rise to heart failure, a leading cause of mortality. Nardosinone is a pharmacologically active compound extracted from the roots of Nardostachys chinensis, a well-known traditional Chinese medicine. In order to investigate the effects of nardosinone on AngII-induced cardiac cell hypertrophy and the related mechanisms, the myoblast cell line H9c2, derived from embryonic rat heart, was treated with nardosinone (25, 50, 100, and 200 ?mol/L) or AngII (1 ?mol/L). Then cell surface area and mRNA expression of classical markers of hypertrophy were detected. The related protein levels in PI3K/Akt/mTOR and MEK/ERK signaling pathways were examined by Western blotting. It was found that pretreatment with nardosinone could significantly inhibit the enlargement of cell surface area induced by AngII. The mRNA expression of ANP, BNP and ?-MHC was obviously elevated in AngII-treated H9c2 cells, which could be effectively blocked by nardosinone at the concentration of 100 ?mol/L. Further study revealed that the protective effects of nardosinone might be mediated by repressing the phosphorylation of related proteins in PI3K/Akt and MEK/ERK signaling pathways. It was suggested that the inhibitory effect of nardosinone on Ang II-induced hypertrophy in H9c2 cells might be mediated by targeting PI3K/Akt and MEK/ERK signaling pathways. PMID:24337842

Du, Meng; Huang, Kun; Gao, Lu; Yang, Liu; Wang, Wen-shuo; Wang, Bo; Huang, Kai; Huang, Dan

2013-12-01

17

Arsenite retards the cardiac differentiation of rat cardiac myoblast H9c2 cells.  

PubMed

It is well known that exposure to inorganic arsenic through groundwater leads not only to cancer and cardiovascular disease, but also to detrimental effects on development. In this study, we investigated the effects of arsenite on the cardiac differentiation of rat myoblast H9c2 cells. The cardiac differentiation of H9c2 cells cultured in media containing 1% fetal bovine serum and all-trans retinoic acid was confirmed by enhanced expression of cardiac troponin T (cTnT), the appearance of multinucleated cells, and cell cycle arrest at G0/G1 phase. Exposure of H9c2 cells to inorganic arsenite (As(III)) during cardiac differentiation suppressed the appearance of the morphological and biological characteristics observed in the cardiac phenotype of H9c2 cells. In addition, As(III) inhibited PKC? phosphorylation, which is detected in early-stage differentiation. These results suggest that As(III) retards the cardiac differentiation of H9c2 cells, at least partly, via the inhibition of PKC? phosphorylation. PMID:23727579

Sumi, Daigo; Abe, Kazusa; Himeno, Seiichiro

2013-06-28

18

Rat Induced Pluripotent Stem Cells Protect H9C2 Cells from Cellular Senescence via a Paracrine Mechanism.  

PubMed

Objectives: Cellular senescence may play an important role in the pathology of heart aging. We aimed to explore whether induced pluripotent stem cells (iPSCs) could inhibit cardiac cellular senescence via a paracrine mechanism. Methods: We collected iPSC culture supernatant, with or without oxidative stress, as conditioned medium (CM) for the rat cardiomyocyte-derived cell line H9C2. Then we treated H9C2 cells, cultured with or without CM, with hypoxia/reoxygenation to induce cellular senescence and measured senescence-associated ?-galactosidase (SA-?-gal) activity, G1 cell proportion and expression of the cell cycle regulators p16(INK4a), p21(Waf1/Cip1) and p53 at mRNA and protein levels in H9C2 cells. In addition, we used Luminex-based analysis to measure concentrations of trophic factors in iPSC-derived CM. Results: We found that iPSC-derived CM reduced SA-?-gal activity, attenuated G1 cell cycle arrest and reduced the expression of p16(INK4a), p21(Waf1/Cip1) and p53 in H9C2 cells. Furthermore, the CM contained more trophic factors, e.g. tissue inhibitor of metalloproteinase-1 and vascular endothelial growth factor, than H9C2-derived CM. Conclusions: Paracrine factors released from iPSCs prevent stress-induced senescence of H9C2 cells by inhibiting p53-p21 and p16-pRb pathways. This is the first report demonstrating that antisenescence effects of stem cell therapy may be a novel therapeutic strategy for age-related cardiovascular disease. © 2014 S. Karger AG, Basel. PMID:24732051

Zhang, Yuzhen; Wang, Dan; Cao, Kejiang; Chen, Minglong; Yang, Xiangjun; Tao, Yanyun

2014-01-01

19

Hydrogen sulphide protects H9c2 cells against chemical hypoxia-induced injury.  

PubMed

1. The aim of the present study was to investigate the effect of hydrogen sulphide (H(2)S) on cobalt chloride (CoCl(2))-induced injury in H9c2 embryonic rat cardiac cells. 2. After 36 h incubation in the presence of 600 micromol/L CoCl(2), reduced cell viability of H9c2 cells was observed, as well as the induction of apoptosis. In addition, CoCl(2) (600 micromol/L) enhanced the production of reactive oxygen species (ROS) and the expression of cleaved caspase 3, induced a loss of mitochondrial membrane potential (MMP) and decreased reduced glutathione (GSH) production. These results suggest that CoCl(2) induces similar responses to hypoxia/ischaemia. 3. Pretreatment of cells with 400 micromol/L NaHS (a H(2)S donor) for 30 min prior to exposure to CoCl(2) (600 micromol/L) significantly protected H9c2 cells against CoCl(2)-induced injury. Specifically, increased cell viability and decreased apoptosis were observed. In addition, NaHS pretreatment blocked the CoCl(2)-induced increases in ROS production and cleaved caspase 3 expression, as well as the decreases in GSH production and loss of MMP. 4. Pretreatment of cells with 2000 micromol/L N-acetylcysteine (NAC), a ROS scavenger, for 1 h prior to CoCl(2) exposure significantly protected H9c2 cells against CoCl(2)-induced injury, specifically enhancing cell viability, decreasing ROS production and preventing loss of MMP. 5. The findings of the present study suggest that H(2)S protects H9c2 cells against CoCl(2)-induced injury by suppressing oxidative stress and caspase 3 activation. PMID:19769612

Chen, Si-Lin; Yang, Chun-Tao; Yang, Zhan-Li; Guo, Rui-Xian; Meng, Jin-Lan; Cui, Yu; Lan, Ai-Ping; Chen, Pei-Xi; Feng, Jian-Qiang

2010-03-01

20

Prazosin induces p53-mediated autophagic cell death in H9C2 cells  

Microsoft Academic Search

Prazosin, a quinazoline-based ?1-adrenoceptor antagonist, is known to induce cell death, and this effect is independent of its ?-blockade activity. However,\\u000a the detailed molecular mechanisms involved are still not fully understood. In this study, we found that prazosin, but not\\u000a doxazosin, could induce patterns of autophagy in H9C2 cells, including intracellular vacuole formation, microtubule-associated\\u000a protein 1 light chain 3 (LC3)

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

2011-01-01

21

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

PubMed Central

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

Jin, Seon Mi

2012-01-01

22

Down-regulation of SM22\\/transgelin gene expression during H9c2 cells differentiation  

Microsoft Academic Search

The embryonic rat ventricle H9c2 cells maintain a proliferative state (P condition) in the presence of 10% FCS. However, by\\u000a reducing serum concentration and in the presence of retinol acetate, proliferation is stopped, myogenic transdifferentiation\\u000a is inhibited while cardiac differentiation is preserved (D condition). Two-dimensional gel electrophoresis and mass spectrometry\\u000a analysis was used to define the modifications of the nuclear

Elisa Bregant; Giovanni Renzone; Renata Lonigro; Nadia Passon; Carla Di Loreto; Maura Pandolfi; Andrea Scaloni; Gianluca Tell; Giuseppe Damante

2009-01-01

23

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

PubMed

Abuse of synthetic drugs is widespread among young people worldwide. In this context, piperazine derived drugs recently appeared in the recreational drug market. Clinical studies and case-reports describe sympathomimetic effects including hypertension, tachycardia, and increased heart rate. Our aim was to investigate the cytotoxicity of N-benzylpiperazine (BZP), 1-(3-trifluoromethylphenyl) piperazine (TFMPP), 1-(4-methoxyphenyl) piperazine (MeOPP), and 1-(3,4-methylenedioxybenzyl) piperazine (MDBP) in the H9c2 rat cardiac cell line. Complete cytotoxicity curves were obtained at a 0-20mM concentration range after 24h incubations with each drug. The EC50 values (?M) were 343.9, 59.6, 570.1, and 702.5 for BZP, TFMPP, MeOPP, and MDBP, respectively. There was no change in oxidative stress markers. However, a decrease in total GSH content was noted for MDBP, probably due to metabolic conjugation reactions. All drugs caused significant decreases in intracellular ATP, accompanied by increased intracellular calcium levels and a decrease in mitochondrial membrane potential that seems to involve the mitochondrial permeability transition pore. The cell death mode revealed early apoptotic cells and high number of cells undergoing secondary necrosis. Among the tested drugs, TFMPP seems to be the most potent cytotoxic compound. Overall, piperazine designer drugs are potentially cardiotoxic and support concerns on risks associated with the intake of these drugs. PMID:24968061

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

2014-08-17

24

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

SciTech Connect

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

Wei, Xing, E-mail: weixing22@163.com [Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078 (China) [Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078 (China); Institute of Cardiovascular Disease, Department of Pathophysiology, School of Medicine, University of South China, 28 Changsheng Xi Road, Hengyang, Hunan 421001 (China); Song, Lan; Jiang, Lei; Wang, Guiliang; Luo, Xinjing; Zhang, Bin [Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078 (China)] [Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078 (China); Xiao, Xianzhong, E-mail: xianzhongxiao@hotmail.com [Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078 (China)] [Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 Xiangya Road, Changsha, Hunan 410078 (China)

2010-03-19

25

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

PubMed

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

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

2014-04-01

26

Chronic-alcohol exposure alters IGF1 signaling in H9c2 cells via changes in PKC delta  

Microsoft Academic Search

Previously, we have demonstrated that chronic-alcohol exposure alters insulin-like growth factor 1 (IGF1) signaling in adult rat heart cells. This report examines the effects of alcohol in vitro on the expression of protein kinase C (PKC) alpha, delta, and epsilon using the embryonic heart cell line, H9c2, and how this may be linked to changes in IGF1 signal transduction. Western

Richard Ila; Michele Solem

2006-01-01

27

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

Microsoft Academic Search

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

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

2007-01-01

28

Constitutive expression and inducibility of CYP1A1 in the H9c2 rat cardiomyoblast cells  

Microsoft Academic Search

Cardiomyocytes are a valuable tool for studying the drug metabolizing enzymes in the heart. However, isolated cardiomyocytes are rather fragile and difficult to isolate. Therefore, there is an urgent need for an in vitro cell line model. The H9c2 cells are commonly used as an in vitro model for studying the cellular mechanisms and signaling pathways involved in drug-induced cardiotoxicity.

Mona E. Aboutabl; Ayman O. S. El-Kadi

2007-01-01

29

Icariin protects rat cardiac H9c2 cells from apoptosis by inhibiting endoplasmic reticulum stress.  

PubMed

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

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

2013-01-01

30

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

PubMed Central

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

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

2013-01-01

31

Antiadrenergic effect of adenosine involves connexin 43 turn-over in H9c2 cells.  

PubMed

Connexin 43 (Cx43) is the major protein of cardiac ventricular gap junctions and is crucial to cell-cell communication and cardiac function. Several authors report that adrenergic stimulation affects Cx43 expression via protein kinase A (PKA) and MAPK-regulated pathways. Adenosine has been shown to exert direct antiadrenergic effects on the heart, protecting it from toxic effects of overstimulation. The aim of our study was to understand the involvement of Cx43 in the anti-adrenergic effect of adenosine on cardiomyocytes. H9c2 cardiomyoblast cells were treated with isoproterenol alone or in association with adenosine. Isoproterenol and adenosine co-treated H9c2 cells showed an increased amount of Cx43 phosphorylated on Ser368. This effect was adenosine A1 receptor-dependent via the activation of the protein kinase C (PKC). Interestingly, the phosphorylation of Cx43 facilitated the degradation of Cx43 through the ubiquitin-proteasome system, as demonstrated by the immunoprecipitation of pCx43 with ubiquitin. On the basis of these results we can hypothesize that the activation of PKC after adenosine A1 receptor stimulation increases Cx43 phosphorylation that is necessary for its ubiquitination and then degradation via the proteasome system. These data better underline new mechanism at the basis of antiadrenergic effects of adenosine. PMID:23834776

Popolo, Ada; Morello, Silvana; Sorrentino, Rosalinda; Pinto, Aldo

2013-09-01

32

Role of large-conductance Ca 2+-activated potassium channels in adenosine A 1 receptor-mediated pharmacological preconditioning in H9c2 cells  

Microsoft Academic Search

Large-conductance Ca2+-activated potassium channels, located on the inner mitochondrial membrane, have recently been implicated in cytoprotection. Therefore, the primary aim of this study was to determine the role of large-conductance Ca2+-activated potassium channels in adenosine A1 receptor-induced pharmacological preconditioning in the rat embryonic cardiomyoblast-derived cell line H9c2. For pharmacological preconditioning, H9c2 cells were exposed to the adenosine A1 receptor agonist

Laurice Fretwell; John M. Dickenson

2009-01-01

33

Downregulation of the cardiotrophin-1 gene expression by valsartan and spironolactone in hypertrophied heart rats in vivo and rat cardiomyocyte H9c2 cell line in vitro: a novel mechanism of cardioprotection.  

PubMed

The incidence, prevalence, and hospitalization rates associated with heart failure (HF) are projected to increase substantially in the world. Among all medications used clinically to treat HF, valsartan (VAL) and spironolactone (SPL) have been shown to reduce morbidity and mortality. Recently, a novel cardiac gene cardiotrophin-1 (CT-1) has been shown to play a crucial role in the pathogenesis of HF. However, the ability of VAL and SPL to modulate the expression of CT-1 has not been investigated yet. Therefore, healthy and isoproterenol (ISO)-induced hypertrophy adult male Wistar albino rats were treated with either VAL or SPL for 14 days. Thereafter, cardiac markers of cardiotoxicity and hypertrophy, creatine kinase, heart weight/body weight ratio, and atrial natriuretic peptide mRNA levels were measured. In addition, CT-1 mRNA and protein levels were determined by real-time polymerase chain reaction and Western blot analysis. Our results showed that the increases in all HF markers, creatine kinase, heart weight/body weight ratio, and atrial natriuretic peptide mRNA levels in ISO-treated rats were significantly restored to their normal levels by VAL and SPL. In addition, induction of cardiac hypertrophy by ISO caused remarkable induction in CT-1 mRNA and protein expression levels by approximately 3.5- and 3-fold, respectively. Importantly, VAL and SPL significantly decreased the induction of CT-1 gene at the mRNA and protein levels in heart hypertrophied rats. On the other hand, treatment of cardiac-derived rat myoblast H9c2 cells with VAL and SPL significantly decreased angiotensin II-induced CT-1 mRNA levels through transcriptional mechanism, as demonstrated by the effect of transcription inhibitor, actinomycin D. In conclusion, VAL and SPL exhibited their cardioprotective effect through inhibiting the expression of CT-1 gene in cardiac hypertrophied rats. PMID:23288202

Al-Mazroua, Haneen A; Al-Rasheed, Nawal M; Korashy, Hesham M

2013-04-01

34

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

SciTech Connect

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.

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

2010-10-01

35

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

PubMed

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

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

2014-06-01

36

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

Microsoft Academic Search

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

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

2011-01-01

37

Determination of the intracellular element concentrations in cultured H9c2 cells by proton microprobe techniques  

NASA Astrophysics Data System (ADS)

The present study was conducted to investigate the applicability of proton microprobe techniques in the assessment of intracellular ion concentrations in individual cardiac-type H9c2 cells. Several methods to minimise the contribution of the extracellular environment were explored. Preliminary results indicate that proton microprobe techniques might be useful to assess intracellular ion concentrations in individual, cultured cells.

Quaedackers, J. A.; van Engeland, S.; Mutsaers, P. H. A.; de Goeij, J. J. M.; Snoeckx, L. H. E. H.; de Voigt, M. J. A.; van der Vusse, G. J.

1999-10-01

38

Globular adiponectin protects H9c2 cells from palmitate-induced apoptosis via Akt and ERK1/2 signaling pathways  

PubMed Central

Background Cardiomyocytes apoptosis is an important contributor to myocardial dysfunction and heart failure. Adiponectin has cardioprotective effects, potential mechanisms behind it are not clear in cardiomyocytes. The purpose of the study was to investigate whether adiponectin can block palmitate-induced apoptosis and the underlying biochemical mechanism in H9c2 cells. Methods H9c2 cells were treated with palmitate presence or absence of 2.5 ?g/mL globular adiponectin. The effect on the cell viability of H9c2 cells was evaluated using MTT assay, and cell apoptosis was determined by Hoechst 33342 staining. Protein expression was measured using the western blot method. Results Our results showed that the palmitate treatment induced apoptosis in H9c2 cells, which was associated with increasing the level of cleaved caspase-3 and cleaved PARP. Meanwhile, palmitate-induced apoptosis increased the protein level of p-ERK1/2, and decreased the protein level of p-Akt significantly. However, levels of both of these proteins were restored to the normal when pretreated with adiponectin, and followed with the decrease of cleaved caspase-3 and cleaved PARP. In line with these results, the protective effect of adiponectin can be blocked by PI3K/Akt inhibitor LY294002, and palmitate-induced apoptosis can be attenuated by ERK1/2 inhibitor U0126. Conclusions Taken together, the present study demonstrated that adiponectin protects H9c2 cells from palmitate-induced apoptosis via PI3K/Akt and ERK1/2 signaling pathways. Our results reveal a link between adiponectin and cardiomyocytes apoptosis, suggesting that adioponectin may be a promising therapeutic for the treatment of lipotoxicity cardiomyopathy.

2012-01-01

39

Hydrogen sulfide protects H9c2 cells against doxorubicin-induced cardiotoxicity through inhibition of endoplasmic reticulum stress.  

PubMed

The roles of hydrogen sulfide (H(2)S) and endoplasmic reticulum (ER) stress in doxorubicin (DOX)-induced cardiotoxicity are still unclear. This study aimed to dissect the hypothesis that H(2)S could protect H9c2 cells against DOX-induced cardiotoxicity by inhibiting ER stress. Our results showed that exposure of H9c2 cells to DOX significantly inhibited the expression and activity of cystathionine-?-lyase (CSE), a synthetase of H(2)S, accompanied by the decreased cell viability and the increased reactive oxygen species (ROS) accumulation. In addition, exposure of cells to H(2)O(2) (an exogenous ROS) mimicked the inhibitory effect of DOX on the expression and activity of CSE. Pretreatment with N-acetyl-L: -cysteine (NAC) (a ROS scavenger) attenuated intracellular ROS accumulation, cytotoxicity, and the inhibition of expression and activity of CSE induced by DOX. Notably, the ER stress-related proteins, including glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were obviously upregulated in DOX-treated H9c2 cells. Pretreatment with sodium hydrosulfide (NaHS, a H(2)S donor) before DOX exposure markedly suppressed DOX-induced overexpressions of GRP78 and CHOP, cytotoxicity and oxidative stress. In conclusion, we have demonstrated that ROS-mediated inhibition of CSE is involved in DOX-induced cytotoxicity in H9c2 cells, and that exogenous H(2)S can confer protection against DOX-induced cardiotoxicity partly through inhibition of ER stress. PMID:22203419

Wang, Xiu-Yu; Yang, Chun-Tao; Zheng, Dong-Dan; Mo, Li-Qiu; Lan, Ai-Ping; Yang, Zhan-Li; Hu, Fen; Chen, Pei-Xi; Liao, Xin-Xue; Feng, Jian-Qiang

2012-04-01

40

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

PubMed

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

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

2011-05-01

41

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

PubMed

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

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

2014-09-01

42

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

Microsoft Academic Search

BACKGROUND: While doxorubicin (DOX) is widely used in cancer chemotherapy, long-term severe cardiotoxicity limits its use. This is the first report of the chemoprotective efficacy of a relatively new thiol antioxidant, N-acetylcysteine amide (NACA), on DOX-induced cell death in cardiomyocytes. We hypothesized that NACA would protect H9c2 cardiomyocytes from DOX-induced toxicity by reducing oxidative stress. Accordingly, we determined the ability

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

2009-01-01

43

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

Microsoft Academic Search

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

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

1998-01-01

44

Proliferation and skeletal myotube formation capability of C2C12 and H9c2 cells on isotropic and anisotropic electrospun nanofibrous PHB scaffolds  

Microsoft Academic Search

This study aims at investigating the behavior in terms of the proliferation and skeletal muscle differentiation capability of two myoblastic cell lines, C2C12 and H9c2, on both isotropic and anisotropic electrospun nanofibrous poly(hydroxybutyrate) (PHB) scaffolds, as well as on PHB films and polystyrene controls. After a careful characterization of the matrices in terms of surface morphology, surface roughness and mechanical

Leonardo Ricotti; Alessandro Polini; Giada G Genchi; Gianni Ciofani; Donata Iandolo; Helena Vazão; Virgilio Mattoli; Lino Ferreira; Arianna Menciassi; Dario Pisignano

2012-01-01

45

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

PubMed Central

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.

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

2013-01-01

46

Cellular and molecular studies of the effects of a selective COX-2 inhibitor celecoxib in the cardiac cell line H9c2 and their correlation with death mechanisms.  

PubMed

Cardiovascular disease is one of the leading causes of death worldwide, and evidence indicates a correlation between the inflammatory process and cardiac dysfunction. Selective inhibitors of cyclooxygenase-2 (COX-2) enzyme are not recommended for long-term use because of potentially severe side effects to the heart. Considering this and the frequent prescribing of commercial celecoxib, the present study analyzed cellular and molecular effects of 1 and 10 µM celecoxib in a cell culture model. After a 24-h incubation, celecoxib reduced cell viability in a dose-dependent manner as also demonstrated in MTT assays. Furthermore, reverse transcription-polymerase chain reaction analysis showed that the drug modulated the expression level of genes related to death pathways, and Western blot analyses demonstrated a modulatory effect of the drug on COX-2 protein levels in cardiac cells. In addition, the results demonstrated a downregulation of prostaglandin E2 production by the cardiac cells incubated with celecoxib, in a dose-specific manner. These results are consistent with the decrease in cell viability and the presence of necrotic processes shown by Fourier transform infrared analysis, suggesting a direct correlation of prostanoids in cellular homeostasis and survival. PMID:24519091

Sakane, K K; Monteiro, C J; Silva, W; Silva, A R; Santos, P M; Lima, K F; Moraes, K C M

2014-01-01

47

Cellular and molecular studies of the effects of a selective COX-2 inhibitor celecoxib in the cardiac cell line H9c2 and their correlation with death mechanisms  

PubMed Central

Cardiovascular disease is one of the leading causes of death worldwide, and evidence indicates a correlation between the inflammatory process and cardiac dysfunction. Selective inhibitors of cyclooxygenase-2 (COX-2) enzyme are not recommended for long-term use because of potentially severe side effects to the heart. Considering this and the frequent prescribing of commercial celecoxib, the present study analyzed cellular and molecular effects of 1 and 10 µM celecoxib in a cell culture model. After a 24-h incubation, celecoxib reduced cell viability in a dose-dependent manner as also demonstrated in MTT assays. Furthermore, reverse transcription-polymerase chain reaction analysis showed that the drug modulated the expression level of genes related to death pathways, and Western blot analyses demonstrated a modulatory effect of the drug on COX-2 protein levels in cardiac cells. In addition, the results demonstrated a downregulation of prostaglandin E2 production by the cardiac cells incubated with celecoxib, in a dose-specific manner. These results are consistent with the decrease in cell viability and the presence of necrotic processes shown by Fourier transform infrared analysis, suggesting a direct correlation of prostanoids in cellular homeostasis and survival.

Sakane, K.K.; Monteiro, C.J.; Silva, W.; Silva, A.R.; Santos, P.M.; Lima, K.F.; Moraes, K.C.M.

2014-01-01

48

Modulation of L-type calcium channel expression during retinoic acid-induced differentiation of H9C2 cardiac cells.  

PubMed

The molecular mechanisms underlying the developmental regulation of L-type voltage-dependent Ca(2+) channels (VDCCs) are still unknown. In this study, we have characterized the expression patterns of skeletal (alpha(1S)) and cardiac (alpha(1C)) L-type VDCCs during cardiogenic differentiation in H9C2 cells that derived from embryonic rat heart. We report that chronic treatment of H9C2 cells with 10 nM all-trans-retinoic acid (all-trans-RA) enhanced cardiac Ca(2+) channel expression, as demonstrated by reverse transcription-polymerase chain reaction, immunoblotting, and indirect immunofluorescence studies, as well as patch-clamp experiments. In addition, RA treatment prevented expression of functional skeletal L-type VDCCs, which were restricted to myotubes that spontaneously appear in control H9C2 cultures undergoing myogenic transdifferentiation. The use of specific skeletal and cardiac markers indicated that RA, by preventing myogenic transdifferentiation, preserves cardiac differentiation of this cell line. Altogether, we provide evidence that cardiac and skeletal subtype-specific L-type Ca(2+) channels are relevant functional markers of differentiated cardiac and skeletal myocytes, respectively. In conclusion, our data demonstrate that in vitro RA stimulates cardiac (alpha(1C)) L-type Ca(2+) channel expression, therefore supporting the hypothesis that the RA pathway might be involved in the tissue specific expression of Ca(2+) channels in mature cardiac cells. PMID:10506158

Ménard, C; Pupier, S; Mornet, D; Kitzmann, M; Nargeot, J; Lory, P

1999-10-01

49

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

PubMed

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 ?M) 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 ?M concentration, which was evident from decreased activity of lactate dehydrogenase (5 ?M ATO + 20 ?g/mL BDE: 6.61 ± 1.97 ?U/mL, respective control group: 16.15 ± 1.92 ?U/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

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

2013-06-01

50

Exenatide protects against hypoxia/reoxygenation-induced apoptosis by improving mitochondrial function in H9c2 cells.  

PubMed

Glucagon-like peptide-1 (GLP-1) analogues might exert the cardioprotective effects via attenuating apoptosis. This study aimed to determine the protective effects and mechanism of exenatide, a GLP-1 analogue, on cardiomyocyte apoptosis using an in vitro model of hypoxia/reoxygenation (H/R). H9c2 cells were employed to establish an in vitro model of H/R. 200 nM exenatide pretreatment significantly reduced apoptosis measured by flow cytometry. To further study the antiapoptotic mechanism of exenatide, we used flow cytometry in combination with laser confocal microscopy to determine the interaction between exenatide and the process of mitochondria-mediated apoptosis. We found that exenatide pretreatment reduced the intracellular reactive oxygen species (ROS) levels and decreased the mitochondrial calcium overload caused by H/R. Furthermore, an increase of total superoxide dismutase (T-SOD) levels, a decrease of malondialdehyde (MDA) levels, a preservation of mitochondrial membrane potential (??m), a reduction of cytochrome-c release, a decline of cleaved caspase-3 expression, and caspase-3 activation were observed in exenatide-pretreated cultures. These results suggest that exenatide exerts a protective effect on preventing against H/R-induced apoptosis. Importantly, the protective effects of exenatide may be attributed to its role in improving mitochondrial function in H9c2 cells subjected to H/R. PMID:24586099

Chang, Guanglei; Zhang, Dongying; Liu, Jian; Zhang, Peng; Ye, Lin; Lu, Kai; Duan, Qin; Zheng, Aihua; Qin, Shu

2014-04-01

51

Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation  

Microsoft Academic Search

Recent experimental data demonstrate cardiovascular effects of the GH secretagogues (GHSs) hexarelin and ghrelin, the proposed natural ligand for the GHS receptor. Moreover, specific cardiac binding sites for GHSs have been suggested. The aim of the present study was to investigate if the natural ligand ghrelin and synthetic GHS peptide hexarelin and analogues have direct effects on the cardiomyocyte cell

I Pettersson; G Muccioli; R Granata; R Deghenghi; E Ghigo; C Ohlsson; J Isgaard

2002-01-01

52

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

PubMed

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

Hong, Show-Jen

2002-10-01

53

Epigenetic regulation of cardiac muscle-specific genes in H9c2 cells by Interleukin18 and histone deacetylase inhibitor m -carboxycinnamic acid bis -hydroxamide  

Microsoft Academic Search

Interleukin-18 (IL-18) elicited a robust hypertrophy response in H9c2 cardiomyocytes as judged by their accelerated rates\\u000a of protein synthesis and increased cell size. Evidently, IL-18 treatment also induced a cardiac hypertrophy-specific program\\u000a of gene expression in H9c2 cardiomyocytes since they elicited enhanced expression of atrial naturetic factor (ANF), desmin,\\u000a and skeletal ?-actin genes accompanied by a canonical switch in the

Gipsy Majumdar; I. Maria Johnson; Santosh Kale; Rajendra Raghow

2008-01-01

54

Cholesterol modulates function of connexin 43 gap junction channel via PKC pathway in H9c2 cells.  

PubMed

It has been shown that cholesterol modulates activity of protein kinase C (PKC), and PKC phosphorylates connexin 43 (Cx43) to regulate its function, respectively. However, it is not known whether cholesterol modulates function of Cx43 through regulating activity of PKC. In the present study, we demonstrated that cholesterol enrichment reduced the dye transfer ability of Cx43 in cultured H9c2 cells. Western blot analysis indicated that cholesterol enrichment enhanced the phosphorylated state of Cx43. Immunofluorescent images showed that cholesterol enrichment made the Cx43 distribution from condensed to diffused manner in the interface between the cells. In cholesterol enriched cells, PKC antagonists partially restored the dye transfer ability among the cells, downregulated the phosphorylation of Cx43 and redistributed Cx43 from the diffused manner to the condensed manner in the cell interface. In addition, reduction of cholesterol level suppressed PKC activity to phosphorylate Cx43 and restored Cx43 function in PKC agonist-treated cells. Furthermore, we demonstrated that cholesterol enrichment upregulated the phosphorylated state of Cx43 at Ser368, while PKC antagonists reversed the effect. Taken together, cholesterol level in the cells plays important roles in regulating Cx43 function through activation of the PKC signaling pathway. PMID:24780378

Zou, Jun; Yue, Xiao-Yang; Zheng, Sheng-Chao; Zhang, Guangwei; Chang, He; Liao, Yan-Chun; Zhang, Ye; Xue, Mao-Qiang; Qi, Zhi

2014-08-01

55

Characterization of adenosine transport in H9c2 cardiomyoblasts  

Microsoft Academic Search

Adenosine plays a significant role in various physiological processes including cardioprotection. Nucleoside transporters modulate adenosine levels in the vicinity of adenosine receptors, which in turn modulate adenosine functional efficacy. In the current study, adenosine transport in the rat heart myoblast cell line H9c2 was characterized. Kinetic analysis of adenosine transport in H9c2 cells revealed a Km of 8.9±0.001 ?M and a

George P. H. Leung; Chung-Ming Tse; Ricky Y. K. Man

2007-01-01

56

Effects of downregulation of microRNA-181a on H2O2-induced H9c2 cell apoptosis via the mitochondrial apoptotic pathway.  

PubMed

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

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

2014-01-01

57

Hypoxia-inducible factor-1alpha is a critical mediator of hypoxia induced apoptosis in cardiac H9c2 and kidney epithelial HK-2 cells  

PubMed Central

Background Hypoxia inducible factor-1 (HIF-1) is a transcription factor that functions to maintain cellular homeostasis in response to hypoxia. There is evidence that HIF-1 can also trigger apoptosis, possibly when cellular responses are inadequate to meet energy demands under hypoxic conditions. Methods Cardiac derived H9c2 and renal tubular epithelial HK-2 cells expressing either the wild type oxygen regulated subunit of HIF-1 (pcDNA3-Hif-1?) or a dominant negative version that lacked both DNA binding and transactivation domains (pcDNA3-DN-Hif-1?), were maintained in culture and exposed to hypoxia. An RNA interference approach was also employed to selectively knockdown expression of Hif-1?. Apoptosis was analyzed in both H9c2 and HK-2 cells by Hoechst and TUNEL staining, caspase 3 activity assays and activation of pro-apoptotic Bcl2 family member Bax. Results Overexpression of pcDNA3-DN-Hif-1? led to a significant reduction in hypoxia -induced apoptosis (17 ± 2%, P < 0.01) in H9c2 cells compared to both control-transfected and wild type Hif-1? transfected cells. Moreover, selective ablation of HIF-1? protein expression by RNA interference in H9c2 cells led to 55% reduction of caspase 3 activity and 46% reduction in the number of apoptotic cells as determined by Hoechst 33258 staining, after hypoxia. Finally, upregulation of the pro-apoptotic protein, Bax, was found in H9c2 cells overexpressing full-length pcDNA3-HA-HIF-1? exposed to hypoxia. In HK-2 cells overexpression of wild-type Hif-1? led to a two-fold increase in Hif-1? levels during hypoxia. This resulted in a 3.4-fold increase in apoptotic cells and a concomitant increase in caspase 3 activity during hypoxia when compared to vector transfected control cells. HIF-1? also induced upregulation of Bax in HK-2 cells. In addition, introduction of dominant negative Hif-1? constructs in both H9c2 and HK-2 -cells led to decreased active Bax expression. Conclusion These data demonstrate that HIF-1? is an important component of the apoptotic signaling machinery in the two cell types.

Malhotra, Ricky; Tyson, David W; Rosevear, Henry M; Brosius, Frank C

2008-01-01

58

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

PubMed Central

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 (??m), 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 ??m, 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.

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

2014-01-01

59

Mitochondrial ATP production is necessary for activation of the extracellular-signal-regulated kinases during ischaemia/reperfusion in rat myocyte-derived H9c2 cells.  

PubMed Central

To search for the stimuli involved in activating the mitogen-activated protein kinases (MAPKs) during ischaemia and reperfusion, we simulated the event in a system in vitro conducive to continuous and non-invasive measurements of several major perturbations that occur at the time: O(2) tension, mitochondrial respiration and energy status. Using H9c2 cells (a clonal line derived from rat heart), we found that activation of the extracellular signal-regulated MAPKs (ERKs) on reoxygenation was abolished if the mitochondria were inhibited prior to and during reoxygenation. Re-introduction of O(2) per se is therefore not sufficient to activate the ERKs. Recovery and maintenance of cellular ATP levels by mitochondrial respiration is necessary, although ATP recovery alone is not sufficient. ERK activation by H(2)O(2), but not phorbol esters, was also sensitive to mitochondrial inhibition. Thus, reoxygenation and H(2)O(2)-mediated oxidative stress share a mechanism of ERK activation that is ATP- or mitochondrion-dependent, and this common feature suggests that the reoxygenation response is mediated by reactive oxygen species. A correlation between ERK activity and ATP levels was also found during the anoxic phase of ischaemia, an effect that was not due to substrate limitation for the kinases. Our results reveal the importance of cellular metabolism in ERK activation, and introduce ATP as a novel participant in the mechanisms underlying the ERK cascade.

Abas, L; Bogoyevitch, M A; Guppy, M

2000-01-01

60

RhoGDI?-induced hypertrophic growth in H9c2 cells is negatively regulated by ZAK  

Microsoft Academic Search

We found that overexpression of RhoGDI?, a Rho GDP dissociation inhibitor, induced hypertrophic growth and suppressed cell cycle progression in a cultured cardiomyoblast cell line. Knockdown of RhoGDI? expression by RNA interference blocked hypertrophic growth. We further demonstrated that RhoGDI? physically interacts with ZAK and is phosphorylated by ZAK in vitro, and this phosphorylation negatively regulates RhoGDI? functions. Moreover, the

Chih-Yang Huang; Li-Chiu Yang; Kuan-Yu Liu; Pao-Hsin Liao; Janet Ing-Yuh Chou; Ming-Yung Chou; Wei-Wen Lin; Jaw-Ji Yang

2009-01-01

61

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

PubMed

TNF-? plays a mediator role in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-? in inflammatory responses has been shown to be mediated through up-regulation of matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-?-induced MMP-9 expression in rat embryonic-heart derived H9c2 cells are largely not defined. We demonstrated that in H9c2 cells, TNF-? induced MMP-9 mRNA and protein expression associated with an increase in the secretion of pro-MMP-9. TNF-?-mediated responses were attenuated by pretreatment with the inhibitor of ROS (N-acetyl-l-cysteine, NAC), NADPH oxidase [apocynin (APO) or diphenyleneiodonium chloride (DPI)], MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), NF-?B (Bay11-7082), or PYK2 (PF-431396) and transfection with siRNA of TNFR1, p47(phox), p42, p38, JNK1, p65, or PYK2. Moreover, TNF-? markedly induced NADPH oxidase-derived ROS generation in these cells. TNF-?-enhanced p42/p44 MAPK, p38 MAPK, JNK1/2, and NF-?B (p65) phosphorylation and in vivo binding of p65 to the MMP-9 promoter were inhibited by U0126, SB202190, SP600125, NAC, DPI, or APO. In addition, TNF-?-mediated PYK2 phosphorylation was inhibited by NAC, DPI, or APO. PYK2 inhibition could reduce TNF-?-stimulated MAPKs and NF-?B activation. Thus, in H9c2 cells, we are the first to show that TNF-?-induced MMP-9 expression is mediated through a TNFR1/NADPH oxidase/ROS/PYK2/MAPKs/NF-?B cascade. We demonstrated that NADPH oxidase-derived ROS generation is involved in TNF-?-induced PYK2 activation in these cells. Understanding the regulation of MMP-9 expression and NADPH oxidase activation by TNF-? on H9c2 cells may provide potential therapeutic targets of chronic heart failure. PMID:23774252

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

2013-10-15

62

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

Microsoft Academic Search

WD40 repeat proteins have a wide range of diverse biological functions including signal transduction, cell cycle regulation, RNA splicing, and transcription. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a novel member of the WD40 repeat proteins superfamily that contains five WD40 repeats. Little is known about its biological role, and the purpose of this study was to determine the

Xing Wei; Lan Song; Lei Jiang; Guiliang Wang; Xinjing Luo; Bin Zhang; Xianzhong Xiao

2010-01-01

63

Ghrelin protects against cobalt chloride-induced hypoxic injury in cardiac H9c2 cells by inhibiting oxidative stress and inducing autophagy.  

PubMed

Ghrelin is a multifunctional peptide that actively protects against cardiovascular ischemic diseases, but the underlying mechanisms are unclear. We used CoCl(2) to mimic hypoxic conditions in cardiac H9c2 cells in order to study the mechanism by which ghrelin protects cardiac myocytes against hypoxic injury by regulating the content of intracellular ROS and autophagy levels. Cell apoptosis and necrosis were evaluated by the flow cytometry assay, Hoechst staining, and LDH activity. Cell viability was detected by the WST-1 assay; ROS levels were assessed using DCFH2-DA; and Nox1, catalase and Mn-SOD were assayed by real-time PCR and activity assays. LC3II was measured by Western blot analysis. We observed that CoCl(2) induced apoptosis and death of H9c2 cells in a dose- and time-dependent manner. This was characterized by an increase in cell apoptosis, LDH activity, ROS content, Nox1 expression, and autophagy levels and a decrease in cell viability, catalase, and Mn-SOD activities. Ghrelin treatment significantly attenuated CoCl(2)-induced hypoxic injury by decreasing cell apoptosis, LDH activity, ROS content, and Nox1 expression and increasing cell viability, autophagy levels, catalase, and Mn-SOD mRNA levels and activities. Further experiments revealed that inhibiting autophagy using 3-MA or AMPK pathway with compound C almost abrogated the induction of ghrelin in autophagy. This was associated with a decrease in cell viability and an increase in LDH activity. Our results indicate that ghrelin protected cardiac myocytes against CoCl(2)-induced hypoxic injury by decreasing Nox1 expression, increasing the expression and activity of endogenous antioxidant enzymes, and inducing protective autophagy in an AMPK-dependent manner. PMID:23000094

Tong, Xin-Xin; Wu, Dan; Wang, Xue; Chen, Hua-Li; Chen, Jia-Xiang; Wang, Xiao-Xiao; Wang, Xu-Lei; Gan, Lu; Guo, Zhi-Yun; Shi, Gui-Xiu; Zhang, Yi-Zheng; Jiang, Wei

2012-12-01

64

On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells.  

PubMed

The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts. Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected. Pulse labeling experiments with [1,3-3H]glycerol and pulse-chase labeling experiments with [1,3-3H]glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C. In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids. Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from [1,3-3H]glycerol indicating the involvement of 1,2-diacyl-sn glycerol. The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels. Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from [methyl-3H]choline was markedly stimulated. These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells. PMID:9823027

Xu, F Y; Kelly, S L; Taylor, W A; Hatch, G M

1998-11-01

65

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

PubMed Central

Background We have shown previously that pan-HDAC inhibitors (HDACIs) m-carboxycinnamic acid bis-hydroxamide (CBHA) and trichostatin A (TSA) attenuated cardiac hypertrophy in BALB/c mice by inducing hyper-acetylation of cardiac chromatin that was accompanied by suppression of pro-inflammatory gene networks. However, it was not feasible to determine the precise contribution of the myocytes- and non-myocytes to HDACI-induced gene expression in the intact heart. Therefore, the current study was undertaken with a primary goal of elucidating temporal changes in the transcriptomes of cardiac myocytes exposed to CBHA and TSA. Results We incubated H9c2 cardiac myocytes in growth medium containing either of the two HDACIs for 6h and 24h and analyzed changes in gene expression using Illumina microarrays. H9c2 cells exposed to TSA for 6h and 24h led to differential expression of 468 and 231 genes, respectively. In contrast, cardiac myocytes incubated with CBHA for 6h and 24h elicited differential expression of 768 and 999 genes, respectively. We analyzed CBHA- and TSA-induced differentially expressed genes by Ingenuity Pathway (IPA), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Core_TF programs and discovered that CBHA and TSA impinged on several common gene networks. Thus, both HDACIs induced a repertoire of signaling kinases (PTEN-PI3K-AKT and MAPK) and transcription factors (Myc, p53, NFkB and HNF4A) representing canonical TGF?, TNF-?, IFN? and IL-6 specific networks. An overrepresentation of E2F, AP2, EGR1 and SP1 specific motifs was also found in the promoters of the differentially expressed genes. Apparently, TSA elicited predominantly TGF?- and TNF-?-intensive gene networks regardless of the duration of treatment. In contrast, CBHA elicited TNF-? and IFN? specific networks at 6 h, followed by elicitation of IL-6 and IFN?-centered gene networks at 24h. Conclusions Our data show that both CBHA and TSA induced similar, but not identical, time-dependent, gene networks in H9c2 cardiac myocytes. Initially, both HDACIs impinged on numerous genes associated with adipokine signaling, intracellular metabolism and energetics, and cell cycle. A continued exposure to either CBHA or TSA led to the emergence of a number of apoptosis- and inflammation-specific gene networks that were apparently suppressed by both HDACIs. Based on these data we posit that the anti-inflammatory and anti-proliferative actions of HDACIs are myocyte-intrinsic. These findings advance our understanding of the mechanisms of actions of HDACIs on cardiac myocytes and reveal potential signaling pathways that may be targeted therapeutically.

2012-01-01

66

Cell hypertrophy and MEK/ERK phosphorylation are regulated by glyceraldehyde-derived AGEs in cardiomyocyte H9c2 cells.  

PubMed

Diabetic cardiomyopathy has been shown to promote hypertrophy, leading to heart failure. Recent studies have reported a correlation between diabetic cardiomyopathy and oxidative stress, suggesting that the accumulation of advanced glycation end products (AGEs) induces the production of reactive oxygen species (ROS). In a clinical setting, AGEs have been shown to increase the risk of cardiovascular disease; however, the relationship between AGEs and cardiac hypertrophy remains unclear. This study sought to identify the role of AGEs in cardiac hypertrophy by treating H9c2 cells with glyceraldehyde-derived AGEs (200 ?g/ml) or H2O2 (50 ?M) for 96 h. Our results demonstrate that AGEs significantly increased protein levels and cell size. These effects were effectively blocked with PD98059 (10 ?M; MEK/ERK inhibitor) pretreatment, suggesting that AGEs caused cell hypertrophy via the MEK/ERK pathway. We then treated cells with AGEs and H2O2 for 0-120 min and employed the Odyssey infrared imaging system to detect MEK/ERK phosphorylation. Our results show that AGEs up-regulated MEK/ERK phosphorylation. However, this effect was blocked by NAC (5 mM; ROS inhibitor), indicating that AGEs regulate MEK/ERK phosphorylation via ROS. Our findings suggest that glyceraldehyde-derived AGEs are closely related to cardiac hypertrophy and further identify a molecular mechanism underlying the promotion of diabetic cardiomyopathy by AGEs. PMID:23288619

Ko, Shun-Yao; Lin, I-Hsuan; Shieh, Tzong-Ming; Ko, Hsin-An; Chen, Hong-I; Chi, Tzong-Cherng; Chang, Shu-Shing; Hsu, Yi-Chiang

2013-07-01

67

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

PubMed Central

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

Jin, Hyou-Ju; Li, Chun-Guang

2013-01-01

68

S -Propargyl-cysteine (SPRC) attenuated lipopolysaccharide-induced inflammatory response in H9c2 cells involved in a hydrogen sulfide-dependent mechanism  

Microsoft Academic Search

The present study attempts to investigate the effects of S-propargyl-cysteine (SPRC), a sulfur-containing amino acid, on lipopolysaccharide (LPS)-induced inflammatory response in H9c2\\u000a cardiac myocytes. We found that SPRC prevented nuclear factor-?B (NF-?B) activation assessed by NF-?B p65 phosphorylation\\u000a and I?B? degradation, suppressed LPS-induced extracellular signal-regulated kinase 1\\/2 (ERK1\\/2) phosphorylation and intracellular\\u000a reactive oxygen species (ROS) production. Furthermore, incubation of H9c2

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

2011-01-01

69

Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions.  

PubMed

The mitochondrial DNA base modification 8-hydroxy 2'-deoxyguanine (8-OHdG) is one of the most common DNA lesions induced by reactive oxygen species (ROS) and is considered an index of DNA damage. High levels of mitochondrial 8-OHdG have been correlated with increased mutation, deletion, and loss of mitochondrial (mt) DNA, as well as apoptosis. 8-Oxoguanosine DNA glycosylase-1 (OGG1) recognizes and removes 8-OHdG to prevent further DNA damage. We evaluated the effects of OGG1 on mtDNA damage, mitochondrial function, and apoptotic events induced by oxidative stress using H9C2 cardiac cells treated with menadione and transduced with either Adv-Ogg1 or Adv-Control (empty vector). The levels of mtDNA 8-OHdG and the presence of apurinic/apyrimidinic (AP) sites were decreased by 30% and 35%, respectively, in Adv-Ogg1 transduced cells (P < 0.0001 and P < 0.005, respectively). In addition, the expression of base excision repair (BER) pathway members APE1 and DNA polymerase ? was upregulated by Adv-Ogg1 transduction. Cells overexpressing Ogg1 had increased membrane potential (P < 0.05) and decreased mitochondrial fragmentation (P < 0.005). The mtDNA content was found to be higher in cells with increased OGG1 (P < 0.005). The protein levels of fission and apoptotic factors such as DRP1, FIS1, cytoplasmic cytochrome c, activated caspase-3, and activated caspase-9 were lower in Adv-Ogg1 transduced cells. These observations suggest that Ogg1 overexpression may be an important mechanism to protect cardiac cells against oxidative stress damage. PMID:24304833

Torres-Gonzalez, Moises; Gawlowski, Thomas; Kocalis, Heidi; Scott, Brian T; Dillmann, Wolfgang H

2014-02-01

70

Ursolic Acid-enriched herba cynomorii extract induces mitochondrial uncoupling and glutathione redox cycling through mitochondrial reactive oxygen species generation: protection against menadione cytotoxicity in h9c2 cells.  

PubMed

Herba Cynomorii (Cynomorium songaricum Rupr., Cynomoriaceae) is one of the most commonly used 'Yang-invigorating' tonic herbs in Traditional Chinese Medicine (TCM). An earlier study in our laboratory has demonstrated that HCY2, an ursolic acid-enriched fraction derived from Herba Cynomorii, increased mitochondrial ATP generation capacity (ATP-GC) and induced mitochondrial uncoupling as well as a cellular glutathione response, thereby protecting against oxidant injury in H9c2 cells. In this study, we demonstrated that pre-incubation of H9c2 cells with HCY2 increased mitochondrial reactive oxygen species (ROS) generation in these cells, which is likely an event secondary to the stimulation of the mitochondrial electron transport chain. The suppression of mitochondrial ROS by the antioxidant dimethylthiourea abrogated the HCY2-induced enhancement of mitochondrial uncoupling and glutathione reductase (GR)-mediated glutathione redox cycling, and also protected against menadione-induced cytotoxicity. Studies using specific inhibitors of uncoupling protein and GR suggested that the HCY2-induced mitochondrial uncoupling and glutathione redox cycling play a determining role in the cytoprotection against menadione-induced oxidant injury in H9c2 cells. Experimental evidence obtained thus far supports the causal role of HCY2-induced mitochondrial ROS production in eliciting mitochondrial uncoupling and glutathione antioxidant responses, which offer cytoprotection against oxidant injury in H9c2 cells. PMID:24473214

Chen, Jihang; Wong, Hoi Shan; Ko, Kam Ming

2014-01-01

71

Transient and sustained oxidative stress differentially activate the JNK1\\/2 pathway and apoptotic phenotype in H9c2 cells  

Microsoft Academic Search

The aim of this study was to investigate the activation of JNK1\\/2 signalling pathway and the respective cellular phenotype\\u000a of H9c2 cardiac myoblasts during two distinct types of oxidative insult. We examined the dose- and time-dependent activation\\u000a of JNK1\\/2 pathway by exogenous H2O2, both under transient and sustained stimulation. At 2 h of either sustained or transient treatment, maximal phosphorylation\\u000a of

Anastasia Pechtelidou; Isidoros Beis; Catherine Gaitanaki

2008-01-01

72

Inhibitory effects of rosmarinic acid on adriamycin-induced apoptosis in H9c2 cardiac muscle cells by inhibiting reactive oxygen species and the activations of c-Jun N-terminal kinase and extracellular signal-regulated kinase  

Microsoft Academic Search

Rosmarinic acid (RA) is a naturally occurring polyphenolic and is found in several herbs in the Lamiaceae family, such as, Perilla frutescens. ADR is a potent anti-tumor drug, but is unfortunately potently cardiotoxic. This study was undertaken to investigate the inhibitory effect of RA on ADR-induced apoptosis in H9c2 cardiac muscle cells at a mechanistic level. In vitro, ADR significantly

Do-Sung Kim; Hyung-Ryong Kim; Eun-Rhan Woo; Seong-Tshool Hong; Han-Jung Chae; Soo-Wan Chae

2005-01-01

73

Schisandrin B-induced increase in cellular glutathione level and protection against oxidant injury are mediated by the enhancement of glutathione synthesis and regeneration in AML12 and H9c2 cells.  

PubMed

To define the relative role of reduced glutathione (GSH) synthesis and regeneration in schisandrin B (Sch B)-induced increase in cellular GSH level and the associated cytoprotection against oxidative challenge, the effects of L-buthionine-[S,R]-sulfoximine (BSO, a specific inhibitor of gamma-glutamate cysteine ligase (GCL)) and 1,3-bis(2-chloroethyl)-1-nitrourea (BCNU, a specific inhibitor of glutathione reductase (GR)) treatments or their combined treatment were examined in control and Sch B-treated AML12 and H9c2 cells, without and/or with menadione intoxication. Both BSO and BCNU treatments reduced cellular GSH level in AML12 and H9c2 cells, with the effect of BSO being more prominent. The GSH-enhancing effect of Sch B was also suppressed by BSO and BCNU treatments, with the effect of the combined treatment with BSO and BCNU being semi-additive. While Sch B treatment increased the GR but not GCL activity in AML12 and H9c2 cells, it increased the cellular cysteine level. BSO treatment also suppressed the Sch B-induced increase in GR activity. BSO or BCNU treatment per se did not cause any detectable cytotoxic effect, as assessed by lactate dehydrogenase leakage, but the combined treatment with BSO and BCNU was cytotoxic, particularly in H9c2 cells. The cytotoxic effect of BSO and BCNU became more apparent following the menadione challenge. The cytoprotection afforded by Sch B pretreatment was partly suppressed by BSO or BCNU treatment, or completely abrogated by the combined treatment with BSO and BCNU. In conclusion, the results indicate that the cytoprotective action of Sch B is causally related to the increase in cellular GSH level, which is likely mediated by the enhancement of GSH synthesis and regeneration. PMID:17119269

Chiu, Po Yee; Ko, Kam Ming

2006-01-01

74

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

PubMed

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

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

2014-07-18

75

Quercetin Inhibits Left Ventricular Hypertrophy in Spontaneously Hypertensive Rats and Inhibits Angiotensin II-Induced H9C2 Cells Hypertrophy by Enhancing PPAR-? Expression and Suppressing AP-1 Activity  

PubMed Central

Background Quercetin is the most abundant flavonoid in fruit and vegetables and is believed to attenuate cardiovascular disease. We hypothesized that quercetin inhibits cardiac hypertrophy by blocking AP-1 (c-fos, c-jun) and activating PPAR-? signaling pathways. Methodology/Principal Findings The aim of this study was to identify the mechanism underlying quercetin-mediated attenuation of cardiac hypertrophy. Quercetin therapy reduced blood pressure and markedly reduced the ratio of left ventricular to body weight (LVW/BW) (P<0.05, vs. spontaneously hypertensive rats (SHRs)). In vitro, quercetin also significantly attenuated Ang II-induced H9C2 cells hypertrophy, as indicated by its concentration dependent inhibitory effects on [3H]leucine incorporation into H9C2 cells (64% reduction) and by the reduced hypertrophic surface area in H9C2 cells compared with the Ang II group (P<0.01, vs. Ang II group). Concurrently, we found that PPAR-? activity was significantly increased in the quercetin-treated group both in vivo and in vitro when analyzed using immunofluorescent or immunohistochemical assays (P<0.05, vs. SHRs or P<0.01, vs. the Ang II group). Conversely, in vivo, AP-1 (c-fos, s-jun) activation was suppressed in the quercetin-treated group, as was the downstream hypertrophy gene, including mRNA levels of ANP and BNP (P<0.05, vs. SHRs). Additionally, both western blotting and real time-PCR demonstrated that PPAR-? protein and mRNA were increased in the myocardium and AP-1 protein and mRNA were significantly decreased in the quercetin-treated group (P<0.05, vs. SHRs). Furthermore, western blotting and real time-PCR analyses also showed that transfection with PPAR-? siRNA significantly increased AP-1 signaling and reversed the effects of quercetin inhibition on mRNA expression levels of genes such as ANP and BNP in hypertrophic H9C2 cells. Conclusions Our data indicate that quercetin may inhibit cardiac hypertrophy by enhancing PPAR-? expression and by suppressing the AP-1 signaling pathway.

Yan, Lei; Zhang, Ji Dong; Wang, Bo; Lv, Yi Jing; Jiang, Hong; Liu, Gui Lin; Qiao, Yun; Ren, Ming; Guo, Xue Feng

2013-01-01

76

Coxsackievirus B3-induced calpain activation facilitates the progeny virus replication via a likely mechanism related with both autophagy enhancement and apoptosis inhibition in the early phase of infection: an in vitro study in H9c2 cells.  

PubMed

Calpain is a family of neutral cysteine proteinase involved in many physiological and pathological processes including virus replication, autophagy and apoptosis. Previous study has indicated the involvement of calpain in pathogenesis of coxsackievirus B3 (CVB3)-induced myocarditis. Besides, many studies demonstrated that host cell autophagy and apoptosis mechanisms participate in virus life cycle. However, role of calpain in CVB3 replication via autophagy/apoptosis mechanisms has not been reported, which was discussed here in H9c2 cardiomyocytes. The data demonstrated that calpain was activated following CVB3 infection. Calpain inhibition decreased autophagy, indicating role of calpain in enhancing autophagy during CVB3 infection. Both calpain activity and autophagy were involved in facilitating CVB3 replication demonstrated by virus titer and CVB3 capsid protein VP1 expression alterations resulting from calpain inhibitor ALLN and autophagy inhibitor 3MA intervention. We also found that both calpain activity and autophagy suppressed caspase3 activity and host cell apoptosis 5-10h post-infection (p.i.). In summary, the present study shows that CVB3 infection of H9c2 cells hinders caspase3 activity provocation and cell apoptosis at least in the early phase of infection (5-10h p.i.) via calpain-induced autophagy enhancement, which might be a mechanism facilitating CVB3 replication in host cells. PMID:24177271

Li, Minghui; Wang, Xinggang; Yu, Yong; Yu, Ying; Xie, Yeqing; Zou, Yunzeng; Ge, Junbo; Peng, Tianqing; Chen, Ruizhen

2014-01-22

77

Hydrogen gas protects against serum and glucose deprivation?induced myocardial injury in H9c2 cells through activation of the NF?E2?related factor 2/heme oxygenase 1 signaling pathway.  

PubMed

Ischemia or hypoxia?induced myocardial injury is closely associated with oxidative stress. Scavenging free radicals and/or enhancing endogenous antioxidative defense systems may be beneficial for the impediment of myocardial ischemic injury. Hydrogen (H2) gas, as a water? and lipid?soluble small molecule, is not only able to selectively eliminate hydroxyl (·OH) free radicals, but also to enhance endogenous antioxidative defense systems in rat lungs and arabidopsis plants. However, thus far, it has remained elusive whether H2 gas protects cardiomyocytes through enhancement of endogenous antioxidative defense systems. In the present study, the cardioprotective effect of H2 gas against ischemic or hypoxic injury was investigated, along with the underlying molecular mechanisms. H9c2 cardiomyoblasts (H9c2 cells) were treated in vitro with a chemical hypoxia inducer, cobalt chloride (CoCl2), to imitate hypoxia, or by serum and glucose deprivation (SGD) to imitate ischemia. Cell viability and intracellular ·OH free radicals were assessed. The role of an endogenous antioxidative defense system, the NF?E2?related factor 2 (Nrf2)/heme oxygenase 1 (HO?1) signaling pathway, was evaluated. The findings revealed that treatment with CoCl2 or SGD markedly reduced cell viability in H9c2 cells. H2 gas?rich medium protected against cell injury induced by SGD, but not that induced by CoCl2. When the cells were exposed to SGD, levels of intracellular ·OH free radicals were markedly increased; this was mitigated by H2 gas?rich medium. Exposure of the cells to SGD also resulted in significant increases in HO?1 expression and nuclear Nrf2 levels, and the HO?1 inhibitor ZnPP IX and the Nrf2 inhibitor brusatol aggravated SGD?induced cellular injury. H2 gas?rich medium enhanced SGD?induced upregulation of HO?1 and Nrf2, and the HO?1 or Nrf2 inhibition partially suppressed H2 gas?induced cardioprotection. Furthermore, following genetic silencing of Nrf2 by RNA interference, the effects of H2 gas on the induction of HO?1 and cardioprotection were markedly reduced. In conclusion, H2 gas protected cardiomyocytes from ischemia?induced myocardial injury through elimination of ·OH free radicals and also through activation of the Nrf2/HO?1 signaling pathway. PMID:24890947

Xie, Qiang; Li, Xue-Xiang; Zhang, Peng; Li, Jin-Cao; Cheng, Ying; Feng, Yan-Ling; Huang, Bing-Sheng; Zhuo, Yu-Feng; Xu, Guo-Hua

2014-08-01

78

c-Src-dependent MAPKs/AP-1 activation is involved in TNF-?-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells.  

PubMed

TNF-? plays a critical mediator in the pathogenesis of chronic heart failure contributing to cardiac remodeling and peripheral vascular disturbances. The implication of TNF-? in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of TNF-?-induced MMP-9 expression are largely unclear in the heart cells. Here, we demonstrated that in rat embryonic-heart derived H9c2 cells, TNF-? could induce MMP-9 mRNA expression associated with an increase in the secretion of MMP-9, determined by real-time PCR, zymography, and promoter activity assays. TNF-?-mediated responses were attenuated by pretreatment with the inhibitor of c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), MEK1/2 (U0126), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) and transfection with siRNA of c-Src, EGFR, PDGFR, p110, Akt, or c-Jun. TNF-? stimulated c-Src, PDGFR, and EGFR phosphorylation, which were reduced by PP1. In addition, TNF-?-stimulated Akt phosphorylation was inhibited by PP1, AG1478, AG1296, or LY294002. We further demonstrated that TNF-? markedly stimulated p38 MAPK, p42/p44 MAPK, and JNK1/2 phosphorylation via a c-Src/EGFR, PDGFR/PI3K/Akt pathway. Finally, we showed that, in H9c2 cells, TNF-?-stimulated AP-1 promoter activity, c-Jun mRNA expression, and c-Jun phosphorylation were attenuated by PP1, AG1478, AG1296, LY294002, SB202190, SP600125, or U0126. These results suggested that TNF-?-induced MMP-9 expression is mediated through a c-Src/EGFR, PDGFR/PI3K/Akt/MAPKs/AP-1 cascade in H9c2 cells. Consequently, MMP-9 induction may contribute to cell migration and cardiovascular inflammation. PMID:23353699

Yang, Chuen-Mao; Lee, I-Ta; Lin, Chih-Chung; Wang, Chao-Hung; Cherng, Wen-Jin; Hsiao, Li-Der

2013-04-15

79

Boerhaavia diffusa L. attenuates angiotensin II-induced hypertrophy in H9c2 cardiac myoblast cells via modulating oxidative stress and down-regulating NF-?? and transforming growth factor ?1.  

PubMed

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

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

2013-10-01

80

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

PubMed Central

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

Valentine, Cathleen D.; Haggie, Peter M.

2011-01-01

81

Ursolic-Acid-Enriched Herba Cynomorii Extract Protects against Oxidant Injury in H9c2 Cells and Rat Myocardium by Increasing Mitochondrial ATP Generation Capacity and Enhancing Cellular Glutathione Redox Cycling, Possibly through Mitochondrial Uncoupling  

PubMed Central

Mitochondrial decay is considered to be a major contributor to aging-related diseases, including neurodegenerative diseases, cardiovascular disorders, and certain metabolic diseases. Therefore, the maintenance of mitochondrial functional capacity and antioxidant status should play an essential role in preventive health. Herba Cynomorii, which is one of the most potent “Yang-invigorating” Chinese tonic herbs, was found to increase mitochondrial ATP generation capacity (ATP-GC) in rat hearts ex vivo. In the present study, we demonstrated that HCY2, an active fraction of Herba Cynomorii, and its major ingredient ursolic acid (UA) could protect against hypoxia/reoxygenation-induced cell apoptosis in H9c2 cells in vitro and also against ischemia/reperfusion-induced injury in rat hearts ex vivo. The cardioprotection was associated with an increase in ATP-GC and an enhancement of glutathione redox cycling. The results suggest that UA may be one of the active ingredients responsible for the cardioprotection afforded by Herba Cynomorii, and this effect may be mediated, at least in part, by enhancement of mitochondrial functional capacity and antioxidant status, possibly through the induction of mitochondrial uncoupling.

Ko, Kam Ming

2013-01-01

82

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

PubMed

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

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

2013-12-01

83

Cardiac Shock Wave Therapy Attenuates H9c2 Myoblast Apoptosis by Activating the AKT Signal Pathway.  

PubMed

Background: Previous studies have demonstrated that Cardiac Shock Wave Therapy (CSWT) improves myocardial perfusion and cardiac function in a porcine model of chronic myocardial ischemia and also ameliorates myocardial ischemia in patients with severe coronary artery disease (CAD). Apoptosis plays a key role in ischemic myocardial pathogenesis. However, it remains unclear whether CSWT is beneficial for ischemia/hypoxia (I/H)-induced myocardial cell apoptosis and by which mechanism CSWT could improve heart function. We put forward the hypothesis that CSWT might protect heart function during ischemia/hypoxia by decreasing apoptosis. Methods: We generated ischemia/hypoxia (I/H)-induced apoptosis in the H9c2 myoblast cell line to examine the CSWT function and possible mechanisms. H9c2 cells were treated under hypoxic serum-starved conditions for 24 h and then treated with or without CSWT (500 shots, 0.06, 0.09, 0.12mJ/mm(2)). The apoptotic cell rate was determined by flow cytometry assay, cell viability was examined by the MTT assay, nuclear fragmentation was detected by Hoechst 33342 staining, and the mitochondrial-mediated intrinsic pathway of apoptosis was assessed by the expression of Bax and Bcl-2 protein and Caspase3 activation. Results: First, apoptosis could be induced by ischemia/hypoxia in H9c2 cells. Second, CSWT attenuates the cell death and decreases the H9c2 cell apoptosis rate induced by ischemia and hypoxia. Third, CSWT suppresses the expression of apoptosis molecules that regulate the intrinsic pathway of apoptosis in H9c2 cells. Fourth, CSWT increases the phosphorylation of AKT, which indicates the activation of the PI3K-AKT pathway. Conclusions: These results indicate that CSWT exerts a protective effect against I/H-induced cell death, potentially by preventing the activation of components of the mitochondrial-dependent intrinsic apoptotic pathway. We also demonstrate that the PI3K-Akt pathway may be involved in the CSWT effects on apoptosis. © 2014 S. Karger AG, Basel. PMID:24802592

Yu, Weiwei; Shen, Tao; Liu, Baoyi; Wang, Shu; Li, Jian; Dai, Dapeng; Cai, Jianping; He, Qing

2014-01-01

84

Metabolic Remodeling During H9c2 Myoblast Differentiation: Relevance for In Vitro Toxicity Studies  

Microsoft Academic Search

H9c2 cells, derived from the ventricular part of an E13 BDIX rat heart, possess a proliferative and relatively undifferentiated\\u000a phenotype but can be readily directed to differentiate under reduced serum conditions originating cells presenting muscle\\u000a features. Skeletal or cardiac phenotypes can be originated depending on whether or not serum reduction is accompanied by a\\u000a daily treatment with all-trans-retinoic acid. In

Sandro L. Pereira; João Ramalho-Santos; Ana F. Branco; Vilma A. Sardão; Paulo J. Oliveira; Rui A. Carvalho

2011-01-01

85

Pro-survival effect of Dock180 overexpression on rat-derived H9C2 cardiomyocytes  

PubMed Central

Background Integrin ?1 subunit and its downstream molecule, focal adhesion kinase (FAK), have been demonstrated to be indispensible to the promotion of cell proliferation and survival and anti-apoptosis in cardiomyocytes via activation of their downstream pro-survival signaling molecule, AKT. As a component of the integrin pathway, Dock180 (dedicator of cytokinesis 1) protein is also thought to be involved in the promotion of cell proliferation and survival and anti-apoptosis in the H9C2 cardiomyocytes. Material/Methods Rat-derived H9C2 cardiomyocytes were transfected with pCXN2-flag-hDock180, a human Dock180 overexpression eukaryotic recombinant plasmid. The rat and human Dock180 mRNA and protein expression, apoptosis and cell proliferation and survival were analyzed in the H9C2 cardiomyocytes treated with either hypoxia/reoxygenation (H/R) or not, respectively. Results Human Dock180 mRNA overexpression could significantly increase the Dock180 protein expression in the H9C2 cardiomyocytes, no matter whether treated with H/R or not. Dock180 overexpression could promote the cell proliferation and survival and anti-apoptosis, and relieve the cell proliferative and survival inhibition and apoptosis induced by H/R in the H9C2 cardiomyocytes via activation of its downstream pro-survival signaling molecule AKT. Conclusions Dock180 could act as a pro-survival molecule in H9C2 cardiomyocytes via activation of its downstream pro-survival signaling molecule, AKT.

Yan, An; Li, Gang; Zhang, Xu; Zhu, Bingbao; Linghu, Hua

2013-01-01

86

Protective effect of components isolated from Lindera erythrocarpa against oxidative stress-induced apoptosis of H9c2 cardiomyocytes.  

PubMed

Eight compounds were isolated from the methanol fraction of Lindera erythrocarpa and assessed for their ability to protect H9c2 cardiomyocytes against oxidative stress-induced cell death. Three of the compounds significantly reduced the release of lactate dehydrogenase from H9c2 cardiomyocytes treated with buthionine-[S,R]-sulfoximine and reduced the uptake of propidium iodide by these cells. These effects were concentration-dependent. The three inhibitory compounds were identified as (-)-epicatechin, avicularin and quercitrin by spectroscopic techniques including one- and two-dimensional NMR and mass spectroscopy. PMID:21412863

Kim, Jeong Ah; Jung, Yi-Sook; Kim, Mi-Young; Yang, Seo Young; Lee, Sohyun; Kim, Young Ho

2011-11-01

87

Assessment of reference genes for real-time quantitative PCR gene expression normalization during C2C12 and H9c2 skeletal muscle differentiation.  

PubMed

Skeletal muscle differentiation occurs during muscle development and regeneration. To initiate and maintain the differentiated state, a multitude of gene expression changes occur. Accurate assessment of these differentiation-related gene expression changes requires good quality template, but more specifically, appropriate internal controls for normalization. Two cell line-based models used for in vitro analyses of muscle differentiation incorporate mouse C2C12 and rat H9c2 cells. In this study, we set out to identify the most appropriate controls for mRNA expression normalization during C2C12 and H9c2 differentiation. We assessed the expression profiles of Actb, Gapdh, Hprt, Rps12 and Tbp during C2C12 differentiation and of Gapdh and Rps12 during H9c2 differentiation. Using NormFinder, we validated the stability of the genes individually and of the geometric mean generated from different gene combinations. We verified our results using Myogenin. Our study demonstrates that using the geometric mean of a combination of specific reference genes for normalization provides a platform for more precise test gene expression assessment during myoblast differentiation than using the absolute expression value of an individual gene and reinforces the necessity of reference gene validation. PMID:24146429

Masilamani, Twinkle J; Loiselle, Julie J; Sutherland, Leslie C

2014-04-01

88

Effects of trichloroethylene and its metabolite trichloroacetic acid on the expression of vimentin in the rat H9c2 cell line  

Microsoft Academic Search

Trichloroethylene (TCE) and its metabolite trichloroacetic acid (TCAA) are environmental contaminants with specific toxicity for the embryonic heart. In an effort to identify the cellular pathways disrupted by TCE and TCAA during heart development, we investigated their effects on expression of vimentin, a marker of cardiac differentiation. Previous studies had shown that the level of vimentin transcript was inhibited in

O. Selmin; P. A. Thorne; P. T. Caldwell; P. D. Johnson; R. B. Runyan

2005-01-01

89

Quantitative proteomic study identified cathepsin B associated with doxorubicin-induced damage in H9c2 cardiomyocytes.  

PubMed

The study was performed to analyze the proteomic profiling of doxorubicin-treated H9c2 cardiomyocytes in order to identify novel protein biomarkers associated with doxorubicin-induced cardiomyopathy. The protein profiling of H9c2 cells in response to doxorubicin at an apoptosis-induced concentration of 0.5 ?M were compared using iTRAQ analysis. Western-blot analysis was used to confirm differentially expressed proteins identified in the proteomic study. A total of 22 differently expressed proteins were identified in doxorubicin-treated H9c2 cells including 15 up-regulated and 7 down-regulated proteins. Gene Ontology (GO) analysis revealed that 10 altered proteins were enriched in the process of apoptosis. We further validated the expression of cathepsin B and its possible regulator nuclear factor kappa B (NF-?B) in H9c2 cells were increased during doxorubicin treatment using Western-blots. Differentially expressed proteins might provide clues to clarify novel mechanisms underlying doxorubicin-induced cardiomyopathy. Our results also suggest that increased cathepsin B expression might be associated with NF-?B up-regulation, and the exact mechanisms need to be clarified. PMID:23337787

Bao, G Y; Wang, H Z; Shang, Y J; Fan, H J; Gu, M L; Xia, R; Qin, Q; Deng, A M

2012-12-01

90

Curcumin Suppresses Gelatinase B Mediated Norepinephrine Induced Stress in H9c2 Cardiomyocytes  

PubMed Central

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

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

2013-01-01

91

Phosphatidylinositol 3Kinase Regulates Differentiation of H9c2 Cardiomyoblasts Mainly through the Protein Kinase B\\/Akt-Independent Pathway  

Microsoft Academic Search

Phosphatidylinositol 3-kinase (PI3-kinase) is known to be a crucial regulator of muscle differentiation. However, its downstream pathway for this function is quite obscure. In this experiment we demonstrated the regulatory mechanism of the differentiation of H9c2 cardiomyoblasts, focusing on PI3-kinase, protein kinase B\\/Akt (PKB\\/Akt) and p42\\/44 mitogen-activated protein kinase (p42\\/44 MAPK). When H9c2 cells stably transfected with a constitutively active

Joung Mok Kim; Moon-young Yoon; Jayoung Kim; Sam Soo Kim; Insug Kang; Joohun Ha; Sung Soo Kim

1999-01-01

92

Signal Transduction of Arginine Vasopressin-Induced Arachidonic Acid Release in H9c2 Cardiac Myoblasts: Role of Ca2+ and the Protein Kinase C-Dependent Activation of p42 Mitogen-Activated Protein Kinase  

Microsoft Academic Search

The mechanism of arginine vasopressin (AVP)-induced arachi- donic acid (AA) release was examined in the cardiac myoblast cell line, H9c2. Stimulation of cells with AVP induced dose-dependent AA release, and this effect was completely inhibited by the V1 receptor antagonist, d(CH)5(Tyr(Me) 2 )AVP. AVP also produced dose-depen- dent stimulation of inositol phosphate formation; this was not affected by pertussis toxin,

WEI-CHYUAN CHEN; CHING-CHOW CHEN

1999-01-01

93

Tetrodotoxin attenuates isoproterenol-induced hypertrophy in H9c2 rat cardiac myocytes.  

PubMed

Cardiac hypertrophy is often associated with an increased sympathetic drive, and both in vitro and in vivo studies have demonstrated the development of cardiomyocyte hypertrophy in response to either ?- or ?- adrenergic stimulation. The present study was carried out to determine whether the reversible sodium channel blocker tetrodotoxin (TTX) exerts a direct anti-hypertrophic effect on isoproterenol (ISO)-induced cell hypertrophy and find the underlying mechanism that regulate [Na(+)]( i ). The experiments were performed on cultured H9c2 cells exposed to ISO (10 ?M) alone or combined with TTX (1 ?M) for 48 h. Our results showed that ISO significantly increased cell surface area by 30 % and atrial natriuretic peptide gene expression by nearly twofold (p < 0.05 for both). These effects were associated with a significant reduction in the gene expression of Na(+)/K(+)-ATPase isoforms ?2 and ?3, whereas the ?1 isoform was unaffected. Conversely, ISO increased Na(+)-H(+) exchanger 1 (NHE-1) gene expression by approximately 40 % and significantly increased [Na(+)]( i ) level by 50 % (p < 0.05 for both). ISO was also found to significantly increase aquaporin 4 gene expression by nearly ninefold (p < 0.05). All these effects were prevented when identical experiments were carried out in the presence of TTX, but the expression of NHE-1. The expression of sodium channel protein type 5 subunit alpha was unaffected by either ISO or TTX. When taken together, these studies show that TTX attenuates the hypertrophic effect of ISO and suggest a possible approach to limiting ISO-induced hypertrophy in clinical treatment. PMID:22941212

Chen, Ming-Zi; Bu, Qing-Ting; Pang, Shu-Chao; Li, Feng-Lan; Sun, Mei-Na; Chu, Er-Fu; Li, Hui

2012-12-01

94

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

PubMed Central

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

ZHOU, HENG; YUAN, YUAN; LIU, YUAN; DENG, WEI; ZONG, JING; BIAN, ZHOU-YAN; DAI, JIA; TANG, QI-ZHU

2014-01-01

95

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

PubMed

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

Zhang, Fangbo; Huang, Bin; Zhao, Ye; Tang, Shihuan; Xu, Haiyu; Wang, Lan; Liang, Rixin; Yang, Hongjun

2013-01-01

96

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

PubMed

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

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

2013-09-01

97

Molecular identification for epigallocatechin-3-gallate-mediated antioxidant intervention on the H2O2-induced oxidative stress in H9c2 rat cardiomyoblasts  

PubMed Central

Background Epigallocatechin-3-gallate (EGCG) has been documented for its beneficial effects protecting oxidative stress to cardiac cells. Previously, we have shown the EGCG-mediated cardiac protection by attenuating reactive oxygen species and cytosolic Ca2+ in cardiac cells during oxidative stress and myocardial ischemia. Here, we aimed to seek a deeper elucidation of the molecular anti-oxidative capabilities of EGCG in an H2O2-induced oxidative stress model of myocardial ischemia injury using H9c2 rat cardiomyoblasts. Results Proteomics analysis was used to determine the differential expression of proteins in H9c2 cells cultured in the conditions of control, 400 ?M H2O2 exposure for 30 min with and/or without 10 to 20 ?M EGCG pre-treatment. In this model, eight proteins associated with energy metabolism, mitochondrial electron transfer, redox regulation, signal transduction, and RNA binding were identified to take part in EGCG-ameliorating H2O2-induced injury in H9c2 cells. H2O2 exposure increased oxidative stress evidenced by increases in reactive oxygen species and cytosolic Ca2+ overload, increases in glycolytic protein, ?-enolase, decreases in antioxidant protein, peroxiredoxin-4, as well as decreases in mitochondrial proteins, including aldehyde dehydrogenase-2, ornithine aminotransferase, and succinate dehydrogenase ubiquinone flavoprotein subunit. All of these effects were reversed by EGCG pre-treatment. In addition, EGCG attenuated the H2O2-induced increases of Type II inositol 3, 4-bisphosphate 4-phosphatase and relieved its subsequent inhibition of the downstream signalling for Akt and glycogen synthase kinase-3? (GSK-3?)/cyclin D1 in H9c2 cells. Pre-treatment with EGCG or GSK-3? inhibitor (SB 216763) significantly improved the H2O2-induced suppression on cell viability, phosphorylation of pAkt (S473) and pGSK-3? (S9), and level of cyclin D1 in cells. Conclusions Collectively, these findings suggest that EGCG blunts the H2O2-induced oxidative effect on the Akt activity through the modulation of PIP3 synthesis leading to the subsequent inactivation of GSK-3? mediated cardiac cell injury.

2014-01-01

98

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

PubMed

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

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

2013-04-30

99

P132NFAT3/GATA4 facilitates the NHE1 mediated induction of osteopontin in H9c2 cardiomyoblast.  

PubMed

Osteopontin (OPN) is a secreted protein involved in cardiac remodeling and hypertrophy induced by the expression of angiotensin 2 (ANG II). Transgenic mice lacking OPN in the heart prevent cardiac hypertrophy and fibrosis mediated by ANG II. ANG II is an activator of the Na+/H+ exchanger isoform 1 (NHE1), a plasma membrane protein that regulates intracellular pH and also implicated in cardiac hypertrophy. Interestingly, transgenic mice expressing active NHE1 developed spontaneous cardiac hypertrophy in association with elevated levels of osteopontin. Whether ANG II induces osteopontin expression via activation of NHE1 remains unknown. ANG II (100 nM, 16h) induces cardiomyocyte hypertrophy associated with an increased OPN protein expression ( ANG II: 138% ±30%, p<0.05). ANG II stimulated cardiomyocyte hypertrophy and OPN protein expression regressed in the presence of the NHE1 inhibitor, EMD (10?M) and calcineurin inhibitor, FK506 (1?M). H9c2 cells infected with the active form of NHE1 showed that NHE1 activation is enough to induce the hypertrophic pathway NFAT3/GATA4 and OPN expression. These effects were prevent by FK506. Our results demonstrate that NHE1 activation, either using an active NHE1 or through induction with ANG II, induces NFAT3 translocation into the nucleus and also demonstrate a significant activation of the transcription factor GATA-4 (NHE1: 149% ±28%, p<0.05). The activation of GATA-4 and OPN protein expression induced by active NHE1 is inhibited by FK506 treatment. These results suggest that NFAT3/GATA4 pathway is a critical component of the NHE1 hypertrophic pathways activated by ANG II in cardiomyoblast and plays a key role in the regulation of cardiomyocyte hypertrophy and OPN protein expression. PMID:25020553

Mlih, M; Abdulrahman, N; Mraiche, F

2014-07-15

100

Glutathione-S-transferase overexpression protects against anthracycline-induced H9C2 cell death  

Microsoft Academic Search

Abstract Anthracyclines (AC) are anti-tumor antibiotics with significant activity against solid and hematologic malignancies. One problem preventing more widespread use has been the development,of cardiac toxicity. Experimental evidence supports oxidant stress as an important trigger and\\/or mediator of AC-induced cardiotoxicity (ACT). Therefore, reducing oxidant stress should be protective against ACT. To determine whether antioxidant protein overexpression can reduce ACT, we

Thomas L'Ecuyer

2004-01-01

101

Hexarelin protects H9c2 cardiomyocytes from doxorubicin-induced cell death  

Microsoft Academic Search

Growth hormone secretagogues (GHSs) are synthetic peptidyl and nonpeptidyl molecules that possess strong growth hormone-releasing\\u000a activity acting on specific pituitary and hypothalamic receptor subtypes. Differently from nonpeptidyl GHSs, peptidyl molecules\\u000a such as hexarelin, a hexapeptide, possess specific high-affinity binding sites in animal and human heart and, after prolonged\\u000a treatment, protect rats in vivo from ischemiainduced myocardial damage. To verify the

Nicoletta Filigheddu; Alberto Fubini; Gianluca Baldanzi; Santina Cutrupi; Corrado Ghè; Filomena Catapano; Fabio Broglio; Amalia Bosia; Mauro Papotti; Giampiero Muccioli; Ezio Ghigo; Romano Deghenghi; Andrea Graziani

2001-01-01

102

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

PubMed

Curcumin derived from the rhizome of turmeric (Curcuma longa L.), is a well known coloring culinary agent, that has therapeutic properties against diverse pathologies such as cancer, atherosclerosis and heart failure. Given the salutary potential of curcumin, deciphering its mode of action particularly in cardiac cells, is of outstanding value. Accumulating evidence implicates curcumin in the regulation of multiple signaling pathways leading to cell survival or apoptosis. Therefore, the present study aimed at elucidating the molecular mechanisms triggered by curcumin in H9c2 cells. Curcumin was found to activate p38-mitogen-activated protein kinase (p38-MAPK) as well as c-jun NH2 terminal kinases (JNKs), in a dose- and time-dependent manner. We also observed curcumin to impair cell survival by promoting apoptosis, evidenced by chromatin condensation, poly(ADP-ribose) polymerase (PARP) and caspase-3 cleavage, as well as Bax translocation and cytochrome c release into the cytosol. Curcumin-induced apoptosis was ascribed to JNKs, since hindering their activity abolished PARP fragmentation. Furthermore, we identified curcumin to exert a pro-oxidative activity, with 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining revealing up-regulation of reactive oxygen species (ROS) levels and anti-oxidants found to abrogate PARP cleavage. In conclusion, curcumin was found to stimulate the apoptotic cell death of H9c2 cells by upregulating ROS generation and triggering activation of JNKs. With reports underscoring the capacity of curcumin to perturb the cellular redox balance ensuring survival or enhancing apoptosis, determination of its mode of action appears to be critical. Future studies should assess the appropriate administration conditions of curcumin, so as to optimize its therapeutic potential against cardiovascular pathologies. PMID:24668280

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

2014-06-01

103

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

PubMed

Ca(+) influx to mitochondria is an important trigger for both mitochondrial dynamics and ATP generation in various cell types, including cardiac cells. Mitochondrial Ca(2+) influx is mainly mediated by the mitochondrial Ca(2+) uniporter (MCU). Growing evidence also indicates that mitochondrial Ca(2+) 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 Ca(2+) uptake pathway in cardiomyocytes. However, it is still unclear which mitochondrial Ca(2+) 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-induced ATP production in cardiac H9c2 myoblasts. PMID:24124188

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; Sheu, Shey-Shing

2013-12-01

104

Antiapoptotic effect of calcitonin gene-related peptide on oxidative stress-induced injury in H9c2 cardiomyocytes via the RAMP1/CRLR complex.  

PubMed

Calcitonin gene-related peptide (CGRP) plays an important role in the mediation of protective effects observed in situations such as ischemic preconditioning in rat hearts. In this study, we investigated in H9c2 rat cardiomyoblasts if the protective effect of CGRP could be linked to an inhibitory effect on the apoptotic pathway. We also determined the specificity of observed effects by treatment with adrenomedullin (ADM) in stress conditions generated by 100 microM hydrogen peroxide. Using MTT assays, we demonstrate that a pretreatment with CGRP decreases by half the loss of cell viability induced by H(2)O(2). CGRP inhibits phosphatidylserine externalization, caspase 3 activation and DNA fragmentation due to oxidative stress. Using RT-PCR, we observed an increase in Bcl-2 mRNA expression induced by CGRP treatment. Dot blotting experiments showed that, in stress conditions, Bcl-2 protein level decreases while Bax is increased. CGRP administration prior to stress prevents these effects. The three-receptor activity modifying protein (RAMP) isotypes were detected by RT-PCR in H9c2 cells and in left ventricle rat tissue, RAMP1 and RAMP3 being the most abundant in both cases. RAMP1 expression was upregulated by CGRP while RAMP3 mRNA level was decreased. Cell viability assessed by MTT indicates that, contrary to CGRP, pretreatment of stressed cells with ADM, a RAMP2 agonist, fails to protect them while treatment with CGRP(8-37) (a RAMP1 and 2 inhibitor) abolished CGRP protective effect. Taken together, these data suggest that CGRP has antiapoptotic properties through the RAMP1/CRLR complex. CGRP could be used to prevent apoptosis in an ischemia-reperfusion context. PMID:16242145

Sueur, Stéphanie; Pesant, Matthieu; Rochette, Luc; Connat, Jean-Louis

2005-12-01

105

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

PubMed

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

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

2014-05-01

106

Dipeptidyl petidase-IV inhibitor (gemigliptin) inhibits tunicamycin-induced endoplasmic reticulum stress, apoptosis and inflammation in H9c2 cardiomyocytes.  

PubMed

The direct effects of dipeptidyl peptidase-IV (DPP-IV) inhibitors on endoplasmic reticulum (ER) stress-induced apoptosis and inflammation in cardiomyocytes have not been elucidated. H9c2 cell viability, which was reduced by tunicamycin, was increased after DPP-IV inhibitor gemigliptin treatment. Gemigliptin significantly decreased the tunicamycin-mediated increase in glucose regulated protein 78 (GRP78) expression and ER stress-mediated signaling molecules such as protein kinase RNA-like endoplasmic reticulum kinase (PERK)/C-EBP homologous protein (CHOP) and inositol-requiring enzyme 1? (IRE1?)/c-Jun N-terminal kinase (JNK)-p38. Furthermore, gemigliptin effectively induced Akt phosphorylation in a dose-dependent manner. Using flow cytometry and Hoechst staining, we showed that treatment with Akt inhibitor significantly blocked the anti-apoptotic effects mediated by gemigliptin. The reduction in tunicamycin-induced GRP78 level and PERK/CHOP pathway activity by gemigliptin was reversed after treatment with Akt inhibitor. In conclusion, gemigliptin effectively inhibited ER stress-induced apoptosis and inflammation in cardiomyocytes via Akt/PERK/CHOP and IRE1?/JNK-p38 pathways, suggesting its direct protective role in cardiovascular diseases. PMID:24813659

Hwang, Hwan-Jin; Jung, Tae Woo; Ryu, Ja Young; Hong, Ho Cheol; Choi, Hae Yoon; Seo, Ji A; Kim, Sin Gon; Kim, Nan Hee; Choi, Kyung Mook; Choi, Dong Seop; Baik, Sei Hyun; Yoo, Hye Jin

2014-07-01

107

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

Microsoft Academic Search

Doxorubicin (DOX) is a widely used chemotherapy agent. The major adverse effect of DOX treatment in cancer patients is the onset of cardiomyopathy and heart failure. Reactive oxygen species (ROS) are proposed to be responsible for DOX cardiotoxicity. Curcumin, a natural compound extracted from Curcuma Longa L., is known for its anti-oxidant properties. It has been identified as increased apoptosis

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

2011-01-01

108

Trichloroethylene Induces Methylation of the Serca2 Promoter in H9c2 Cells and Embryonic Heart  

Microsoft Academic Search

Trichloroethylene (TCE) is a halogenated hydrocarbon used as a solvent in industrial settings and in house-cleaning products.\\u000a Exposure to TCE has been linked to increased risk for congenital heart malformations in both human and animal models. Previous\\u000a studies showed TCE exposure reduced the expression and function of the ATP-dependent calcium pump, Serca2a, which is important for regulating calcium flux in

Brittany Palbykin; Jamie Borg; Patricia T. Caldwell; Josh Rowles; Andreas J. Papoutsis; Donato F. Romagnolo; Ornella I. Selmin

2011-01-01

109

Rat H9c2 cardiac myocytes are sensitive to arsenite due to a modest activation of transcription factor Nrf2  

Microsoft Academic Search

The mechanism underlying the hepatotoxicity induced by arsenic exposure is well investigated. However, little is known about\\u000a the detailed mechanisms of arsenic-induced cardiotoxicity or cardiac factors involved in high sensitivity to arsenicals in\\u000a spite of the fact that arsenic trioxide, which is used to treat acute promyelocytic leukemia, causes cardiotoxicity. Here,\\u000a we show that rat H9c2(2-1) cardiac myocytes exhibit high

Daigo Sumi; Takahiko Sasaki; Hideki Miyataka; Seiichiro Himeno

110

Isorhamnetin inhibits H?O?-induced activation of the intrinsic apoptotic pathway in H9c2 cardiomyocytes through scavenging reactive oxygen species and ERK inactivation.  

PubMed

As a traditional Chinese medicine, the sea buckthorn (Hippophae rhamnoides L.) has a long history in the treatment of ischemic heart disease and circulatory disorders. However, the active compounds responsible for and the underlying mechanisms of these effects are not fully understood. In this article, isorhamnetin pretreatment counteracted H(2)O(2)-induced apoptotic damage in H9c2 cardiomyocytes. Isorhamnetin did not inhibit the death receptor-dependent or extrinsic apoptotic pathways, as characterized by its absence in both caspase-8 inactivation and tBid downregulation along with unchanged Fas and TNFR1 mRNA levels. Instead, isorhamnetin specifically suppressed the mitochondria-dependent or intrinsic apoptotic pathways, as characterized by inactivation of caspase-9 and -3, maintenance of the mitochondrial membrane potential (??m), and regulation of a series of Bcl-2 family genes upstream of ??m. The anti-apoptotic effects of isorhamnetin were linked to decreased ROS generation. H(2)O(2) activated ERK and p53, whereas isorhamnetin inhibited their activation. ERK overexpression overrode the isorhamnetin-induced inhibition of the intrinsic apoptotic pathway in H9c2 cardiomyocytes, which indicated that an ERK-dependent pathway was involved. Furthermore, N-acetyl cysteine (a potent ROS scavenger) could attenuate the H(2)O(2)-induced apoptosis. However, PD98059 (an ERK-specific inhibitor) could not effectively antagonize ROS generation, which indicates that ROS may be an upstream inducer of ERK. In conclusion, isorhamnetin inhibits the H(2)O(2)-induced activation of the intrinsic apoptotic pathway via ROS scavenging and ERK inactivation. Therefore, isorhamnetin is a promising reagent for the treatment of ROS-induced cardiomyopathy. PMID:21948481

Sun, Bing; Sun, Gui-Bo; Xiao, Jing; Chen, Rong-Chang; Wang, Xin; Wu, Ying; Cao, Li; Yang, Zhi-Hong; Sun, Xiao-Bo

2012-02-01

111

Effects of RACK1 on cell migration and IGF-I signalling in cardiomyoctes are not dependent on an association with the IGF-IR  

Microsoft Academic Search

RACK1 can act as a scaffolding protein to integrate IGF-IR and integrin signalling in transformed cells but its actions in regulating IGF-IR signalling in non-transformed cells are less well understood. Here, we investigated the function of RACK1 in the non-transformed cardiomyocyte cell line H9c2. Overexpression of RACK1 in H9c2 cells was sufficient to increase cell size, increase adhesion to collagen

Helen C. O'Donovan; Patrick A. Kiely; Rosemary O'Connor

2007-01-01

112

Stable overexpression of human metallothionein-IIA in a heart-derived cell line confers oxidative protection  

Microsoft Academic Search

Metallothionein (MT) is a metal binding protein and cardioprotective. In order to understand the molecular mechanisms underlying the role of MT in the heart, in the current study we established a stable MT-IIA over-expressing cardiac cell line, and evaluated its anti-oxidative property. Rat heart-derived H9c2 cell line was stably transfected with a vector in which the human MT-IIA gene was

Wanli Xue; Qiuqu Liu; Lu Cai; Zhilun Wang; Wenke Feng

2009-01-01

113

Anthracycline-induced cardiac injury using a cardiac cell line: potential for gene therapy studies.  

PubMed

Anthracyclines are effective antitumor agents whose chief limitation has been cardiotoxicity directly related to free radical production. Therefore, strategies designed to selectively overexpress antioxidant proteins in the heart could protect against drug-induced toxicity and allow higher doses of chemotherapy. However, to date an adequate cardiac model system that is susceptible to anthracycline injury and can express foreign genes in a controlled fashion has been lacking. Developing a cardiac model system would permit examination of the relationship between the expression level of a potentially protective foreign gene and the degree of protection from injury. In this study we have examined the potential of the H9C2 rat cardiac myocyte cell line in this regard. H9C2 cells differentiate in a reproducible fashion, as shown by progressive increases in muscle tropomyosin-expressing cells, the organization of this thin filament protein, and the percentage of muscle cells contained within myotubes. Exposure of this cell line to the anthracycline doxorubicin produces cell injury as indicated by release of the intracellular enzyme lactate dehydrogenase into the culture medium. This injury is preceded by generation of reactive oxygen species, indicated by fluorescence after loading with carboxy-dichlorodihydrofluorescein diacetate. Stable transfection of H9C2 cells with a plasmid producing a tetracycline transactivator protein allows foreign genes to be expressed at a level tightly controlled by the concentration of tetracycline in the culture medium. Since H9C2 cells differentiate, can be injured by anthracycline exposure, and can express foreign genes at controllable levels, this is a suitable system in which to design genetic approaches to prevent this important clinical problem. PMID:11708868

L'Ecuyer, T; Horenstein, M S; Thomas, R; Vander Heide, R

2001-11-01

114

Activation of AMP-Activated Protein Kinase Contributes to Doxorubicin-Induced Cell Death and Apoptosis in Cultured Myocardial H9c2 Cells  

Microsoft Academic Search

Despite its potent antitumor effect, clinical use of Doxorubicin is limited because of serious side effects including myocardial\\u000a toxicity. Understanding the cellular mechanism involved in this process in a better manner is beneficial for optimizing Doxorubicin\\u000a treatment. In the current study, the authors focus on the AMP-activated protein kinase (AMPK) in the said process. In this\\u000a study, the authors discovered

Min-Bin Chen; Xiao-Yang Wu; Jin-Hua Gu; Qing-Tao Guo; Wen-Xiang Shen; Pei-Hua Lu

2011-01-01

115

TBP-associated factor 1 overexpression induces tolerance to Doxorubicin in confluent H9c2 cells by an increase in cdk2 activity and cyclin E expression  

Microsoft Academic Search

Doxorubicin (DOX) is a DNA topoisomerase II inhibitor widely used in anticancer treatment, however, it can lead to irreversible cardiac damage with severe debilitation. TBP-binding associated factor 1 (TAF1) is increased in DOX damaged hearts in vivo and in cardiomyocytes in vitro. To identify the functional role for TAF1 in DOX-treated heart we overexpressed wild type and mutant TAF1 in

Nicolas Servant; Daniela Marcantonio; John P. H. Th'ng; Lorraine E. Chalifour

2004-01-01

116

Cell lines.  

PubMed

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

Cherbas, Lucy; Gong, Lei

2014-06-15

117

Differential responses to docosahexaenoic acid in primary and immortalized cardiac cells.  

PubMed

The importance of dietary polyunsaturated fatty acids (PUFAs) in the reduction of cardiovascular disease has been recognized for many years. Docosahexaenoic acid (22:6n3, DHA) is an n-3 PUFA known to affect numerous biological functions and provide cardioprotection; however, the exact molecular and cellular protective mechanism(s) remain unknown. In contrast, DHA also possesses many anti-tumorgenic properties including suppressing cell growth and inducing apoptosis. In the present study, we investigated the effect of DHA toward H9c2 cells (an immortalized cardiac cell line) and neonatal primary cardiomyocytes (NCM). Cells were treated with 0?M, 10?M or 100?M DHA for upto 48h. Cell viability and mitochondrial activity were assayed at different time points. DHA caused a significant time- and dose-dependent decrease in cell viability and mitochondrial activity in H9c2 cells but not NCM. In addition, DHA decreased levels of TGF-?1 but increased IL-6 release in H9c2 cells. Significant induction of apoptosis was observed only in H9c2 cells, which involved activation of caspase-8 and -3 activities with a marked release of cytochrome c from mitochondria. DHA-induced severe mitochondrial damage resulting in a fragmented and punctated morphology with corresponding loss of mitochondrial membrane potential within 3h, prior to activation of caspases and cytochrome c release at 6h in H9c2 cells. Our data indicate that DHA treatment targets mitochondria, triggering collapse of mitochondrial membrane potential, increasing cellular stress and mitochondrial fragmentation resulting in apoptosis in immortalized cardiac cells, H9c2, but not neonatal primary cardiomyocyte. PMID:23523905

Qadhi, Rawabi; Alsaleh, Nasser; Samokhvalov, Victor; El-Sikhry, Haitham; Bellenger, Jérôme; Seubert, John M

2013-06-01

118

Mitochondrial dysfunction caused by saturated fatty acid loading induces myocardial insulin-resistance in differentiated H9c2 myocytes: a novel ex vivo myocardial insulin-resistance model.  

PubMed

Heart failure (HF) is often accompanied with metabolic disorders and insufficient energy production. Some previous studies have suggested an elevated serum free fatty acid (FA) due to chronic adrenergic stimulation induces myocardial insulin-resistance, which further impairs myocardial energy production. Because little is known about the pathogenesis of FA-induced cardiac insulin-resistance, we established an ex vivo cardiac insulin-resistant model and investigated the relationship between insulin-resistance and mitochondrial dysfunction. The ex vivo insulin-resistant myocytes, which was produced by treating differentiated H9c2 myocytes with palmitate (saturated FA; 0.2mM) for 24h, exhibited insulin-signaling deficiency and attenuated 2-deoxy-d-glucose (2-DG) uptake. When myocytes were pretreated with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP, a ROS scavenger; 200 ?M), the insulin-signaling deficiency by palmitate was restored, whereas the attenuated 2-DG uptake was remained. In contrast to TMPyP, the pretreatment with perhexiline (a mitochondrial FA uptake inhibitor; 2 ?M) restored the insulin-signaling deficiency and the attenuated 2-DG uptake by palmitate. Perhexiline restored the depolarized mitochondrial membrane potential (??m) and the reduced intracellular ATP by palmitate, and thereby improved the impaired GLUT4 recruitment to plasma membrane after insulin, whereas TMPyP failed to do so. These results suggested that the mitochondrial dysfunction by saturated FA loading and consequent intracellular energy shortage induced myocardial insulin-resistance in our ex vivo insulin-resistant model. PMID:23416068

Nobuhara, Mamoru; Saotome, Masao; Watanabe, Tomoyuki; Urushida, Tsuyoshi; Katoh, Hideki; Satoh, Hiroshi; Funaki, Makoto; Hayashi, Hideharu

2013-04-15

119

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

Microsoft Academic Search

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

Josephine C. Adams

1997-01-01

120

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

NASA Technical Reports Server (NTRS)

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.

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

1989-01-01

121

Expression of inducible stress protein 70 in rat heart myogenic cells confers protection against simulated ischemia-induced injury.  

PubMed Central

Myocardial ischemia markedly increases the expression of several members of the stress/heat shock protein (HSP) family, especially the inducible HSP70 isoforms. Increased expression of HSP70 has been shown to exert a protective effect against a lethal heat shock. We have examined the possibility of using this resistance to a lethal heat shock as a protective effect against an ischemic-like stress in vitro using a rat embryonic heart-derived cell line H9c2 (2-1). Myogenic cells in which the heat shock proteins have been induced by a previous heat shock are found to become resistant to a subsequent simulated ischemic stress. In addition, to address the question of how much does the presence of the HSP70 contribute to this protective effect, we have generated stably transfected cell lines overexpressing the human-inducible HSP70. Embryonal rat heart-derived H9c2(2-1) cells were used for this purpose. This stably transfected cell line was found to be significantly more resistant to an ischemic-like stress than control myogenic cells only expressing the selectable marker (neomycin) or the parental cell line H9c2(2-1). This finding implicates the inducible HSP70 protein as playing a major role in protecting cardiac cells against ischemic injury. Images

Mestril, R; Chi, S H; Sayen, M R; O'Reilly, K; Dillmann, W H

1994-01-01

122

Tick Cell Lines.  

National Technical Information Service (NTIS)

This invention relates to new continuous cell lines from embryonic tissues of ticks (Acari Ixodidae); the use of such cell lines for replicating selected microorganisms; and the use of the replicated microorganisms for diagnosis, prophylaxis and control o...

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

1981-01-01

123

Effect of gamma irradiation on spleen cell function and cytotoxicity of doxorubicin  

Microsoft Academic Search

The present study was attempted to evaluate the effects of gamma-irradiated doxorubicin (IRD) on spleen cell proliferation, cytokines release (IFN-? and IL-2) and lung metastasis in mice. Gamma irradiation induced degradation of doxorubicin molecule and cytotoxicity on melanoma (B16BL6) and myoblast (H9c2) cell lines were determined by high performance liquid chromatography (HPLC) and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazole) assay, respectively. Non-irradiated

Ju-Woon Lee; Nak-Yun Sung; Jae-Kyung Kim; Jae-Hun Kim; Hanumantha Rao Balaji Raghavendran; Yung-Choon Yoo; Mee-Hye Shin; Myung-Woo Byun

2008-01-01

124

Glutamate-induced ATP synthesis: relationship between plasma membrane Na+/Ca2+ exchanger and excitatory amino acid transporters in brain and heart cell models.  

PubMed

It is known that glutamate (Glu), the major excitatory amino acid in the central nervous system, can be an essential source for cell energy metabolism. Here we investigated the role of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) and the excitatory amino acid transporters (EAATs) in Glu uptake and recycling mechanisms leading to ATP synthesis. We used different cell lines, such as SH-SY5Y neuroblastoma, C6 glioma and H9c2 as neuronal, glial, and cardiac models, respectively. We first observed that Glu increased ATP production in SH-SY5Y and C6 cells. Pharmacological inhibition of either EAAT or NCX counteracted the Glu-induced ATP synthesis. Furthermore, Glu induced a plasma membrane depolarization and an intracellular Ca(2+) increase, and both responses were again abolished by EAAT and NCX blockers. In line with the hypothesis of a mutual interplay between the activities of EAAT and NCX, coimmunoprecipitation studies showed a physical interaction between them. We expanded our studies on EAAT/NCX interplay in the H9c2 cells. H9c2 expresses EAATs but lacks endogenous NCX1 expression. Glu failed to elicit any significant response in terms of ATP synthesis, cell depolarization, and Ca(2+) increase unless a functional NCX1 was introduced in H9c2 cells by stable transfection. Moreover, these responses were counteracted by EAAT and NCX blockers, as observed in SH-SY5Y and C6 cells. Collectively, these data suggest that plasma membrane EAAT and NCX are both involved in Glu-induced ATP synthesis, with NCX playing a pivotal role. PMID:23913256

Magi, Simona; Arcangeli, Sara; Castaldo, Pasqualina; Nasti, Annamaria Assunta; Berrino, Liberato; Piegari, Elena; Bernardini, Renato; Amoroso, Salvatore; Lariccia, Vincenzo

2013-10-01

125

A correlation between cytotoxicity and reductase-mediated metabolism in cell lines treated with doxorubicin and daunorubicin.  

PubMed

The role of metabolism in daunorubicin (DAUN)- and doxorubicin (DOX)-associated toxicity in cancer patients is dependent on whether the parent drugs or major metabolites, doxorubicinol (DOXol) and daunorubicinol (DAUNol), are the more toxic species. Therefore, we examined whether an association exists between cytotoxicity and the metabolism of these drugs in cell lines from nine different tissues. Cytotoxicity studies using MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] cell viability assays revealed that four cell lines [HepG2 (liver), HCT-15 (colon), NCI-H460 (lung), and A-498 (kidney)] were more tolerant to DAUN and DOX than the five remaining cell lines [H9c2 (heart), PC-3 (prostate), OVCAR-4 (ovary), PANC-1 (pancreas), and MCF-7 (breast)], based on significantly higher LC50 values at incubation times of 6, 24, and 48 hours. Each cell line was also assessed for its efficiency at metabolizing DAUN and DOX. The four drug-tolerant cell lines converted DAUN/DOX to DAUNol/DOXol more rapidly than the five drug-sensitive cell lines. We also determined whether exposure to DAUN or DOX induced an increase in metabolic activity among any of these nine different cell types. All nine cell types showed a significant increase in their ability to metabolize DAUN or DOX in response to pre-exposure to the drug. Western blot analyses demonstrated that the increased metabolic activity toward DAUN and DOX correlated with a greater abundance of eight aldo-keto and two carbonyl reductases following exposure to either drug. Overall, our findings indicate an inverse relationship between cytotoxicity and DAUN or DOX metabolism in these nine cell lines. PMID:23995598

Bains, Onkar S; Szeitz, András; Lubieniecka, Joanna M; Cragg, Gina E; Grigliatti, Thomas A; Riggs, K Wayne; Reid, Ronald E

2013-11-01

126

Cell line provenance  

Microsoft Academic Search

Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology.\\u000a However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere\\u000a in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and\\u000a contamination, given the will to apply it. Combined with proper

R. Ian Freshney

2002-01-01

127

Stable transfection of UCP1 confers resistance to hypoxia\\/reoxygenation in a heart-derived cell line  

Microsoft Academic Search

Mitochondrial uncoupling proteins, which secure physiological uncoupling of oxidative phosphorylation, have been proposed to serve as an oxidative-stress compensatory mechanism. Here, heart-derived H9c2 cells acquired improved resistance to injury upon transfection of the prototypic uncoupling protein UCP1. Following hypoxia\\/reoxygenation, stable overexpression of UCP1 provided enhanced cardioblast survival with preserved mitochondrial structure and function, while limiting reactive oxygen species formation. Thus,

Martin Bienengraeber; Cevher Ozcan; Andre Terzic

2003-01-01

128

Plant cell lines in cell morphogenesis research.  

PubMed

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

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

2014-01-01

129

Cell Type-Specific Activation of the Cytomegalovirus Promoter by Dimethylsulfoxide and 5-Aza-2?-deoxycytidine  

PubMed Central

The cytomegalovirus promoter is a very potent promoter commonly used for driving the expression of transgenes, though it gradually becomes silenced in stably transfected cells. We examined the methylation status of the cytomegalovirus promoter in two different cell lines and characterized its mechanisms of activation by dimethylsulfoxide and 5-Aza-2?-deoxycytidine. The cytomegalovirus promoter stably transfected into Chinese hamster ovary cells is suppressed by DNA methylation-independent mechanisms, which is different from the rat embryonic cardiomyoblast H9c2-Fluc.3 cells in which the cytomegalovirus promoter is silenced by methylation. Dimethylsulfoxide and 5-Aza-2?-deoxycytidine can activate the cytomegalovirus promoter in both cell types by overlapping mechanisms. Dimethylsulfoxide activates the cytomegalovirus promoter in Chinese hamster ovary cells by promoting histone acetylation and the activation of p38 mitogen-activated protein kinase and nuclear factor ?B signaling pathways, while 5-Aza-2?-deoxycytidine increases histone acetylation and activates the nuclear factor ?B but not the p38 mitogen-activated protein kinase pathway. In H9c2-Fluc.3 cells, both agents promote demethylation of the cytomegalovirus promoter, and enhance its activity exclusively through activation of the nuclear factor ?B pathway and to a lesser extent of the p38 mitogen-activated protein kinase pathway. Our findings suggest that suppression and activation of the cytomegalovirus promoter are cell type-specific. These results may be used for developing strategies to enhance the expression of transgenes and the production of recombinant proteins encoded by transgenes controlled by a cytomegalovirus promoter.

Radhakrishnan, Prakash; Basma, Hesham; Klinkebiel, David; Christman, Judith; Cheng, Pi-Wan

2014-01-01

130

Comparative analysis of ?B-crystallin expression in heat-stressed myocardial cells in vivo and in vitro.  

PubMed

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

Tang, Shu; Lv, Yingjun; Chen, Hongbo; Adam, Abdelnasir; Cheng, Yanfen; Hartung, Jörg; Bao, Endong

2014-01-01

131

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

PubMed Central

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

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

2014-01-01

132

Molluscan cells in culture: primary cell cultures and cell lines  

PubMed Central

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.

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

2013-01-01

133

Generating Mammalian Stable Cell Lines by Electroporation  

PubMed Central

Expression of functional, recombinant mammalian proteins often requires expression in mammalian cells (see Single Cell Cloning of a Stable Mammalian Cell Line). If the expressed protein needs to be made frequently, it can be best to generate a stable cell line instead of performing repeated transient transfections into mammalian cells. Here, we describe a method to generate stable cell lines via electroporation followed by selection steps. This protocol will be limited to the CHO dhfr– Urlaub et al. (1983) and LEC1 cell lines, which in our experience perform the best with this method.

Longo, Patti A.; Kavran, Jennifer M.; Kim, Min-Sung; Leahy, Daniel J.

2014-01-01

134

How Embryonic Stem Cell Lines are Made  

NSDL National Science Digital Library

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

2012-03-23

135

Molecular Characterization of Putative Chordoma Cell Lines  

PubMed Central

Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.

Bruderlein, Silke; Sommer, Joshua B.; Meltzer, Paul S.; Li, Sufeng; Osada, Takuya; Ng, David; Moller, Peter; Alcorta, David A.; Kelley, Michael J.

2010-01-01

136

CellLineMiner: a knowledge portal for human cell lines  

PubMed Central

Experimental models of human tissues and disease phenotypes frequently rely upon immortalized cell lines, which are easily accessible and simple to use due to their infinite capability of cell division. For decades, cell lines have been used to investigate cellular mechanisms of disease and the efficacy of drugs, most prominently for human cancers. However, the large body of knowledge with respect to human cell lines exists primarily in an unstructured fashion, that is, as free text in the scientific literature. Here we present CellLineMiner, a novel text mining-based web database that provides a comprehensive view of human cell line knowledge. The application offers a simple search in all indexed cell lines, accompanied by a rapid display of all identified literature associations. The CellLineMiner is intended to serve as a knowledge resource companion to the cellular model systems used in biomedical research. Availability CellLineMiner is accessible at http://dev.pubgene.com/cellmine

Nakken, Sigve; Johansen, Morten; Fillebeen, Julien; Berge, Ole Petter; Kirker?d, Harald; Jenssen, Tor-Kristian; Hovig, Eivind

2012-01-01

137

Human Esophageal Epithelial Cell Lines.  

National Technical Information Service (NTIS)

Human esophageal epithelial cells having replicative capacity in cell culture that is enhanced compared to normal cells and are unable to produce tumors is disclosed. Normal human esophagus tissue from two autopsy specimens was explanted in serum-free med...

G. D. Stoner R. R. Reddel C. C. Harris R. Roger

1989-01-01

138

Regulation of the human atrial myosin light chain 1 promoter by Ca2+-calmodulin-dependent signaling pathways  

Microsoft Academic Search

We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 pro- moter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling

Christiane Woischwill; Peter Karczewski; Holger Bartsch; Hans-Peter Luther; Monika Kott; Hannelore Haase; Ingo Morano

2005-01-01

139

Renal Cell Carcinoma (ccRCC) Human Cell Line  

Cancer.gov

A renal cell carcinoma (RCC) cell line designated UOK171 has been developed from the resected tumor of a patient diagnosed with stage IV high nuclear grade clear cell type renal cell carcinoma (ccRCC). The UOK171 cell line was immortalized spontaneously by mincing the resected tumor into pieces followed by propagation of the cells over more than twenty generations. One of the most prominent characteristics of this cell line is its intact, nonmutated von Hippel-Lindau (VHL) tumor suppressor gene.

140

Thyroid cancer cell lines: an overview.  

PubMed

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution. PMID:23162534

Saiselet, Manuel; Floor, Sébastien; Tarabichi, Maxime; Dom, Geneviève; Hébrant, Aline; van Staveren, Wilma C G; Maenhaut, Carine

2012-01-01

141

Thyroid cancer cell lines: an overview  

PubMed Central

Human thyroid cancer cell lines are the most used models for thyroid cancer studies. They must be used with detailed knowledge of their characteristics. These in vitro cell lines originate from differentiated and dedifferentiated in vivo human thyroid tumors. However, it has been shown that mRNA expression profiles of these cell lines were closer to dedifferentiated in vivo thyroid tumors (anaplastic thyroid carcinoma, ATC) than to differentiated ones. Here an overview of the knowledge of these models was made. The mutational status of six human thyroid cancer cell lines (WRO, FTC133, BCPAP, TPC1, K1, and 8505C) was in line with previously reported findings for 10 genes frequently mutated in thyroid cancer. However, the presence of a BRAF mutation (T1799A: V600E) in WRO questions the use of this cell line as a model for follicular thyroid carcinoma (FTC). Next, to investigate the biological meaning of the modulated mRNAs in these cells, a pathway analysis on previously obtained mRNA profiles was performed on five cell lines. In five cell lines, the MHC class II pathway was down-regulated and in four of them, ribosome biosynthesis and translation pathways were up-regulated. mRNA expression profiles of the cell lines were also compared to those of the different types of thyroid cancers. Three datasets originating from different microarray platforms and derived from distinct laboratories were used. This meta-analysis showed a significant higher correlation between the profiles of the thyroid cancer cell lines and ATC, than to differentiated thyroid tumors (i.e., PTC or FTC) specifically for DNA replication. This already observed higher correlation was obtained here with an increased number of in vivo tumors and using different platforms. In summary, this would suggest that some papillary thyroid carcinoma or follicular thyroid carcinoma (PTC or FTC) cell lines (i.e., TPC-1) might have partially lost their original DNA synthesis/replication regulation mechanisms during their in vitro cell adaptation/evolution.

Saiselet, Manuel; Floor, Sebastien; Tarabichi, Maxime; Dom, Genevieve; Hebrant, Aline; van Staveren, Wilma C. G.; Maenhaut, Carine

2012-01-01

142

Chromosomes of human hepatoma cell lines  

Microsoft Academic Search

The karyotypes of three human hepatoma cell lines Hep G2, Hep 3B and PLC\\/PRF\\/5 were investigated by G- and C-banding techniques. In addition to ploidy changes, typical for most carcinoma cell lines, certain markers were found that remained stable throughout passage of these cultures. Chromosome I is involved in multiple translocations, resulting in at least three copies of the chromosome

Daniela Simon; David P. Aden; Barbara B. Knowles

1982-01-01

143

Cell-host, LINE and environment  

PubMed Central

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.

Del Re, Brunella; Giorgi, Gianfranco

2013-01-01

144

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

Microsoft Academic Search

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http:\\/\\/bioinformatics.istge.it\\/hypercldb\\/) is a hyper- text version of CLDB that improves data accessibil- ity by also allowing information retrieval

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

2009-01-01

145

Generation of rabbit pluripotent stem cell lines.  

PubMed

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology. PMID:22925641

Tancos, Z; Nemes, C; Polgar, Z; Gocza, E; Daniel, N; Stout, T A E; Maraghechi, P; Pirity, M K; Osteil, P; Tapponnier, Y; Markossian, S; Godet, M; Afanassieff, M; Bosze, Z; Duranthon, V; Savatier, P; Dinnyes, A

2012-11-01

146

Human cell line authentication: the critical first step in any project using human cell lines.  

PubMed

Short tandem repeat (STR) typing is a standard procedure used in many laboratories for the authentication of human cell lines. This technology, which is based on the informativeness of known polymorphism of numerous loci to uniquely identify a human cell line, has allowed for direct-amplification of human DNA stored on FTA(®) paper. We describe an application of this technology to create a unique STR profile by direct amplification of HCT 116 (ATCC(®) CCL-247™) cell line DNA, a cell line commonly used in colon research. The ability to perform direct-amplification of DNA opens up the possibility of using FTA(®) paper as a way to maintain long-term storage of DNA samples from a cell line and other human tissues, such as buccal cells. PMID:23296621

McLaren, Robert S; Reid, Yvonne; Storts, Douglas R

2013-01-01

147

Umbelliprenin Induces Apoptosis in CLL Cell Lines.  

PubMed

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

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

2012-01-01

148

Umbelliprenin Induces Apoptosis in CLL Cell Lines  

PubMed Central

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

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

2012-01-01

149

CHARACTERIZATION OF A UNIQUE MUSCLE CELL LINE  

PubMed Central

A clonal cell line derived from a mouse neoplasm is described which shares many properties with smooth muscle. The cells have electrically excitable membranes capable of generating overshooting action potentials, and they contract both spontaneously and with electrical stimulation. They respond to the iontophoretic application of acetylcholine with a depolarizing response, and to norepinephrine with a hyperpolarizing response. Electron microscopy reveals that the cells have a morphology similar in many, but not all, respects to that of smooth muscle cells in vivo. The cells secrete soluble collagen-like molecules in addition to several proteins of undefined function. Finally, there is an increase in the specific activities of creatine phosphokinase and myokinase associated with increased cell density and the cessation of cell division.

Schubert, David; Harris, A. John; Devine, Carrick E.; Heinemann, Stephen

1974-01-01

150

Catecholamines in a macrophage cell line  

Microsoft Academic Search

This study provides the first evidence for catecholamine synthesis and release in the RAW264.7 cell line, an important macrophage model. Although catecholamines were low in unstimulated cells, activation with lipopolysaccharide (LPS) induced tyrosine hydroxylase (TH) mRNA and increased extracellular norepinephrine and intracellular dopamine within 48 h. The catecholamine synthesis inhibitor ?-methyl-para-tyrosine (?-mpt) decreased extracellular norepinephrine levels, suggesting release and rapid

Scott W Brown; Randall T Meyers; Karen M Brennan; Julie M Rumble; Nedathur Narasimhachari; Edmund F Perozzi; John J Ryan; Jennifer K Stewart; Krista Fischer-Stenger

2003-01-01

151

Radiation sensitivity of Merkel cell carcinoma cell lines  

SciTech Connect

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.

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others] [Queensland Institute of Medical Research (Australia); and others

1995-07-30

152

Granulocyte growth modulators elaborated by human cell lines  

Microsoft Academic Search

Two human monocyte-like cell lines have been established which produce colony stimulating activity (CSA) and colony inhibitory activity (CIA) for man and other species. Cell line CSA has been partially characterized. Cell line CIA, a low molecular weight hydrophobic molecule has been partially purified and shown to inhibit not only CFU-C growth but the growth of various lymphoid cell lines.

J. F. DiPersio; J. K. Brennan; M. A. Lichtman

1978-01-01

153

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

SciTech Connect

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.

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

2011-04-01

154

MHC class I bound peptides of a colon carcinoma cell line, a Ki-ras gene-targeted progeny cell line and a B cell line  

Microsoft Academic Search

MHC class I associated peptides on cancer cells represent potential targets for CD8+ cytotoxic T cell activity against tumor cells. We eluted the naturally bound MHC class I peptides of a colon carcinoma cell line and compared them to peptides isolated from a B cell line and a slow-growing activated Ki-ras-disrupted colon cancer cell line. While we failed to detect

Christopher J Savoie; Nobuhiro Kamikawaji; Tohru Sudo; Masanori Furuse; Senji Shirasawa; Takeshi Tana; Takehiko Sasazuki

1998-01-01

155

Investigating citrullinated proteins in tumour cell lines  

PubMed Central

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

2013-01-01

156

Establishment of a follicular lymphoma cell line (FLK-1) dependent on follicular dendritic cell-like cell line HK  

Microsoft Academic Search

A novel cell line, FLK-1, was established from bone marrow cells of a patient with follicular lymphoma by means of co-culture with follicular dendritic cell (FDC)-like cell line HK. Immunophenotypic analysis showed that FLK-1 expressed CD10, CD19, CD20, CD38, IgG and HLA-DR, which is a typical feature of germinal center B cells. Cytogenetic analysis of FLK-1 demonstrated t(14;18)(q32;q21) translocation involving

Y Kagami; J Jung; YS Choi; K Osumi; S Nakamura; Y Morishima; M Seto

2001-01-01

157

Colonic myofibroblast cell line stimulates colonoid formation.  

PubMed

A stable and efficient system for the culture of murine colon epithelial cells or crypts is required to facilitate studies of the dynamics and factors affecting colon stem cell niche and crypt formation. Survival of colonic epithelial cells or crypts in vitro was not established until recently, when it was found that exogenous Wnt3A and R-spondin could promote cell survival and formation of spheroids (colonospheres) or some advanced organoids with well-developed crypts (colonoids). However, after 6-8 days in these culture conditions, only small numbers of colonospheres form organoids with crypt-like structures (colonoids). This study describes the use of a myofibroblast cell line and a coculture system that increases the efficiency of colonoid formation from isolated crypts. The enhanced coculture system has significantly improved colonoid-forming efficiency compared with results from previous systems. Crypt formation can be detected as early as day 2. The coculture system will facilitate the characterization of the colon stem cell niche and the changes that occur as a result of perturbations or mutations in colon stem or epithelial cells, such as those that favor precancerous adenoma or cancer. PMID:24481605

Hirokawa, Yumiko; Yip, Kelvin Hon Yan; Tan, Chin Wee; Burgess, Antony W

2014-04-01

158

Establishment of stable cell lines of Drosophila germ-line stem cells  

PubMed Central

Each Drosophila ovariole has three independent sets of stem cells: germ-line stem cells (GSCs) and escort stem cells, located at the anterior tip of the germarium, and somatic stem cells (SSCs), located adjacent to the newly formed 16-cell cysts. Decapentaplegic (Dpp) is required to maintain the anterior stem cells, whereas Hedgehog is required for maintenance and cell division of the SCCs. In an effort to establish a new in vitro system to analyze intrinsic and extrinsic factors regulating the division and differentiation of GSCs of Drosophila, we tested various culture conditions for growing GSCs, derived from bag of marbles (bam) mutant ovaries. We have shown that bam? GSCs can be maintained and promoted to divide in vitro in media containing Dpp. These cells retain the morphological features of GSCs, i.e., expression of Vasa and Nanos and spectrosomes, even after several months of culture. Somatic cells are induced to grow in culture by the presence of sonic Hedgehog. The somatic cells produce Dpp. GSCs associate with the somatic cells via DE-cadherin, features that are also prominent at the niche of a normal germarium. Finally, we have established stable cell cultures consisting of GSCs and sheets of somatic cells, which are dependent on the addition of fly extract. A somatic cell line, lacking GSCs, has also been established. These cells are thought to be descendants of SCCs. Our in vitro system may provide the opportunity to manipulate GSCs genetically and to analyze the interaction of germ-line stem cells and soma.

Niki, Yuzo; Yamaguchi, Takafumi; Mahowald, Anthony P.

2006-01-01

159

Establishment of stable cell lines of Drosophila germ-line stem cells.  

PubMed

Each Drosophila ovariole has three independent sets of stem cells: germ-line stem cells (GSCs) and escort stem cells, located at the anterior tip of the germarium, and somatic stem cells (SSCs), located adjacent to the newly formed 16-cell cysts. Decapentaplegic (Dpp) is required to maintain the anterior stem cells, whereas Hedgehog is required for maintenance and cell division of the SCCs. In an effort to establish a new in vitro system to analyze intrinsic and extrinsic factors regulating the division and differentiation of GSCs of Drosophila, we tested various culture conditions for growing GSCs, derived from bag of marbles (bam) mutant ovaries. We have shown that bam(-) GSCs can be maintained and promoted to divide in vitro in media containing Dpp. These cells retain the morphological features of GSCs, i.e., expression of Vasa and Nanos and spectrosomes, even after several months of culture. Somatic cells are induced to grow in culture by the presence of sonic Hedgehog. The somatic cells produce Dpp. GSCs associate with the somatic cells via DE-cadherin, features that are also prominent at the niche of a normal germarium. Finally, we have established stable cell cultures consisting of GSCs and sheets of somatic cells, which are dependent on the addition of fly extract. A somatic cell line, lacking GSCs, has also been established. These cells are thought to be descendants of SCCs. Our in vitro system may provide the opportunity to manipulate GSCs genetically and to analyze the interaction of germ-line stem cells and soma. PMID:17056713

Niki, Yuzo; Yamaguchi, Takafumi; Mahowald, Anthony P

2006-10-31

160

Cytosine methylation profiling of cancer cell lines.  

PubMed

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

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

2008-03-25

161

Cytosine methylation profiling of cancer cell lines  

PubMed Central

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.

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

2008-01-01

162

CellLineNavigator: a workbench for cancer cell line analysis  

PubMed Central

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

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

2013-01-01

163

GPVI oligomerisation in cell lines and platelets  

PubMed Central

Summary Background Glycoprotein VI (GPVI) is a physiological receptor for collagen expressed at the surface of platelets and megakaryocytes. Constitutive dimerisation of GPVI has been proposed as necessary for the interaction with collagen, although direct evidence of dimerisation has not been reported in cell lines or platelets. Objectives To investigate oligomerisation of GPVI in transfected cell lines and in platelets under nonstimulated conditions. Methods and Results By using a combination of molecular and biochemical techniques, we demonstrate that GPVI association occurs at the surface of transfected 293T cells under basal conditions, through an interaction at the extra-cellular domain of the receptor. Bioluminescence resonance energy transfer was used to confirm oligomerisation of GPVI under these conditions. A chemical cross-linker was used to detect constitutive oligomeric forms of GPVI at the surface of platelets, which contain the FcR ?-chain. Conclusions The present results directly demonstrate GPVI-FcR ?-chain oligomerisation at the surface of the platelet, and thereby add to the growing evidence that oligomerisation of GPVI may be a pre-requisite for binding of the receptor to collagen, and therefore for proper functioning of platelets upon vascular damage.

2007-01-01

164

Scalable cell alignment on optical media substrates.  

PubMed

Cell alignment by underlying topographical cues has been shown to affect important biological processes such as differentiation and functional maturation in vitro. However, the routine use of cell culture substrates with micro- or nano-topographies, such as grooves, is currently hampered by the high cost and specialized facilities required to produce these substrates. Here we present cost-effective commercially available optical media as substrates for aligning cells in culture. These optical media, including CD-R, DVD-R and optical grating, allow different cell types to attach and grow well on them. The physical dimension of the grooves in these optical media allowed cells to be aligned in confluent cell culture with maximal cell-cell interaction and these cell alignment affect the morphology and differentiation of cardiac (H9C2), skeletal muscle (C2C12) and neuronal (PC12) cell lines. The optical media is amenable to various chemical modifications with fibronectin, laminin and gelatin for culturing different cell types. These low-cost commercially available optical media can serve as scalable substrates for research or drug safety screening applications in industry scales. PMID:23601659

Anene-Nzelu, Chukwuemeka G; Choudhury, Deepak; Li, Huipeng; Fraiszudeen, Azmall; Peh, Kah-Yim; Toh, Yi-Chin; Ng, Sum Huan; Leo, Hwa Liang; Yu, Hanry

2013-07-01

165

Peroxisome proliferator-activated receptor y (PPARy) activation protects H9c2 cardiomyoblasts from oxidative stress-induced apoptosis  

Microsoft Academic Search

Objective: Activation of peroxisome proliferator-activated receptor a (PPARa) and PPARg plays beneficial roles in cardiovascular disorders such as atherosclerosis and heart reperfusion. Although PPARa and g have been documented to reduce oxidative stress in the vasculature and the heart, the role of PPARy remains poorly studied. Methods and results: We focused on PPARy function in the regulation of oxidative stress-induced

Matthieu Pesant; Stephanie Sueur; Patrick Dutartre; Mireille Tallandier; Paul A. Grimaldi; Luc Rochette; Jean-Louis Connat

2006-01-01

166

On the Ontology Based Representation of Cell Lines  

PubMed Central

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

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

2012-01-01

167

CALCIUM OXALATE CRYSTAL ATTACHMENT TO CULTURED KIDNEY EPITHELIAL CELL LINES  

Microsoft Academic Search

PurposeCultured kidney epithelial cell lines have frequently been used in urolithiasis research, and in particular in studies related to the interactions between stone crystals and cell membranes. There is evidence that when epithelial cell lines are transformed or serially passed to immortalize them, they experience changes in both cell physiology and morphology. Stone research utilizing cell cultures is frequently necessary

MICHAEL W. BIGELOW; JOHN H. WIESSNER; JACK G. KLEINMAN; NEIL S. MANDEL

1998-01-01

168

Catecholamines in a macrophage cell line.  

PubMed

This study provides the first evidence for catecholamine synthesis and release in the RAW264.7 cell line, an important macrophage model. Although catecholamines were low in unstimulated cells, activation with lipopolysaccharide (LPS) induced tyrosine hydroxylase (TH) mRNA and increased extracellular norepinephrine and intracellular dopamine within 48 h. The catecholamine synthesis inhibitor alpha-methyl-para-tyrosine (alpha-mpt) decreased extracellular norepinephrine levels, suggesting release and rapid turnover of newly synthesized norepinephrine. High concentrations of dopamine or norepinephrine (>/=100 microM) decreased proliferation and increased apoptosis of macrophages. These anti-proliferative effects were prevented by simultaneous treatment with the anti-oxidant ascorbic acid. Pre-incubation with a glutathione synthesis inhibitor (L-buthionine-[S,R]-sulfoximine [L-BSO]) increased sensitivity to catecholamine-stimulated apoptosis, suggesting that glutathione protects macrophages from both endogenous and exogenous catecholamines. PMID:12576223

Brown, Scott W; Meyers, Randall T; Brennan, Karen M; Rumble, Julie M; Narasimhachari, Nedathur; Perozzi, Edmund F; Ryan, John J; Stewart, Jennifer K; Fischer-Stenger, Krista

2003-02-01

169

Tetanus toxin as a marker for small-cell lung cancer cell lines  

Microsoft Academic Search

Tetanus toxin labeling of human lung cancer cell lines was investigated using direct and indirect immunofluorescence and immunohistochemical staining. Cells of characterized permanent cell lines, eight small-cell lung cancer (SCLC) cell lines of classic subtype, six SCLC cell lines of variant subtype and seven non-small-cell lung cancer (NSCLC) cell lines, were incubated with a saturating concentration of tetanus toxin. For

Jochen Heymanns; Kurt Neumann; Klaus Havemann

1989-01-01

170

Detection algorithm for the validation of human cell lines.  

PubMed

Cell lines are an important tool in understanding all aspects of cancer growth, development, metastasis and tumor cell death. There has been a dramatic increase in the number of cell lines and diversity of the cancers they represent; however, misidentification and cross-contamination of cell lines can lead to erroneous conclusions. One method that has gained favor for authenticating cell lines is the use of short tandem repeats (STR) to generate a unique DNA profile. The challenge in validating cell lines is the requirement to compare the large number of existing STR profiles against cell lines of interest, particularly when considering that the profiles of many cell lines have drifted over time and original samples are not available. We report here methods that analyze the variations and the proportional changes extracted from tetra-nucleotide repeat regions in the STR analysis. This technique allows a paired match between a target cell line and a reference database of cell lines to find cell lines that match within a user designated percentage cut-off quality matrix. Our method accounts for DNA instability and can suggest whether the target cell lines are misidentified or unstable. PMID:22419365

Eltonsy, Névine; Gabisi, Vivian; Li, Xuesong; Russe, K Blair; Mills, Gordon B; Stemke-Hale, Katherine

2012-09-15

171

Personalized chemotherapy profiling using cancer cell lines from selectable mice  

PubMed Central

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.

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

172

DNA profiling and characterization of animal cell lines.  

PubMed

The history of the culture of animal cell lines is littered with published and much unpublished experience with cell lines that have become switched, mislabelled, or cross-contaminated during laboratory handling. To deliver valid and good quality research and to avoid waste of time and resources on such rogue lines, it is vital to perform some kind of qualification for the provenance of cell lines used in research and particularly in the development of biomedical products. DNA profiling provides a valuable tool to compare different sources of the same cells and, where original material or tissue is available, to confirm the correct identity of a cell line. This chapter provides a review of some of the most useful techniques to test the identity of cells in the cell culture laboratory and gives methods which have been used in the authentication of cell lines. PMID:24297409

Stacey, Glyn N; Byrne, Ed; Hawkins, J Ross

2014-01-01

173

Match criteria for human cell line authentication: where do we draw the line?  

PubMed

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ?80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines. PMID:23136038

Capes-Davis, Amanda; Reid, Yvonne A; Kline, Margaret C; Storts, Douglas R; Strauss, Ethan; Dirks, Wilhelm G; Drexler, Hans G; MacLeod, Roderick A F; Sykes, Gregory; Kohara, Arihiro; Nakamura, Yukio; Elmore, Eugene; Nims, Raymond W; Alston-Roberts, Christine; Barallon, Rita; Los, Georgyi V; Nardone, Roland M; Price, Paul J; Steuer, Anton; Thomson, Jim; Masters, John R W; Kerrigan, Liz

2013-06-01

174

Functional Analysis of an Established Mouse Vascular Endothelial Cell Line  

Microsoft Academic Search

Background: In vitrostudies using cell lines are useful for the understanding of cellular mechanisms. The purpose of our study is to develop a new immortalized aortic vascular endothelial cell (EC) line that retains endothelial characteristics and can facilitate the study of ECs. Methods: A mouse aortic vascular EC line (MAEC) was established from p53-deficient mouse aorta and cultured for over

Tatsuaki Nishiyama; Kenji Mishima; Fumio Ide; Koichi Yamada; Kumi Obara; Aki Sato; Noriko Hitosugi; Hiroko Inoue; Kazuo Tsubota; Ichiro Saito

2007-01-01

175

Cell line misidentification: the beginning of the end  

Microsoft Academic Search

Cell lines are used extensively in research and drug development as models of normal and cancer tissues. However, a substantial proportion of cell lines is mislabelled or replaced by cells derived from a different individual, tissue or species. The scientific community has failed to tackle this problem and consequently thousands of misleading and potentially erroneous papers have been published using

2010-01-01

176

Differential signaling of the GnRH receptor in pituitary gonadotrope cell lines and prostate cancer cell lines  

PubMed Central

The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKC?, PKC?II and PKC? is much higher in ?T3-1 and L?T2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKC?. Activation of PKC?, PKC?II and PKC? by GnRH is relatively transient in ?T3-1 and L?T2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca2+ ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in L?T2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells.

Sviridonov, Ludmila; Dobkin-Bekman, Masha; Shterntal, Boris; Przedecki, Fiorenza; Formishell, Linor; Kravchook, Shani; Navi, Liat Rahamim-Ben; Bar-Lev, Tali Hana; Kazanietz, Marcelo G.; Yao, Zhong; Seger, Rony; Naor, Zvi

2014-01-01

177

Effect of melatonin on cytotoxicity of doxorubicin toward selected cell lines (human keratinocytes, lung cancer cell line A-549, laryngeal cancer cell line Hep-2).  

PubMed

The pineal hormone melatonin (MLT) has been recognised as a substance capable of alleviating in vivo nephro-, cardio- and myelotoxicity of doxorubicin (DOX) and of other anthracyclines in animal models. However, few data are available on the effects of MLT on cytotoxicity of antineoplastic drugs toward tumor cells in vitro. The present study aimed at the evaluation of effects of MLT and of DOX on selected cell lines. The experiments were conducted on human keratinocytes (primary culture), non-small cell lung cancer (A-549) and laryngeal cancer cell lines (HEp-2). In keratinocytes and in A-549 cells, MLT used at pharmacological concentrations (0.1 and 1.0 mM) was observed to intensify apoptotic lesions. MLT exerted no clear-cut effects on the HEp-2 cell line. In contrast, DOX at concentrations of 0.1 and 1.0 microg/ml intensified apoptosis and augmented the frequency of necrotic lesions in cell nuclei in all the examined cell lines. MLT intensified cytotoxicity of DOX in all cell lines, significantly decreasing cell numbers and promoting apoptosis. The effect was MLT concentration-dependent. MLT decreased the proportion of cells with necrotic lesions. PMID:17591362

Fic, Magdalena; Podhorska-Okolow, Marzena; Dziegiel, Piotr; Gebarowska, Elzbieta; Wysocka, Teresa; Drag-Zalesinska, Malgorzata; Zabel, Maciej

2007-01-01

178

Biological behaviors and proteomics analysis of hybrid cell line EAhy926 and its parent cell line A549  

Microsoft Academic Search

BACKGROUND: It is well established that cancer cells can fuse with endothelial cells to form hybrid cells spontaneously, which facilitates cancer cells traversing the endothelial barrier to form metastases. However, up to now, little is known about the biologic characteristics of hybrid cells. Therefore, we investigate the malignant biologic behaviors and proteins expression of the hybrid cell line EAhy926 with

Ze Jun Lu; Ya Qiong Ren; Guo Ping Wang; Qi Song; Mei Li; Sa Sa Jiang; Tao Ning; Yong Song Guan; Jin Yang; Feng Luo

2009-01-01

179

Polyamine Synthesis in Maize Cell Lines 1  

PubMed Central

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

Hiatt, Andrew

1989-01-01

180

Investigation of Radiosensitivity Gene Signatures in Cancer Cell Lines  

PubMed Central

Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n?=?16] and head and neck [n?=?11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2 median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins.

Hall, John S.; Iype, Rohan; Senra, Joana; Taylor, Janet; Armenoult, Lucile; Oguejiofor, Kenneth; Li, Yaoyong; Stratford, Ian; Stern, Peter L.; O'Connor, Mark J.; Miller, Crispin J.; West, Catharine M. L.

2014-01-01

181

Continuous human cell lines and method of making same  

DOEpatents

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.

Stampfer, Martha R. (Oakland, CA)

1989-01-01

182

Continuous human cell lines and method of making same  

DOEpatents

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.

Stampfer, M.R.

1985-07-01

183

Shortest path based splitting line finding for touching cells  

NASA Astrophysics Data System (ADS)

A shortest path based algorithm is proposed in this paper to find splitting lines for touching cells. Firstly, an initial splitting line is obtained through the distance transform of a marker image and the watershed algorithm. Then, the initial splitting line is separated into different line segments if necessary, and the start and end points of these line segments act as the start and end points of shortest path. Finally, the shortest path algorithm is used to find the splitting line between the start and end points, and the final result of touching cells splitting can be formed by the contour of the touching cells and the splitting lines. Experimental results show that the proposed algorithm is efficient for different types of touching cells.

Bai, Xiangzhi; Sun, Changming; Wang, Peng; Zhou, Fugen

2013-10-01

184

The cell line data base and the new catalogue: Detailed information on 2650 human and animal cell lines  

Microsoft Academic Search

The Cell Line Data Base (CLDB), set up within the Interlab Project, is a relational database containing data on 2650 Human and animal cell lines which are available in labs and cell banks all over Europe. The second edition of the catalogue, directly generated from the database, has been produced, and will be published in the first months of 1993.

A. Manniello; O. Aresu; B. Parodi; P. Romano; B. Iannotta; G. Rondanina; L. Viegi; T. Ruzzon

1993-01-01

185

Differentiation of Embryonic Stem Cell Lines Generated from Adult Somatic Cells by Nuclear Transfer  

Microsoft Academic Search

Embryonic stem (ES) cells are fully pluripotent in that they can differentiate into all cell types, including gametes. We have derived 35 ES cell lines via nuclear transfer (ntES cell lines) from adult mouse somatic cells of inbred, hybrid, and mutant strains. ntES cells contributed to an extensive variety of cell types, including dopaminergic and serotonergic neurons in vitro and

Teruhiko Wakayama; Viviane Tabar; Ivan Rodriguez; Anthony C. F. Perry; Lorenz Studer; Peter Mombaerts

2001-01-01

186

Complement-Fixing Antigens in Burkitt Lymphoma Cell Lines  

PubMed Central

The complement-fixing (CF) activity of antigens from cultured Burkitt lymphoma cells was determined by using normal American sera as the source of antibody. Approximately 75% of the sera fixed complement with the positive cell lines. These lines contained the herpes-like virus detectable by electron microscopy. The content of CF antigen depended on the cell line used but appeared to be independent of the number of cells which produced Henle's immunofluorescence (IF) antigen. Only sera that reacted in the IF test also contained CF antibodies to the crude cell extracts.

McCormick, Kenneth J.; Stenback, Wayne A.; Mumford, David M.; Trentin, John J.

1971-01-01

187

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls  

PubMed Central

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

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

2013-01-01

188

Regulated expression of erythropoietin by two human hepatoma cell lines  

SciTech Connect

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.

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

1987-11-01

189

Replicative Capacity of MERS Coronavirus in Livestock Cell Lines.  

PubMed

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

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

2014-02-01

190

GREG cells, a dysferlin-deficient myogenic mouse cell line  

SciTech Connect

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.

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

2012-01-15

191

HLA Homozygous Stem Cell Lines Derived from Human Parthenogenetic Blastocysts  

Microsoft Academic Search

Individual HLA homozygous parthenogenetic human stem cell (hpSC-Hhom) lines have the potential for cell-based therapy in a significant number of individuals, provided the HLA haplotype is prevalent. We report the successful derivation of four stable hpSC-Hhom lines from both HLA homozygous and HLA heterozygous donors. Of these, the hpSC-Hhom-4 line carries the HLA haplotype found most commonly within the U.S.

E. S. Revazova; N. A. Turovets; O. D. Kochetkova; L. S. Agapova; J. L. Sebastian; M. V. Pryzhkova; V. Iu. Smolnikova; L. N. Kuzmichev; J. D. Janus

2008-01-01

192

Phenotype and Genotype of Pancreatic Cancer Cell Lines  

PubMed Central

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.

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

193

Undermethylation of specific LINE-1 sequences in human cells producing a LINE-1-encoded protein.  

PubMed

Nucleotide sequences near the 5' ends of some long interspersed elements-1 (LINE-1) from Homo sapiens (L1Hs) are undermethylated in cell lines which produce a L1Hs-encoded protein. In contrast, these sequences are methylated in cell lines with little or no detectable L1Hs expression. The fact that the 5' end of L1Hs is differentially methylated in cells exhibiting different levels of L1Hs expression suggests that the methylation state of this region plays a role in L1Hs expression. PMID:7693554

Thayer, R E; Singer, M F; Fanning, T G

1993-11-15

194

Regulatory networks define phenotypic classes of human stem cell lines  

PubMed Central

Stem cells are defined as self-renewing cell populations that can differentiate into multiple distinct cell types. However, hundreds of different human cell lines from embryonic, fetal, and adult sources have been called stem cells, even though they range from pluripotent cells, typified by embryonic stem cells, which are capable of virtually unlimited proliferation and differentiation, to adult stem cell lines, which can generate a far more limited repertory of differentiated cell types. The rapid increase in reports of new sources of stem cells and their anticipated value to regenerative medicine1, 2 have highlighted the need for a general, reproducible method for classification of these cells3. We report here the creation and analysis of a database of global gene expression profiles (“Stem Cell Matrix”) that enables the classification of cultured human stem cells in the context of a wide variety of pluripotent, multipotent, and differentiated cell types. Using an unsupervised clustering method4, 5 to categorize a collection of ~150 cell samples, we discovered that pluripotent stem cell lines group together, while other cell types, including brain-derived neural stem cell lines, are very diverse. Using further bioinformatic analysis6 we uncovered a protein-protein network (“PluriNet”) that is shared by the pluripotent cells (embryonic stem cells, embryonal carcinomas, and induced pluripotent cells). Analysis of published data showed that the PluriNet appears to be a common characteristic of pluripotent cells, including mouse ES and iPS cells and human oocytes. Our results offer a new strategy for classifying stem cells and support the idea that pluripotence and self-renewal are under tight control by specific molecular networks.

Muller, Franz-Josef; Laurent, Louise C.; Kostka, Dennis; Ulitsky, Igor; Williams, Roy; Lu, Christina; Park, In-Hyun; Rao, Mahendra S.; Shamir, Ron; Schwartz, Philip H.; Schmidt, Nils O.; Loring, Jeanne F.

2008-01-01

195

Further characterization of the first seminoma cell line TCam-2.  

PubMed

Testicular germ cell tumors of adolescents and adults (TGCTs) can be classified into seminomatous and nonseminomatous tumors. Various nonseminomatous cell lines, predominantly embryonal carcinoma, have been established and proven to be valuable for pathobiological and clinical studies. So far, no cell lines have been derived from seminoma which constitutes more than 50% of invasive TGCTs. Such a cell line is essential for experimental investigation of biological characteristics of the cell of origin of TGCTs, i.e., carcinoma in situ of the testis, which shows characteristics of a seminoma cell. Before a cell line can be used as model, it must be verified regarding its origin and characteristics. Therefore, a multidisciplinary approach was undertaken on TCam-2 cells, supposedly the first seminoma cell line. Fluorescence in situ hybridization, array comparative genomic hybridization, and spectral karyotyping demonstrated an aneuploid DNA content, with gain of 12p, characteristic for TGCTs. Genome wide mRNA and microRNA expression profiling supported the seminoma origin, in line with the biallelic expression of imprinted genes IGF2/H19 and associated demethylation of the imprinting control region. Moreover, the presence of specific markers, demonstrated by immunohistochemistry, including (wild type) KIT, stem cell factor, placental alkaline phosphatase, OCT3/4 (also demonstrated by a specific Q-PCR) and NANOG, and the absence of CD30, SSX2-4, and SOX2, confirms that TCam-2 is a seminoma cell line. Although mutations in oncogenes and tumor suppressor genes are rather rare in TGCTs, TCam-2 had a mutated BRAF gene (V600E), which likely explains the fact that these cells could be propagated in vitro. In conclusion, TCam-2 is the first well-characterized seminoma-derived cell line, with an exceptional mutation, rarely found in TGCTs. PMID:18050305

de Jong, Jeroen; Stoop, Hans; Gillis, Ad J M; Hersmus, Remko; van Gurp, Ruud J H L M; van de Geijn, Gert-Jan M; van Drunen, Ellen; Beverloo, H Berna; Schneider, Dominik T; Sherlock, Jon K; Baeten, John; Kitazawa, Sohei; van Zoelen, E Joop; van Roozendaal, Kees; Oosterhuis, J Wolter; Looijenga, Leendert H J

2008-03-01

196

Characterization of Liposarcoma Cell Lines for Preclinical and Biological Studies  

PubMed Central

Liposarcoma cell lines represent in vitro models for studying disease mechanisms at the cellular level and for preclinical evaluation of novel drugs. To date there are a limited number of well-characterized models available. In this study, nine immortal liposarcoma cell lines were evaluated for tumor-forming ability, stem cell- and differentiation potential, and metastatic potential, with the aim to generate a well-characterized liposarcoma cell line panel. Detailed stem cell and differentiation marker analyses were also performed. Five of the liposarcoma cell lines were tumorigenic, forming tumors in mice. Interestingly, tumor-forming ability correlated with high proliferative capacity in vitro. All the cell lines underwent adipocytic differentiation, but the degree varied. Surprisingly, the expression of stem cell and differentiation markers did not correlate well with function. Overall, the panel contains cell lines suited for in vivo analyses (LPS141, SA-4, T778, SW872, and LISA-2), for testing novel drugs targeting cancer stem cells (LPS141) and for studying tumor progression and metastasis (T449 and T778).

Stratford, Eva W.; Castro, Russell; Daffinrud, Jeanette; Skarn, Magne; Lauvrak, Silje; Munthe, Else; Myklebost, Ola

2012-01-01

197

Development and characterization of immortalized ovine endometrial cell lines.  

PubMed

The objective of this study was to generate immortalized endometrial epithelial and stromal cell lines from the ovine uterus. Luminal (LE) and glandular epithelial (GE) cells and stromal (ST) cells were enzymatically isolated from the uterus of a Day 5 cyclic ewe (estrus on Day 0), and primary cultures were immortalized by transduction with a retroviral vector (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analogue G418 (0.6-0.8 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for more than 40 passages. Phase-contrast microscopy revealed that LE and GE cells exhibited a cobblestone morphology whereas immortalized ST cells were spindle shaped. The epithelial origin of LE and GE was confirmed by positive cytokeratin immunostaining, and ST cells were vimentin positive. All cell lines were negative for smooth muscle alpha-actin staining. Western blot analyses of cell extracts revealed the presence of signal transducers and activators of transcription (STAT) proteins 1, 2, and 3. In the LE cells, interferon tau (IFNtau) induced nuclear translocation of STAT proteins 1 and 2 and up-regulated several IFN-inducible genes, including STATs 1, 2, and 3 and ubiquitin cross-reactive protein (UCRP/ISG17). In the LE cell line, IFN regulatory factor one was transiently up-regulated and then down-regulated by IFNtau. Immunostaining revealed the presence of nuclear estrogen receptor and progesterone receptor in all cell lines. These ovine endometrial cell lines provide useful in vitro model systems for the study of hormone and cytokine action, signal transduction pathways, cell-cell interactions, and gene expression in specific cell types of the ovine endometrium. PMID:10529281

Johnson, G A; Burghardt, R C; Newton, G R; Bazer, F W; Spencer, T E

1999-11-01

198

Murine bone marrow cell line producing colony-stimulating factor  

SciTech Connect

A cell line (H-1) derived from the adherent layer of a 14-wk-old Dexter bone marrow culture has been maintained as cloned and uncloned lines through 21 passages at the time of these studies. These cell lines develop many fat droplets as they age and become confluent. The uncloned line produces increasing amounts of colony-stimulating activity as the cells become confluent. Feeder-layers or supernatants from the nonconfluent or confluent fat-laden cells stimulate the formation of greater numbers of colonies derived from cultures of colony-forming units (CFU) than does medium from L cell culture containing colony-stimulating factor (CSF). Antibody to the CSF-containing medium from L cell culture neutralizes the colony-stimulating activity, thus showing immunologic similarity to a known molecular species that stimulates colony production in a CFU culture that produces granulocyte or macrophage populations, or both.

Harigaya, K. (Brookhaven National Lab., Upton, NY); Cronkite, E.P.; Miller, M.E.; Shadduck, R.K.

1981-11-01

199

Generation of mesenchymal stem cell lines from murine bone marrow.  

PubMed

Mesenchymal stem cells (MSC), because of their multipotency and ease of purification and amplification, are an ideal stem cell source for cell therapies. Bone-marrow-derived stem cells (BMSC) can be used to develop MSC-like immortalized cell lines with large proliferation and differentiation potentialities. Their immortalized status prevents the maintenance of MSC function and characters; this can be negated by modifying the isolation and maintenance protocol. Adult murine BMSC were isolated and maintained in media without additional growth factors together with passage-dependent reseeding following trypsinization. Cells maintained over 25 passages were considered as putative cell lines and characterized. The phenotypic and genotypic characteristics and multilineage differentiation potential of the cells were assessed by morphological, phenotypic, and molecular assays at various passages. The putative BMSC cell lines showed the characteristics of MSC and were able to maintain these characteristics, even after immortalization. The phenotypic data demonstrated difference among two cell lines; this was further validated by the difference in their multilineage differentiation potential following specific induction. More importantly, no changes were observed in the genotypic level in comparison with control cells, even after more than 50 passages. Our protocol thus advances the isolation and maintenance of BMSC and the development of putative BMSC cell lines that maintain characteristics of MSC, including multilineage differentiation potential, after more than 40 passages. PMID:22836234

Sreejit, P; Dilip, K B; Verma, R S

2012-10-01

200

Quantitative methods to characterize morphological properties of cell lines.  

PubMed

Descriptive terms are often used to characterize cells in culture, but the use of nonquantitative and poorly defined terms can lead to ambiguities when comparing data from different laboratories. Although recently there has been a good deal of interest in unambiguous identification of cell lines via their genetic markers, it is also critical to have definitive, quantitative metrics to describe cell phenotypic characteristics. Quantitative metrics of cell phenotype will aid the comparison of data from experiments performed at different times and in different laboratories where influences such as the age of the population and differences in culture conditions or protocols can potentially affect cellular metabolic state and gene expression in the absence of changes in the genetic profile. Here, we present examples of robust methodologies for quantitatively assessing characteristics of cell morphology and cell-cell interactions, and of growth rates of cells within the population. We performed these analyses with endothelial cell lines derived from dolphin, bovine and human, and with a mouse fibroblast cell line. These metrics quantify some characteristics of these cells lines that clearly distinguish them from one another, and provide quantitative information on phenotypic changes in one of the cell lines over large number of passages. PMID:22619183

Mancia, Annalaura; Elliott, John T; Halter, Michael; Bhadriraju, Kiran; Tona, Alessandro; Spurlin, Tighe A; Middlebrooks, Bobby L; Baatz, John E; Warr, Gregory W; Plant, Anne L

2012-07-01

201

Small RNAs: Keeping Stem Cells in Line  

PubMed Central

Stem cells and RNA silencing have emerged as areas of intense interest for both basic and clinical research. Recently these fields have converged with reports implicating small regulatory RNAs in the maintenance and pluripotency of stem cells.

Stadler, Bradford M.; Ruohola-Baker, Hannele

2010-01-01

202

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

PubMed

In the first 50 years of cell culture, the development of new cell lines was mainly based on trial and error. Due to the understanding of the molecular networks of aging, senescence, proliferation, and adaption by mutation, the generation of new cell lines with physiologic properties has become more systematic. This endeavor has been supported by the availability of new technological achievements and increasing knowledge about the biology of cell differentiation and cell-cell communication. Here, we review some promising developments that are contributing toward this goal. These include molecular tools frequently used for the immortalization process. In addition to these broadly acting immortalization regimens, we focus on the developments of cell type-specific immortalization and on the methodologies of how to control the growth of newly established cell lines. PMID:23863696

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

2013-12-01

203

Development of a conditionally immortalized human pancreatic ? cell line  

PubMed Central

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

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

2014-01-01

204

UOK 268 Cell Line for Hereditary Leiomyomatosis and Renal Cell Carcinoma  

Cancer.gov

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

205

Antineoplastic activity of rinvanil and phenylacetylrinvanil in leukaemia cell lines  

PubMed Central

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.

LUVIANO, AXEL; AGUINIGA-SANCHEZ, ITZEN; DEMARE, PATRICIA; TIBURCIO, REYNALDO; LEDESMA-MARTINEZ, EDGAR; SANTIAGO-OSORIO, EDELMIRO; REGLA, IGNACIO

2014-01-01

206

Search for the critical characteristics of phenotypically different B cell lines, Burkitt lymphoma cells and lymphoblastoid cell lines, which determine differences in their functional interaction with allogeneic lymphocytes  

Microsoft Academic Search

Summary Burkitt lymphoma (BL) lines can be grouped according to phenotypic characteristics. Group I cells exhibit the phenotype of resting B cells and grow as single cells. Such lines can be Epstein-Barr-virus(EBV)-negative or -positive. Group II and group III cells are always EBV-positive, they express B cell activation markers, grow in aggregates and resemble in varying degrees lymphoblastoid cell lines

Javier Avila-Carifio; Sigurbjörg Torsteinsdottir; Barbro Ehlin-Henriksson; Maria G. Masucci; Eva Klein

1991-01-01

207

[DNA fingerprinting analysis of silkworm embryo cell lines].  

PubMed

DNA extraction and the polymerase chain reaction (PCR) were used on DNA genomes study of cell lines of Bombyx mori. DNA polymorphic marker analysis was conducted and DNA fingerprint of cell lines of Bombyx mori. was carried out using ISSR and RAPD. Primers that can reliably find polymorphic bands were screened out. 26 ISSR primers were selected from them any available, and 797 polymorphic bands were abtained through PCR amplification in 9 samples, including 3 embryo cell lines of Bombyx mori (BmE-SWU1, BmE-SWU2, BmE-SWU3), 5 passage cell lines (BmE, BmN, Sf9, Sf21, Hi5) and the embryos from which BmE-SWU1 originated. The ration of polymorphic bands was 89.9%. 43 RAPD primers were selected out through PCR amplification, and 1205 polymorphic bands were obtained in 9 samples. The ration of polymorphic bands was 76.6%. There were many DNA polymorphic bands differences in the cell lines of Bombyx mori. The special DNA markers of the 3 embryo cell lines were found respectively. The similarity index Nei and genetic distance of the 9 samples were calculated and the phylogeny tree of 9 samples was constructed by UPGMA. Results showed that 2 groups were divided,one group including the 3 embryo cell lines and the embryo of XQ has close relative. Another group constructed by five insect cell lines came from different species, their genetic distance was closer than the 3 embryo cell lines. PMID:17348206

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

2006-12-01

208

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

Microsoft Academic Search

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

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

1984-01-01

209

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

PubMed

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

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

2014-01-01

210

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

NASA Astrophysics Data System (ADS)

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

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

211

Engineering cultured insulin-secreting pancreatic B-cell lines  

Microsoft Academic Search

Despite many triumphs, a significant limitation of the usefulness of many of the available B-cell lines for the study of\\u000a insulin secretion are either inappropriate or lack of responsiveness to glucose. Commonly employed cell lines generated prior\\u000a to the 1990s following X-ray irradiation (RINm5F cells) or simian virus 40 B-cell transformation (HIT-T15 cells and BTC) fall\\u000a into this category. More

Neville H. McClenaghan; P. R. Flatt

1999-01-01

212

Characterization of a hormone-producing ovarian carcinoma cell line.  

PubMed

An ovarian carcinoma cell line (OTN 11) was produced from the ascitic fluid of a patient with a moderately to well differentiated papilliferous cystadenocarcinoma of the ovary. The cell line was characterized using electron microscopy karyotyping, immunohistochemical techniques with monoclonal antibodies against keratins as epithelial markers, and the monoclonal antibodies OV-TL 3 and OC 125 as ovarian carcinoma markers. These techniques revealed the epithelial and adenocarcinomatous nature of the cell line and the presence of ovarian carcinoma-related surface markers. The adenocarcinomatous nature of the cell line also became apparent after heterotransplantation of cell suspensions into nude mice and nude rats, in which adenomatous tumor structures were formed. These xenografts had the same ultrastructural and immunohistochemical properties as the cell line. Despite the adenocarcinomatous character of the tumor the cultured cells release estradiol into the culture medium. We may conclude that OTN 11 is an ovarian carcinoma cell line which has retained highly differentiated functions, such as the production of an ovarian hormone. PMID:2463217

Poels, L G; Jap, P H; Ramaekers, F F; Scheres, J M; Thomas, C M; Vooijs, P G; Croes, H J; Mungyer, G

1989-02-01

213

Vascular mimicry in cultured head and neck tumour cell lines  

PubMed Central

Introduction Vascuologenesis is the de novo establishment of blood vessels and vascular networks from mesoderm-derived endothelial cell precursors (angioblasts). Recently a novel mechanism, by which some genetically deregulated and aggressive tumour cells generate "micro-vascular" channels without the participation of endothelial cells and independent of angiogenesis, has been proposed. This has been termed "vasculogenic mimicry" and has implications beyond angiogenesis and adds another layer of complexity to the current concept for the generation of tumour micro-circulation. We suggest this is common phenomenon in head and neck squamous cell carcinoma (HNSCC) cell lines and other aggressive tumour cell lines. We present experimental evidence of vasculogenic mimicry in HNSCC cell lines and compare them with other tumours and a positive control vascular cell line. Materials and methods The cell lines used were HUVEC, HN 2a, 2b (primary and metastatic tongue base squamous carcinoma cell line), HCT116 (colonic carcinoma cell line) and DU145 (prostate carcinoma cell line). Pilot experiments were undertaken to assess growth of a bank of tumour cell lines on (growth factor reduced) matrigel (Sigma) with standard media (DMEM with 10% Fetal Calf Serum). A functional growth assay was performed by preparing the appropriate cell suspension in serum free medium plated onto either bare plastic or a well pre-coated with growth factor reduced type 4 collagen analogues. Phase contrast photomicrographs were taken at 4 hours and 24 hours. Image analysis was performed; particular features of interest were two dimensional area (surrogate of growth and migration), branch points and end point measurements (surrogate of intercellular complexity). Results There were observable differences in growth of the cells on laboratory plastic and collagen matrix. Tumour cells formed capillary like networks similar to HUVEC cells. Metastatic HNSCC cells lines were found to have vasculogenic properties similar to HUVEC cell lines when compared to cell lines from their corresponding primary tumour. The endothelial growth factor antibodies used did not inhibit or stimulate cell growth when compared to control but did discourage vascular mimicry. Other tumour cell lines also displayed this property. Discussion Tumour "vasculogenic mimicry" must still be regarded as a controversial issue whose existence is not proven. The clinical importance of this phenomenon however, is that it does explain the lack of complete efficacy of current anti-angiogenic treatments due to the added layer of complexity. It provides a feasible mechanism of early tumour vascular supply which can co-exist and incorporate with later angiogenic mechanisms. We suggest that "vasculogenic mimicry" maybe a common neoplastic phenomena which appears to also be dictated by the cells micro-environment. Its existence also suggests a further process that of the development of tumour mosaic vessels as the neo-vasculature integrates with the existing endothelial lined systems.

2011-01-01

214

Characteristics of nine newly derived mesothelioma cell lines.  

PubMed

This report characterizes nine new cell lines derived from patients with malignant pleural mesothelioma. The lines were initiated between July 1990 and July 1992 from solid tumors (5 lines) or effusions (4 lines) and had proliferated for a period of at least 2 months without senescence. They were characterized by cell size, doubling time, immunohistochemical analyses, electron microscopy, and chromosomal karyotyping. Growth factor/cytokine elaboration was determined using enzyme-linked immunoassays. The established lines were similar in morphology to their parent tumor (ie, epithelial or sarcomatoid). Cell sizes ranged from 59 to 81 microns, and the doubling times varied from 31 to 65 hours. The lines stained with cytokeratin and showed expected negative staining for adenomarkers including B72.3 and carcinoembryonic antigen. All cell lines exhibited aneuploidy, with modal chromosome numbers between 40 and 81 and had multiple chromosomal aberrations. Significant production of granulocyte-monocyte colony-stimulating factor, leukemia inhibitory factor, platelet-derived growth factor, and interleukin-6 was seen. These new cell lines derived from human mesotheliomas can now be used to aid in the design of innovative treatment strategies. PMID:7695406

Pass, H I; Stevens, E J; Oie, H; Tsokos, M G; Abati, A D; Fetsch, P A; Mew, D J; Pogrebniak, H W; Matthews, W J

1995-04-01

215

[The effects of actovegin on cell proliferation of permanent lines].  

PubMed

The influence of Actovegin on proliferation activity and mitotic regimen of cells of permanent lines PK-15-IEKVM and BHK-21 clone 13/04 was investigated. Addition of Actovegin into growth media containing bovine serums of different components and concentrations stimulates cell proliferation. Conclusion has been made that Actovegin can be used in cell culture biotechnology. PMID:18411759

Gulevski?, A K; Trifonova, A V; Lavrik, A A

2008-01-01

216

Development of multidrug resistance in a canine lymphoma cell line.  

PubMed

New multidrug resistant cell lines developed from the canine B cell lymphoma cell line (GL-1) were characterized in terms of chemosensitivity to some antineoplastics and P-glycoprotein (Pgp) expression. GL-1 was continuously exposed to a culture medium containing gradually increasing levels of doxorubicin and the cells that could grow in the presence of doxorubicin were obtained. Chemosensitivity of these cells to various antineoplastics were investigated with or without verapamil, which reversed Pgp-mediated drug resistance. The expression of Pgp on the cells was also examined by Western blot analysis. As a result, three kinds of resistant cell lines, designated as GL-DOX60, 300, and 4000 were obtained. These cell lines showed stable proliferation in the medium containing 60, 300, and 4000 ng/ml, respectively. These cells were much more resistant to vincristine than doxorubicin. This resistance was strongly reversed by the presence of verapamil. On the other hand, cisplatin was effective enough in killing these derived cells. In the Western Blot analysis, some bands that reacted to the anti-human Pgp monoclonal antibodies were observed in GL-DOX4000. The cells derived from GL-1 have multidrug resistance potential mediated by canine Pgp. The cells produced in this experimental trial are considered to be useful models for various investigations on canine multidrug resistance. PMID:15766940

Uozurmi, K; Nakaichi, M; Yamamoto, Y; Une, S; Taura, Y

2005-06-01

217

Establishment and characterization of an oral melanoma cell line (ME)  

Microsoft Academic Search

A new cell line, ME, has been established from a melanoma of the palatal mucosa. The cultured monolayer of cells was fusiform and melanin-producing. The cells were highly tumorigenic and metastatic in nude mice. The xenographic tumors resembled the original tumor in morphology, melanin production, and the expression of S-100 and HMB-45 antigens. The metaphase karyotype of ME indicated multiple

Kuo-Wei Chang; Shu-Chun Lin; Shou-Yee Chao; Po-Cheung Kwan; Chun-Po Chiu; Yong-Kie Wong

2001-01-01

218

Evolution and ultrastructure of the bovine spermatogonia precursor cell line  

Microsoft Academic Search

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

Karl-Heinz Wrobel; Daniela Bickel; Richard Kujat; Margit Schimmel

1995-01-01

219

Clonal cell lines from the rat central nervous system  

Microsoft Academic Search

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

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

1974-01-01

220

Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines  

Microsoft Academic Search

Purpose: Doxorubicin (DOX) is a commonly used anticancer drug which causes DNA damage and kills cancer cells mainly by apoptosis. However, the process leading to killing of cancer cells and the molecular basis of resistance to DOX are not well understood. To evaluate the role of p53 and the cellular effects of DOX on hepatoma cell lines, we examined three

Terence Lee; Tracy Lau; Irene Ng

2002-01-01

221

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

PubMed

L1 retro-elements comprise 17% of the human genome. Approximately 100 copies of these autonomous mobile elements are active in our DNA and can cause mutations, gene disruptions, and genomic instability. Therefore, human cells control the activities of L1 elements, in order to prevent their deleterious effects through different mechanisms. However, some toxic agents increase the retrotransposition activity of L1 elements in somatic cells. In order to identify specific effects of neurotoxic metals on L1 activity in neuronal cells, we studied the effects of mercury and cobalt on L1-retroelement activity by measuring levels of cellular transcription, protein expression, and genomic retrotransposition in a neuroblastoma cell line compared with the effects in three non-neuronal cell lines. Our results show that mercury increased the expression of L1 RNA, the activity of the L1 5'UTR, and L1 retrotransposition exclusively in the neuroblastoma cell line but not in non-neuronal cell lines. However, cobalt increased the expression of L1 RNA in neuroblastoma cells, HeLa cells, and wild-type human fibroblasts, and also increased the activity of the L1 5'UTR as well as the SV40 promoter in HeLa cells but not in neuroblastoma cells. Exposure to cobalt did not result in increased retrotransposition activity in HeLa cells or neuroblastoma cells. We conclude that non-toxic levels of the neurotoxic agent mercury could influence DNA by increasing L1 activities, specifically in neuronal cells, and may make these cells susceptible to neurodegeneration over time. PMID:24240092

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

2014-01-01

222

MORPHOMETRIC SUBTYPING FOR A PANEL OF BREAST CANCER CELL LINES  

SciTech Connect

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.

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

2009-05-08

223

Effects of ethanol on an intestinal epithelial cell line  

SciTech Connect

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.

Nano, J.L.; Cefai, D.; Rampal, P. (Laboratoire de Gastroenterologie et de Nutrition, U.E.R. de Medecine, Nice (France))

1990-02-01

224

DNA Fingerprinting of the NCI-60 Cell Line Panel  

PubMed Central

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

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

2009-01-01

225

Cancer Stem Cell-Like Side Population Cells in Clear Cell Renal Cell Carcinoma Cell Line 769P  

PubMed Central

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. Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells as compared with other four 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. 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.

Yao, Zhi Jun; Chen, Xu; Guo, Sheng Jie; Mao, Xiao Peng; Wang, Dao Hu; Chen, Jun Xing; Qiu, Shao Peng

2013-01-01

226

Human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50 are admixtures of the human colon carcinoma cell line HCT 116  

Microsoft Academic Search

In two recently described human oesophageal adenocarcinoma cell lines JROECL 47 and JROECL 50, derived from one tumour, we detected identical E-cadherin and ?-catenin gene mutations as in colon carcinoma cell line HCT 116. We demonstrate by HLA-typing, mutation analysis and microsatellite analysis that cell lines JROECL 47 and JROECL 50 are admixtures of the human colon adenocarcinoma cell line

B P L Wijnhoven; M G J Tilanus; A G Morris; S J Darnton; H W Tilanus; W N M Dinjens

2000-01-01

227

Sensing cell line to dioxin-type chemicals  

NASA Astrophysics Data System (ADS)

Detection dioxins-type chemicals by a sensing cell line containing luciferase reporter gene under the control of dioxin responsive elements was compared with the traditional ethoxyresorufin O-deethylase (EROD) induction. The result suggested that the luciferase induction was 30-fold more sensitive than EROD induction, the detection time was shorter for 68-hour and the detection procedure was also simpler. The data showed the sensing cell line can screen lots of samples and quickly semi-quantitate.

Zhang, Zhiren; Xu, Shunqing; Zhou, Yikai; Cai, Xiaokun; Liu, Zhiwei

2001-09-01

228

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

Cancer.gov

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

229

Isoenzyme patterns in soybean- Nicotiana somatic hybrid cell lines  

Microsoft Academic Search

Zymograms obtained by polyacrylamide gel electrophoresis of alcohol dehydrogenase (EC 1.1.1.1.) and aspartate aminotransferase (EC 2.6.1.1.) from 5 different soybean-Nicotiana hybrid cell lines showed enzymatic characteristics derived from both parents. Variations in the zymogram of the cell lines were observed during a culture period of 8 months (more than 100 generations). These variations may be related to chromosomal loss from

L. R. Wetter

1977-01-01

230

Reliable in vitro studies require appropriate ovarian cancer cell lines  

PubMed Central

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.

2014-01-01

231

Modulation of melphalan cytotoxic activity in human melanoma cell lines.  

PubMed

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

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

1996-07-01

232

Mammalian cell line developments in speed and efficiency.  

PubMed

Mammalian cell expression systems are the dominant tool today for producing complex biotherapeutic proteins. In this chapter, we discuss the basis for this dominance, and further explore why the Chinese hamster ovary (CHO) cell line has become the prevalent choice of hosts to produce most recombinant biologics. Furthermore, we explore some of the innovations that are currently in development to improve the CHO cell platform, from cell line specific technologies to overarching technologies that are designed to improve the overall workflow of bioprocess development. PMID:24196317

Estes, Scott; Melville, Mark

2014-01-01

233

Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines  

PubMed Central

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.

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

2012-01-01

234

Dengue2 virus infection of human mononuclear cell lines and establishment of persistent infections  

Microsoft Academic Search

Summary Twenty three human mononuclear cell lines including ten myelomonocytic cell lines, eight B cell lines and five T cell lines, were examined to determine whether they could be infected with dengue-2 virus. All the cell lines were infected with dengue-2 virus as determined by immunofluorescent staining and by virus titration of culture supernatant fluids. K 562, Jiyoye and Jurkat,

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

1990-01-01

235

Comparative analysis of cell death induction by Taurolidine in different malignant human cancer cell lines  

PubMed Central

Background Taurolidine (TRD) represents an anti-infective substance with anti-neoplastic activity in many malignant cell lines. So far, the knowledge about the cell death inducing mechanisms and pathways activated by TRD is limited. The aim of this study was therefore, to perform a comparative analysis of cell death induction by TRD simultaneously in different malignant cell lines. Materials and methods Five different malignant cell lines (HT29/Colon, Chang Liver/Liver, HT1080/fibrosarcoma, AsPC-1/pancreas and BxPC-3/pancreas) were incubated with increasing concentrations of TRD (100 ?M, 250 ?M and 1000 ?M) for 6 h and 24 h. Cell viability, apoptosis and necrosis were analyzed by FACS analysis (Propidiumiodide/AnnexinV staining). Additionally, cells were co-incubated with the caspase Inhibitor z-VAD, the radical scavenger N-Acetylcystein (NAC) and the Gluthation depleting agent BSO to examine the contribution of caspase activation and reactive oxygen species in TRD induced cell death. Results All cell lines were susceptible to TRD induced cell death without resistance toward this anti-neoplastic agent. However, the dose response effects were varying largely between different cell lines. The effect of NAC and BSO co-treatment were highly different among cell lines - suggesting a cell line specific involvement of ROS in TRD induced cell death. Furthermore, impact of z-VAD mediated inhibition of caspases was differing strongly among the cell lines. Conclusion This is the first study providing a simultaneous evaluation of the anti-neoplastic action of TRD across several malignant cell lines. The involvement of ROS and caspase activation was highly variable among the five cell lines, although all were susceptible to TRD induced cell death. Our results indicate, that TRD is likely to provide multifaceted cell death mechanisms leading to a cell line specific diversity.

2010-01-01

236

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

Cancer.gov

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

237

Lack of functional erythropoietin receptors of cancer cell lines.  

PubMed

Erythropoietin (Epo) therapy reduces red cell transfusion requirements and improves the quality of life of anemic cancer patients receiving chemotherapy. However, there is concern that Epo may promote tumor growth. We investigated by real-time RT-PCR, immunofluorescence microscopy, Western blotting and cell growth analysis whether human cancer cell lines (SH-SY5Y, MCF7, HepG2, U2-OS, HeLa, HEK293T, RCC4, HCT116, 7860wt and SW480) possess functional Epo receptors (EpoR). We detected EpoR mRNA in all cell lines. Neither hypoxia nor Epo treatment altered the level of EpoR mRNA expression. Four commonly used commercial antibodies proved to be unsuitable for immunoblot procedures because they cross-reacted with several proteins unrelated with EpoR. Depending on the antibody used, EpoR was localized to the plasma membrane, the cytoplasm or the nucleus. Experiments with small interfering RNA showed that EpoR protein was not expressed by the tumor cells except by UT7/Epo leukemia cells, which served as an EpoR positive control line, and by cells transfected with the human EpoR gene. Apart from UT7/Epo, none of the tumor cell lines responded to Epo treatment with phosphorylation of signaling molecules or with cell proliferation. PMID:17990315

Laugsch, Magdalena; Metzen, Eric; Svensson, Tanja; Depping, Reinhard; Jelkmann, Wolfgang

2008-03-01

238

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

PubMed Central

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

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

1991-01-01

239

Stem-like Cells in Bladder Cancer Cell Lines with Differential Sensitivity to Cisplatin  

PubMed Central

Background Recurrence is a common problem in bladder cancer; this has been attributed to cancer stem cells. In this study, we characterized potential cancer stem cell populations isolated from three cell lines that demonstrate different responses to cisplatin. Materials and Methods The ALDEFLUOR® assay was used to isolate cells from TCCSUP, T24, and 5637 cell lines, and these cells were evaluated for their ability to form colonies, differentiate, migrate and invade. Results The cell lines demonstrate a spectrum of aldehyde dehydrogenase high (ALDHHigh)populations that correlate with resistance to cisplatin. In the two resistant cell lines, T24 and 5637, the ALDHHigh cells demonstrate increased colony formation, migration, invasion, and ability to differentiate. The resistant T24 and 5637 cell lines may serve as models to investigate alternative therapies for bladder cancer.

Sarachine Falso, Miranda J.; Buchholz, Bruce A.; deVere White, Ralph W.

2013-01-01

240

Generation of stable Drosophila cell lines using multicistronic vectors  

PubMed Central

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

Gonzalez, Monika; Martin-Ruiz, Itziar; Jimenez, Silvia; Pirone, Lucia; Barrio, Rosa; Sutherland, James D.

2011-01-01

241

[Effect of NKG2D in eliminating hematological malignant cell lines by natural killer cells].  

PubMed

The aim of this study was to clarify whether NKG2D plays an activating role in eliminating hematological malignant cells lines by natural killer (NK) cells. Several hematological malignant cell lines (K562, NB4, Kasumi-1 THP-1, MV-4-11, MOLT-4, Jurkat, RS4; 11, Raji) were used as target cells. The expression levels of major histocompatibility complex class I (MHC I)-related molecules A/B (MICA, MICB), whose corresponding ligand was NKG2D, were detected in target cells by flow cytometry. Firstly, the target cell lines were co-incubated with carboxyfluorescein succinimidyl ester (CFSE) for 30 min. In the meanwhile, NK92MI, a kind of NK cell line, was co-incubated respectively with isotype control antibody or blocking antibody, the latter could block NKG2D specifically. Then, NK92MI cells were co-cultured with different target cell lines. After incubation for 2 h, the apoptotic ratio of each target cell line was detected by flow cytometry. The results demonstrated that there was a significant reduction of the apoptotic ratio in Kasumi-1, an acute myeloid leukemia cell line, when NK92MI cells were incubated with NKG2D blocking antibody previously. In contrast, the apoptotic ratio of other cell lines varied minimally. It is concluded that NKG2D can activate NK cells through inducing cytotoxicity to certain target cells. PMID:22541085

Wang, Wei; Gao, Li; Ma, Yi-Gai

2012-04-01

242

Novel cell lines established from pediatric brain tumors.  

PubMed

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

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

2012-04-01

243

Novel cell lines established from pediatric brain tumors  

PubMed Central

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

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

2012-01-01

244

Three-dimensional cultured glioma cell lines  

NASA Technical Reports Server (NTRS)

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.

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

1991-01-01

245

Human embryonic stem cell lines derived from single blastomeres.  

PubMed

The derivation of human embryonic stem (hES) cells currently requires the destruction of ex utero embryos. A previous study in mice indicates that it might be possible to generate embryonic stem (ES) cells using a single-cell biopsy similar to that used in preimplantation genetic diagnosis (PGD), which does not interfere with the embryo's developmental potential. By growing the single blastomere overnight, the resulting cells could be used for both genetic testing and stem cell derivation without affecting the clinical outcome of the procedure. Here we report a series of ten separate experiments demonstrating that hES cells can be derived from single blastomeres. In this proof-of-principle study, multiple biopsies were taken from each embryo using micromanipulation techniques and none of the biopsied embryos were allowed to develop in culture. Nineteen ES-cell-like outgrowths and two stable hES cell lines were obtained. The latter hES cell lines maintained undifferentiated proliferation for more than eight months, and showed normal karyotype and expression of markers of pluripotency, including Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, nanog and alkaline phosphatase. These cells retained the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. The ability to create new stem cell lines and therapies without destroying embryos would address the ethical concerns of many, and allow the generation of matched tissue for children and siblings born from transferred PGD embryos. PMID:16929302

Klimanskaya, Irina; Chung, Young; Becker, Sandy; Lu, Shi-Jiang; Lanza, Robert

2006-11-23

246

Isolation and characterization of cancer stem-like cells from MHCC97H Cell Lines  

Microsoft Academic Search

ObjectiveTo identify and isolate CD133 positive cancer stem-like cells (CD133+ cells) from the highly invasive human hepatocellular carcinoma cell line(MHCC97H), and examine their potential for clonogenicity and tumorigenicity.

Shanyong Yi; Kejun Nan; Aihua Yuan; Chuangxin Lu

2009-01-01

247

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

Microsoft Academic Search

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

Yoshinobu Matsuo; Hans G Drexler

1998-01-01

248

Growth inhibition of newly established human glioma cell lines by leukemia inhibitory factor  

Microsoft Academic Search

We have established three new cell lines deriving from malignant human gliomas. The cell lines were described in terms of both morphology and growth characteristics. Most cells in all three cell lines expressed the neuroepithelial marker protein GFAP. In terms of growth characteristics, the cells showed only slight differences. The cell lines showed no expression of the neural form of

Hartmut Halfter; Joachim Kremerskothen; Jürgen Weber; Ursula Hacker-Klom; Angelika Barnekow; E. Bernd Ringelstein; Florian Stögbauer

1998-01-01

249

HEL Cells: A New Human Erythroleukemia Cell Line with Spontaneous and Induced Globin Expression  

Microsoft Academic Search

A new human erythroleukemia cell line has been established. This line, designated HEL, is capable of spontaneous and induced globin synthesis, producing mainly Ggamma and Agamma chains. Embryonic chains (? , zeta ) and alpha chains are detectable in very small amounts; beta chains are undetectable. This line provides a new model system for studying aspects of erythroid cell differentiation

Paul Martin; Thalia Papayannopoulou

1982-01-01

250

Membrane Nanotubes in Urothelial Cell Line T24  

Microsoft Academic Search

Membrane nanotubes (also referred as tunnelling nanotubes—TNTs, nanotu- bules, cytonemes), that directly connect separated neighboring cells, may offer a very specific and effective way of intercellular transport and communication. Our experiments on T24 cell line show that TNTs can be divided into two types with respect to their biochemical and biophysical characteristics and the nature of their formation. As type

CHAPTER T HREE; Marusa Lokar; Sarka Perutkova; Veronika Kralj-Iglic; Peter Veranic

251

Chapter 3 Membrane Nanotubes in Urothelial Cell Line T24  

Microsoft Academic Search

Membrane nanotubes (also referred as tunnelling nanotubes—TNTs, nanotubules, cytonemes), that directly connect separated neighboring cells, may offer a very specific and effective way of intercellular transport and communication. Our experiments on T24 cell line show that TNTs can be divided into two types with respect to their biochemical and biophysical characteristics and the nature of their formation. As type I

Maruša Lokar; Šárka Perutková; Veronika Kralj-Igli?; Aleš Igli?; Peter Verani?

2009-01-01

252

In-line load cell for flexible strength member materials  

Microsoft Academic Search

An in-line load cell for flexible strength member materials is presented. An aluminum cylindrical body section houses two steel pins that perpendicularly pass through the longitudinal axis of the body section. Both steel pins extend from either side of the body section to provide four wrapping points for a flexible strength member to be tested. The load cell is placed

Joseph Liguore

1992-01-01

253

Epithelial Cell Line Expressing a Cystic Fibrosis Phenotype.  

National Technical Information Service (NTIS)

An airway epithelial cell line (CF/T43) was developed by infecting cultured cystic fibrosis (CF) airway epithelial cells with the pZIPneoSV(X)1/SV40T retrovirus and selecting for Genetian (G418) resistance and ion transport properties. The distinctive chl...

A. M. Jetten J. R. Yankaskas

1989-01-01

254

Differences in Arachidonic Acid Metabolism by Human Myelomonocytic Cell Lines.  

National Technical Information Service (NTIS)

The production of arachidonic acid metabolites by the HL60, ML3, and U937 human phagocyte cell lines was determined after incubation with interferon-gamma (IFN-gamma, 500 U/ml) or vehicle for 4 days. Cells were prelabeled with tritiated arachidonic acid, ...

M. C. Madden S. Becker H. S. Koren M. Friedman

1992-01-01

255

Elimination of mycoplasma from human B-lymphoblastoid cell lines.  

PubMed

Intraperitoneal passage of human B-lymphoblastoid cell lines in nude mice was examined as a means of mycoplasma eradication. Recovery of viable cells from the mice was facilitated by immediate plating on feeder layers of human foreskin fibroblasts. In all cases, nude mouse passage for as little as 5 days was totally effective in removing all contaminating mycoplasma. PMID:6983518

Howell, D N; Machamer, C E; Cresswell, P

1982-11-01

256

Generation and characterization of a mouse lymphatic endothelial cell line  

Microsoft Academic Search

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

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

2006-01-01

257

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 ...

258

Taurine chloramine induces apoptosis in human osteosarcoma cell lines.  

PubMed

Although combination of surgery with chemotherapy has noticeably improved the survival rate of osteosarcoma patients, the application of anticancer drugs is still associated with significant adverse reactions, for instance acquisition of drug-resistant phenotypes, necessitating the development of new chemotherapeutical agents. Therefore, the aim of this study was to research, if taurine chloramine (NCT) induces apoptosis in the osteosarcoma cell lines HOS, MG-63, and SAOS-2. Proliferation of osteosarcoma cells was detected with the "EZ4U Cell Proliferation and Cyotoxicity Assay" showing a time- and dose-dependent cytotoxic effect of NCT on these cell lines. After 3 h of incubation all cell lines showed significantly less cells at 5.5 mM NCT solutions, after 6 h at concentrations of 1.1 and 2.2 mM. Acridine-orange fluorescence nuclear staining showed characteristic features of apoptosis. DNA fragmentation was detected via ELISA, showing significant results for HOS and MG-63 after 6 h at an NCT concentration of 3.3 mM. Results of JC-1 mitochondrial FACS analysis presented a significant increase in apoptotic cells after 6 h at 3.3 mM for the tested cell lines. Summarized, the results of this study indicate that NCT is a promising agent in osteosarcoma therapy. PMID:22674504

Pilz, Magdalena; Holinka, Johannes; Vavken, Patrick; Marian, Brigitte; Krepler, Petra

2012-12-01

259

Pluripotency network in porcine embryos and derived cell lines.  

PubMed

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals. PMID:22827355

Brevini, Tal; Pennarossa, G; Maffei, S; Gandolfi, F

2012-08-01

260

Differential effects of monastrol in two human cell lines.  

PubMed

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

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

2004-08-01

261

Metabolic characteristics of different malignant cancer cell lines.  

PubMed

Extracellular AMP inhibits cell proliferation of MCF-7 and MDA-MB-453 whereas cell proliferation of the highly malignant Novikoff cell line is not affected. In medium with low glucose supply MDA-MB-453 cells grow well, Novikoff cells are slightly inhibited and MCF-7 cells are totally unable to grow. Isoelectric focusing revealed that a glyclytic enzyme complex exists in all three cell lines. In addition to the glycolytic enzymes, c-Raf-kinase, adenylate kinase, and nucleoside diphosphate kinase are also found within the complex. The differences in glucose in dependence of the three cell lines can be explained by the different constitutions of shuttle enzymes. MDA-MB-453 and Novikoff cells contain cytsolic glycerol 3-phosphate dehydrogenase which is associated with glyceraldehyde 3-phosphate dehydrogenase within the glycolytic enzyme complex and which is responsible for the transport of cytoslic hydrogen in the mitochondria. MCF-7 and Novikoff cells contain the pI 7.8 form of malate dehydrogenase which couples glycolysis with glutaminolysis. PMID:9858895

Mazurek, S; Grimm, H; Wilker, S; Leib, S; Eigenbrodt, E

1998-01-01

262

Basic protocols for zebrafish cell lines: maintenance and transfection.  

PubMed

Cell lines derived from zebrafish embryos show great potential as cell culture tools to study the regulation and function of the vertebrate circadian clock. They exhibit directly light-entrainable rhythms of clock gene expression that can be established by simply exposing cultures to light-dark cycles. Mammalian cell lines require treatments with serum or activators of signaling pathways to initiate transient, rapidly dampening clock rhythms. Furthermore, zebrafish cells grow at room temperature, are viable for long periods at confluence, and do not require a CO2-enriched atmosphere, greatly simplifying culture conditions. Here we describe detailed methods for establishing zebrafish cell cultures as well as optimizing transient and stable transfections. These protocols have been successfully used to introduce luciferase reporter constructs into the cells and thereby monitor clock gene expression in vivo. The bioluminescence assay described here lends itself particularly well to high-throughput analysis. PMID:17417032

Vallone, Daniela; Santoriello, Cristina; Gondi, Srinivas Babu; Foulkes, Nicholas S

2007-01-01

263

Reconstruction of endometrium from human endometrial side population cell lines.  

PubMed

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 (ER?) 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

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

2011-01-01

264

Establishment, Immortalisation and Characterisation of Pteropid Bat Cell Lines  

PubMed Central

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.

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

265

Non-targeted radiation effects in vertebrate cell lines  

NASA Astrophysics Data System (ADS)

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

Ryan, Lorna

266

Artificial islets from hybrid spheroids of three pancreatic cell lines.  

PubMed

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

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

2014-05-01

267

Nitric Oxide-Dependent Apoptosis in Ovarian Carcinoma Cell Lines  

Microsoft Academic Search

Objective. In a recent study, we found different profiles of inducible nitric oxide synthase (iNOS) gene expression in the ovarian carcinoma cell lines OVCAR-3, HOC-7, and 2774 following stimulation by proinflammatory cytokines. The present study was performed to determine whether nitric oxide (NO) synthesis correlates with programmed cell death in these cells.Methods. NO-Dependent apoptosis was detected by DNA fragmentation analysis

Josef Rieder; Rolf Jahnke; Michaela Schloesser; Maja Seibel; Monika Czechowski; Christian Marth; Georg Hoffmann

2001-01-01

268

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

PubMed

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

Szepes, Mónika; Janicsek, Zsófia; Benk?, Zsolt; Cselenyák, Attila; Kiss, Levente

2013-01-01

269

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

PubMed Central

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

SZEPES, MONIKA; JANICSEK, ZSOFIA; BENKO, ZSOLT; CSELENYAK, ATTILA; KISS, LEVENTE

2012-01-01

270

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

Microsoft Academic Search

Animal insulinoma cell lines are widely used to study physiological and pathophysiological mechanisms involved in glucose metabolism and to establish in vitro models for studies on ‚-cells. In contrast, human insulinoma cell lines are rarely used because of diYculties in obtaining and culturing them for long periods. The aim of our study was to investigate, under diVerent experimental conditions, the

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

1999-01-01

271

Adaptation of an insect cell line ( Agallia constricta ) in a mammalian cell culture medium  

Microsoft Academic Search

Summary  An established insect cell line (AC20) from the leafhopperAgallia constricta has been adapted to a mammalian cell culture medium based on the formulation of two commercially available media. The cell\\u000a population doubling time of the adapted line in this medium is approximately 45 hr at 30C.

Arthur H. Mc Intosh; K. Maramorosch; C. Rechtoris

1973-01-01

272

Second-line treatment outcomes after first-line sunitinib therapy in metastatic renal cell carcinoma.  

PubMed

This study was conducted to evaluate the treatment outcomes associated with common second-line targeted therapies given after first-line sunitinib for metastatic renal cell carcinoma (mRCC). The sample comprised patients with mRCC (n = 257) who were receiving second-line everolimus, sorafenib, or temsirolimus between April 1, 2008, and February 29, 2011, after first-line sunitinib treatment. The patients were followed-up from the start of second-line treatment until treatment failure (defined as advancement to a third-line therapy or to mortality) or the last observation in the medical and pharmacy databases. Treatment failure was observed in 38.5% (n = 99) of cases: 20.2% of patients (n = 52) advanced a line of treatment; and 18.3% of patients (n = 47) died. Kaplan-Meier analysis indicated a statistical difference in time to treatment failure among the 3 second-line targeted therapies (log-rank test, P = .045). The estimated 1-year cumulative probabilities of treatment failure were 49.9% for everolimus, 68.4% for sorafenib, and 71.4% for temsirolimus. In a multivariate Cox proportional hazards model, a higher adjusted risk of treatment failure vs. everolimus was observed for both temsirolimus (hazard ratio [HR] 2.05 [95% CI, 1.26-3.35]; P = .004) and sorafenib (HR 1.77 [95% CI, 1.02-3.07]; P = .043). The results of this real-world data analysis suggest that the risk of second-line treatment failure after first-line sunitinib was significantly higher with temsirolimus and sorafenib compared with everolimus. PMID:22682982

Chen, Chi-Chang; Hess, Gregory P; Liu, Zhimei; Gesme, Dean H; Agarwala, Sanjiv S; Garay, Carlos C; Hill, Jerrold W; Guo, Amy

2012-12-01

273

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

PubMed

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

Wrzosek, Antoni

2014-07-01

274

Establishment and characterization of five human small cell lung cancer cell lines from early tumor xenografts.  

PubMed

Five small-cell lung carcinoma (SCLC) cell lines were established from xenografted tumor lines. These tumor lines were established after transplantation into nude mice of primary tumors or metastatic foci obtained surgically, from untreated (IRSC-2M, IRSC6M, IRSC-10M and IRSC-61M) or treated patients (IRSC-74M). They were then set-up in culture as parallel cell lines. Histologically, these tumor lines were classified as being of the classic (IRSC-2M, IRSC-10M and IRSC-61M) or intermediate type (IRSC-6M and IRSC74M). Four of these 5 SCLC cell lines grew as floating cell aggregates, while one (IRSC6M) grew as an adherent cell monolayer. Growth rates were slow (doubling times ranged between 120 and 194 h) but could be accelerated (67 to 144 h) by cultivating cells in medium mixed (v/v) with self-conditioned medium. Electron microscopical examination revealed that all SCLC cell lines contained dense core granules, characteristic of their neuroendocrine origin. These cell lines formed colonies in agarose with colony forming efficiencies ranging from 0.02-0.36%. The classic-type cell lines retained their tumorigenic capacity when re-injected intracranially into naive nude mice, whereas the intermediatetype cells did not. Cytogenetic analysis confirmed the human origin of SCLC xenografts and cultured cell lines. Various numerical and structural chromosome abnormalities were found, with deletion in the short arm of chromosome 3 being the most common (4 of the 5 cell lines). Deletions in or loss of the chromosome 10 were also observed. Oncogene expression was studied in 3 representative cell lines (IRSC-10M, IRSC-2M and IRSC-74M). L-myc was overexpressed only in IRSC-74M, while the GRP gene was overexpressed in the classic (IRSC-2M and IRSC-10M) but not in the intermediate-type cells (IRSC-74M). The Ki-ras oncogene was overexpressed in the 3 cell lines, while c-myc, N-myc, Ha-ras, N-ras, erb B2 and sis were not detected in any of them. The 3 cell lines weakly expressed the MDR1 gene, while the GST-pi gene was not expressed. These cell lines constitute a multifaceted well-characterized in vitro model for studying the biology of these phenotypically diverse cancer cells. PMID:7847823

Arvelo, F; Poupon, M F; Le Chevalier, T

1994-01-01

275

Identity of some human bladder cancer cell lines.  

PubMed

Recent reports on transfection of mouse cells with DNA from the established human urinary bladder cancer cell lines T24, J82 and EJ (MGH-U1), and the presence of an identical genetic modification in T24 and EJ cells have led us to examine the identity of these and other cultures of urothelial origin. By the criteria of HLA-A-B-C typing 7 and isozyme analysis, we conclude that EJ (MGH-U1) and some cultures of J82 are in fact T24 cells. However, five other bladder cancer cell lines, J82 (CO'T), RT4, RT112, TCCSuP and SCaBER, are clearly distinct from T24 by HLA typing (ref. 7) and/or isozyme patterns. PMID:6823318

O'Toole, C M; Povey, S; Hepburn, P; Franks, L M

1983-02-01

276

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

PubMed

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

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

1977-02-01

277

The ‘monocytic’ cell line I937 displays typical B cell characteristics  

Microsoft Academic Search

The monocytic cell line I937 was derived from U937 by sorting for cells with high expression of MHC class II molecules. Further analysis of these class II molecules revealed the presence of the HLA-DR3 subtype suggesting that the cell line was a potential candidate for testing antigen presentation to T cells restricted by HLA-DR3. We found that the T cell

I. G. Reischl; E.-M. Pöllabauer; S. Peiritsch; S. Schlager; P. Gladstone; W. C. Vooijs; M. Woisetschläger; G. C. Mudde

1996-01-01

278

Cell surfactomes of two endometrial epithelial cell lines that differ in their adhesiveness to embryonic cells.  

PubMed

Adhesiveness of the endometrial epithelium to an embryo plays a critical role in the initiation of pregnancy. Loss or gain of adhesiveness also dictates the potential of endometrial epithelial cells to metastasize, an event that can result from certain genetic insults. A proteomics-based exploration of the "adhesiveness" these epithelial cells was employed that could identify targets that could disrupt embryo-endometrium interactions and/or metastasis of endometrial cancer cells. The present study defined the surfactomes of two human endometrial epithelial cell lines known for their differential adhesiveness to embryonic cells. Comparative, two-dimensional electrophoretic analysis of the surfactomes of RL95-2 (exhibiting higher adhesiveness to the embryonic cell line JAr) and HEC-1A (exhibiting reduced adhesiveness to JAr cells) revealed 55 differentially enriched proteins. Of these, 10 proteins were identified by MALDI-TOF/TOF or LC-MS/MS. TUBB2C, ADAMTS3, and elongation factor beta were more abundant on the HEC-1A cell surface whereas HSP27, HSPA9, GP96, CRT, Tapasin-ERP57, PDI, and ?-actin were more abundant on the RL95-2 cell surface. Nano LC-MS/MS was also employed to generate a more comprehensive surfactomes of RL95-2 and HEC-1A. The study also demonstrated a pro-adhesive role of CRT and HSPA9 and an anti-adhesive role of TUBB2C populations found on the cell surface. In brief, this study identifies the cell-surface protein complements of two human endometrial epithelial cell lines, and reveals the role of three proteins in heterotypic cell adhesion. PMID:24415223

Bhagwat, Sonali R; Redij, Tejashree; Phalnikar, Kruttika; Nayak, Sumeet; Iyer, Swati; Gadkar, Sushama; Chaudhari, Uddhav; Kholkute, Sanjeeva D; Sachdeva, Geetanjali

2014-04-01

279

Xenobiotic metabolism and mutation in a human lymphoblastoid cell line.  

PubMed

Aryl hydrocarbon hydroxylase-1 (AHH-1) cells are a human lymphoblastoid cell line competent in some aspects of xenobiotic metabolism. This cell line contains stable mixed function oxidase activity which is inducible by polycyclic aromatic hydrocarbons (PAHs) but not by phenobarbital or Arochlor 1254. Two substrates for the cellular mixed function oxidase activity, benzo[a]pyrene (B[a]P) and 7-ethoxyresorufin, have been examined. The basal and induced activities have different kinetic parameters toward these two substrates. In contrast, basal and induced activities had similar sensitivities to two cytochrome P-450 suicide substrates. B[a]P metabolism and mutagenicity were studied in this cell line. AHH-1 cells were found to produce predominantly B[a]P phenols and quinones. The major phenol metabolite cochromatographed with authentic 9-hydroxy B[a]P. AHH-1 cells were capable of forming glucuronic acid conjugates of B[a]P phenols; the major product after hydrolysis cochromatographed with 3-hydroxy B[a]P standard. AHH-1 cells did not contain detectable epoxide hydrolase activity using B[a]P-4,5-oxide as substrate. This observation is consistent with the absence of trans-dihydrodiol B[a]P metabolites in the metabolic profile. B[a]P-induced mutagenicity at the hypoxanthine guanine phosphoribosyl transferase (hgprt) locus in AHH-1 cells was found to be linearly related to phenol production during treatment and inhibited by alpha-naphthoflavone (ANF). PMID:4006009

Crespi, C L; Altman, J D; Marletta, M A

1985-05-01

280

Expression of the somatostatin gene in human astrocytoma cell lines.  

PubMed Central

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

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

1996-01-01

281

Glucose induces apoptosis of cardiomyocytes via microRNA1 and IGF-1  

Microsoft Academic Search

Glucose toxicity is an important initiator of cardiovascular disease, contributing to the development of cardiomyocyte death and diabetic complications. The present study investigated whether high glucose state could induce apoptosis of rat cardiomyocyte cell line H9C2 through microRNA regulated insulin-like growth factor (IGF-1) signaling pathway. Our data showed that H9C2 cells exposed to high glucose have increased miR-1 expression level,

Xi-Yong Yu; Yao-Hua Song; Yong-Jian Geng; Qiu-Xiong Lin; Zhi-Xin Shan; Shu-Guang Lin; Yangxin Li

2008-01-01

282

Neuroectoderm-associated antigens on Ewing's sarcoma cell lines.  

PubMed

The histogenesis of Ewing's sarcoma, the second most frequent primary bone tumor in humans, remains controversial. Ten Ewing cell lines were analyzed by immunological methods. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. They included ganglioside GD2, a marker of neuroectodermal tissues and tumors, and an acidic glycolipid detected by monoclonal antibody HNK-1 in the nervous system. The P61 rat monoclonal antibody that reacts with a peptide moiety of neural cell adhesion molecule (N-CAM) and a rabbit antiserum raised to purified mouse N-CAM also stained Ewing cells. Flow cytometry analysis performed using these reagents allowed the definition of four distinct Ewing phenotypes: all reagents equally stained group 1 lines; group 2 lines were strongly reactive with anti-N-CAM reagents, by contrast with a fainter staining with HNK-1 and anti-GD2 antibodies; all reagents but P61 were strongly reactive with group 3 lines; in group 4, Ewing lines were stained by P61 but only poorly by the anti-N-CAM antiserum. Several antibodies to melanoma and neuroblastoma associated antigens including two monoclonal antibodies to the nerve growth factor receptor were also found to react with Ewing cells. By contrast, all antibodies detecting antigens specifically expressed in hematopoietic cell lineages were totally unreactive. HLA class II antigens were never detected while the level of expression of class I antigens varied to a large extent. Ewing cells are characterized by a specific t(11;22)(q23-24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor. Thus, Ewing's sarcoma cells share antigenic and karyotypic features with derivatives of the neuroectoderm possibly indicating a related histogenesis. PMID:3024814

Lipinski, M; Braham, K; Philip, I; Wiels, J; Philip, T; Goridis, C; Lenoir, G M; Tursz, T

1987-01-01

283

Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies  

PubMed Central

Cell death is a fundamentally important problem in cell lines used by the biopharmaceutical industry. Environmental stress, which can result from nutrient depletion, by-product accumulation and chemical agents, activates through signalling cascades regulators that promote death. The best known key regulators of death process are the Bcl-2 family proteins which constitute a critical intracellular checkpoint of apoptosis cell death within a common death pathway. Engineering of several members of the anti-apoptosis Bcl-2 family genes in several cell types has extended the knowledge of their molecular function and interaction with other proteins, and their regulation of cell death. In this review, we describe the various modes of cell death and their death pathways at molecular and organelle level and discuss the relevance of the growing knowledge of anti-apoptotic engineering strategies to inhibit cell death and increase productivity in mammalian cell culture.

Krampe, Britta

2010-01-01

284

Molecular cytogenetic analysis of breast cancer cell lines  

PubMed Central

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

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

2000-01-01

285

[Cytogenetic studies on 3 esophageal cancer cell lines].  

PubMed

The cytogenetic studies on three esophageal cancer cell lines established in China were done. Thirty metaphases showing suitable chromosome length and good G-banding pattern from each of the cell lines were chosen and subjected to karyological analysis. The typical karyotypes from these cell lines were listed, and the variation of chromosome number as well as the morphological characteristics, possible sources and the incidence of the marker chromosomes were analysed. Eca 109 is the cell line first established and used extensively in China. A strictly regular karyotype pattern was found in it: the modal number of chromosomes being 63-64; the number of each type of chromosome varying between 1-5; a distal deletion of short arm of chromosome No. 1 being discernible in all metaphases, with break sites located within 1p22-1p33. Also a distal deletion of long arm of chromosome No. 4 was usually visible. There were seven marker chromosomes with high incidence. Among them, M1 marker chromosome was a large subacrocentric chromosome which was observed in the early passages of this cell line. The chromosome number of Ec 17 cell line was usually subtetraploid. In addition to the numerical variation in some of the chromosomes, six marker chromosomes were usually observed. Among them, M1 involved reciprocal translocation between chromosome No. 1 and No. 4. M3 of Ec 17 was in correspondence with M5 of Eca 109. Both were Rob (13q;14q). The chromosome number of Ec 56 was usually subtetraploid, and in addition to the numerical variation in some of the chromosomes, seven marker chromosomes were usually observed. PMID:3477417

Li, S D; Zhang, L R; Luo, X; Ao, P

1987-03-01

286

Generation of cell lines for monoclonal antibody production.  

PubMed

Monoclonal antibodies (mAbs) represent the largest group of therapeutic proteins with 30 products approved in the USA and hundreds of therapies currently undergoing clinical trials. The complex nature of mAbs makes their development as therapeutic agents constrained by numerous criteria such as quality, safety, regulation, and quantity. Identification of a clonal cell line expressing high levels of mAb with adequate quality attributes and generated in compliance with regulatory standards is a necessary step prior to a program moving to large-scale production for clinical material. This chapter outlines the stable transfection technology that generates clonal cell lines for commercial manufacturing processes. PMID:24515472

Alvin, Krista; Ye, Jianxin

2014-01-01

287

Isolation of three stem cell lines from human sacrococcygeal teratomas.  

PubMed

Sacrococcygeal teratomas (SCTs) are benign tumours of the newborn with absolute indication for surgery directly after birth. We recently described the presence of stem cells positive for the stem cell markers nanog and Oct4 in SCTs. Here we report the isolation of three stem cell lines from three different SCTs. Cells were propagated in mesenchymal or in embryonic stem cell medium. Non-clonal homogeneous stem cell lines were obtained after two to three passages and characterized in vitro by immunocytochemistry, RT-PCR, western blot, FACS analysis, and metaphase spreads. The differentiation potential was tested in vitro and in vivo. The isolated cell lines, which we refer to as human sacrococcygeal teratoma stem cells (hSctSCs), express nanog, Oct4 and stella, and are negative for malignancy markers alpha-fetoprotein and carcinoembryonic antigen. They can be induced in vitro to express neuronal, osteogenic, and chondrogenic traits. After grafting in vivo, spontaneous integration into the neural crest of the chick embryo and teratoma formation in the nude mouse were obtained. Our results indicate that SCTs are derived from remnants of the epiblast-derived primitive streak, which in the human embryo normally regresses but forms teratomas in children affected with SCT. The hSctSCs therefore may be comparable to mouse epiblast-derived stem cells (EpiSCs) and share characteristic features with human embryonic stem (hES) cells. Thus, SCT tissue obtained after surgery appears to be a novel source for the generation of human stem cells without the ethical implications associated with hES cells. PMID:19142973

Busch, Christian; Bareiss, Petra M; Sinnberg, Tobias; Just, Lothar; Wehrmann, Manfred; Fuchs, Jörg; Garbe, Claus; Drews, Ulrich

2009-03-01

288

In Vitro Expression of the Endothelial Phenotype: Comparative Study of Primary Isolated Cells and Cell Lines, Including the Novel Cell Line HPMEC-ST1.6R  

Microsoft Academic Search

Endothelial cell lines are commonly used in in vitro studies to avoid problems associated with the use of primary endothelial cells such as the presence of contaminating cells, the difficulty in obtaining larger numbers of cells, as well as the progressive loss of cell viability and expression of endothelial markers in the course of in vitro propagation. We have analyzed

Ronald E. Unger; Vera Krump-Konvalinkova; Kirsten Peters; C. James Kirkpatrick

2002-01-01

289

Establishment of new multidrug-resistant human osteosarcoma cell lines.  

PubMed

Multidrug-resistant clones of human osteosarcoma MNNG/HOS and MG63 cells were isolated by stepwise selection on exposure to increasing doses of doxorubicin (DXR). The final clones MNNG/HOS/DXR1000 and MG63/DXR1000, established after ethylmethane sulfonate mutagenesis, showed 96-fold and 121-fold higer resistance to DXR than their parental cell lines. They were also cross-resistant to vincristine, but not to cisplatinum or methotrexate. The levels of multidrug-resistance-1 (MDR1) mRNA expression increased gradually according to the concentration of DXR in both cell lines. Although the parental MNNG/HOS cells expressed a low level of MDR1 mRNA, the parental MG63 cells showed no MDR1 expression. The IC50 values of MNNG/HOS and its resistant variant to DXR were higher than those of MG63 and its resistant clone. Multidrug-resistant associated protein (MRP) mRNA expression was detected in MNNG/HOS or MG63 parental cell lines, and in their resistant variants. MG63 and its resistant variants revealed stable expression of MRP, whereas the resistant phenotype of MNNG/HOS showed decreased MRP expression, compared to its parental cell line. No alteration in the levels of hepatocyte growth factor (HGF) or its receptor c-MET was recognized between parental lines and their resistant variants. The results indicate that our DXR-resistant variants of MNNG/HOS and MG63 reveal a classical MDR phenotype and can offer a model with which to investigate the mechanisms of multidrug resistance in osteosarcoma. PMID:10854558

Oda, Y; Matsumoto, Y; Harimaya, K; Iwamoto, Y; Tsuneyoshi, M

2000-01-01

290

Mechanisms of resistance to azacitidine in human leukemia cell lines.  

PubMed

The DNA methylation inhibitor azacitidine (5-azacytidine) is used against myelodysplastic syndrome and acute myeloid leukemia, but drug resistance is an ongoing, intractable problem. To investigate resistance mechanisms, we generated two azacitidine-resistant cell lines, THP-1/AR and HL60/AR, and studied genetic disparities between them and their corresponding parental lines. In cells treated with azacitidine, significant mitotic variations were noted in parental cells which were absent in resistant cells, suggesting that resistance arises from negating azacitidine-mediated activation of apoptosis signaling and reestablishing G2/M checkpoint. Importantly, both resistant cell lines have common point mutations in the uridine-cytidine kinase 2 (UCK2) gene, which encodes the rate-limiting enzyme of the azacitidine activation pathway. Forced expression of mutated UCK2 in parental THP-1 cells abrogated azacitidine-induced apoptosis, whereas overexpression of wild type UCK2 in resistant THP-1/AR cells restored sensitivity to azacitidine, implying that UCK2 gene mutations perturb azacitidine activation and advance azacitidine resistance. Our study provides new insights into azacitidine resistance and establishes models useful in developing effective strategies to overcome it. PMID:24368162

Sripayap, Piyanuch; Nagai, Tadashi; Uesawa, Mitsuyo; Kobayashi, Hiroyuki; Tsukahara, Tomonori; Ohmine, Ken; Muroi, Kazuo; Ozawa, Keiya

2014-04-01

291

Cellular and Phenotypic Characterization of Canine Osteosarcoma Cell Lines  

PubMed Central

Canine and human osteosarcoma (OSA) have many similarities, with the majority of reported cases occurring in the appendicular skeleton, gender predominance noted, high rate of metastasis at the time of presentation, and a lack of known etiology for this devastating disease. Due to poor understanding of the molecular mechanisms underlying OSA, we have characterized seven different OSA canine cell lines: Abrams, D17, Grey, Hughes, Ingles, Jarques, and Marisco and compared them to U2, a human OSA cell line, for the following parameters: morphology, growth, contact inhibition, migrational tendencies, alkaline phosphatase staining, heterologous tumor growth, double-strand DNA breaks, and oxidative damage. All results demonstrated the positive characteristics of the Abrams cell line for use in future studies of OSA. Of particular interest, the robust growth of a subcutaneous tumor and rapid pulmonary metastasis of the Abrams cell line in an immunocompromised mouse shows incredible potential for the future use of Abrams as a canine OSA model. Further investigations utilizing a canine cell model of OSA, such as Abrams, will be invaluable to understanding the molecular events underlying OSA, pharmaceutical inhibition of metastasis, and eventual prevention of this devastating disease.

Legare, Marie E.; Bush, Jamie; Ashley, Amanda K.; Kato, Taka; Hanneman, William H.

2011-01-01

292

Evaluating cell lines as tumour models by comparison of genomic profiles  

PubMed Central

Cancer cell lines are frequently used as in vitro tumour models. Recent molecular profiles of hundreds of cell lines from The Cancer Cell Line Encyclopedia and thousands of tumour samples from the Cancer Genome Atlas now allow a systematic genomic comparison of cell lines and tumours. Here we analyse a panel of 47 ovarian cancer cell lines and identify those that have the highest genetic similarity to ovarian tumours. Our comparison of copy-number changes, mutations and mRNA expression profiles reveals pronounced differences in molecular profiles between commonly used ovarian cancer cell lines and high-grade serous ovarian cancer tumour samples. We identify several rarely used cell lines that more closely resemble cognate tumour profiles than commonly used cell lines, and we propose these lines as the most suitable models of ovarian cancer. Our results indicate that the gap between cell lines and tumours can be bridged by genomically informed choices of cell line models for all tumour types.

Domcke, Silvia; Sinha, Rileen; Levine, Douglas A.; Sander, Chris; Schultz, Nikolaus

2013-01-01

293

Metallothionein prevention of arsenic trioxide-induced cardiac cell death is associated with its inhibition of mitogen-activated protein kinases activation in vitro and in vivo.  

PubMed

Cardiotoxicity induced by arsenic trioxide has become a serious blockade of clinical applications of this effective anticancer agent. The general mechanism responsible for arsenic cardiotoxicity has been attributed to its induction of oxidative stress. Metallothionein (MT) has been extensively proven to be a potent endogenous antioxidant that protects heart against oxidative stress-induced cardiac damage. To investigate whether and how MT protects against arsenic cardiotoxicity, MT-overexpressing H9c2 (MT-H9c2) cardiac cells and transgenic (MT-TG) mice with their corresponding controls were exposed to the clinical relevant dose of arsenic trioxide. Cardiac cell apoptosis was detected by molecular indices, including the cleavage of caspase 3 and caspase 12, Bax/Bcl2 expression ratio, CHOP expression and/or confirmed by a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Arsenic trioxide dose- and time-dependently induced cardiac cell death in H9c2 cells with a significant activation of major MAPK subfamily members such as ERK1/2, JNK and p38, but not in MT-H9c2 cells. Importantly, the protective effect of MT on arsenic trioxide-induced apoptotic cell death was completely recaptured in the heart of MT-TG with a significant prevention of MAPKs activation. These results indicate that arsenic trioxide-upregulated MAPKs might play important role in arsenic trioxide-induced apoptotic cell death in cardiac cells both in vivo and in vitro, and MT's suppression of arsenic trioxide apoptotic effect was associated with the inhibition of MAPK activation. Therefore, selective elevation of cardiac MT levels with pharmacological approaches may be a potential strategy for the prevention of arsenic cardiotoxicity. PMID:23664956

Miao, Xiao; Tang, Zefang; Wang, Yonggang; Su, Guanfang; Sun, Weixia; Wei, Wei; Li, Wei; Miao, Lining; Cai, Lu; Tan, Yi; Liu, Qiuju

2013-07-18

294

Decorin Suppresses Bone Metastasis in a Breast Cancer Cell Line  

Microsoft Academic Search

Decorin, the prototype of an expanding family of small leucine-rich proteoglycans, is involved in a number of cellular processes including matrix assembly, fibrillogenesis and the control of cell proliferation. In this study, we investigated the role of decorin in suppressing tumor aggressiveness and bone metastases. We used a metastatic breast cancer cell line, MDA-MB-231, to show that decorin causes marked

Kentaro Araki; Hiroki Wakabayashi; Ken Shintani; Joji Morikawa; Akihiko Matsumine; Katsuyuki Kusuzaki; Akihiro Sudo; Atsumasa Uchida

2009-01-01

295

Celecoxib increases retinoid sensitivity in human colon cancer cell lines  

Microsoft Academic Search

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs.\\u000a This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity\\u000a in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using\\u000a the MTT assay. Apoptosis was detected via Annexin-V\\/PI staining

Jian-Pei Liu; Hong-Bo Wei; Zong-Heng Zheng; Wei-Ping Guo; Jia-Feng Fang

2010-01-01

296

Characterization of Butyrate Uptake by Nontransformed Intestinal Epithelial Cell Lines  

Microsoft Academic Search

Butyrate (BT) is one of the main end products of anaerobic bacterial fermentation of dietary fiber within the human colon.\\u000a Among its recognized effects, BT inhibits colon carcinogenesis. Our aim was to characterize uptake of BT by two nontransformed\\u000a intestinal epithelial cell lines: rat small intestinal epithelial (IEC-6) and fetal human colonic epithelial (FHC) cells.\\u000a Uptake of 14C-BT by IEC-6

Pedro GoncalvesJoao; João R. Araújo; Fátima Martel

2011-01-01

297

The NCI60 human tumour cell line anticancer drug screen  

Microsoft Academic Search

The US National Cancer Institute (NCI) 60 human tumour cell line anticancer drug screen (NCI60) was developed in the late 1980s as an in vitro drug-discovery tool intended to supplant the use of transplantable animal tumours in anticancer drug screening. This screening model was rapidly recognized as a rich source of information about the mechanisms of growth inhibition and tumour-cell

Robert H. Shoemaker

2006-01-01

298

Conditionally Immortalized Neural Cell Lines: Potential Models for the Study of Neural Cell Function  

PubMed

Studies on primary cell cultures have contributed significantly to our understanding of neural cell function. Nevertheless, for many studies the value of these primary cell cultures has been limited by the time the cultures survive in vitro, the quantity of cellular material available for analysis, and the need to prepare the cells on a regular basis from fresh tissue. Techniques for immortalizing cells have existed for some time, but the repertoire of immortalizing genes has grown significantly. This has expanded our ability to generate useful cell lines of specific neural types that are better models of the in vivo phenotype than previously. The constitutive expression of oncogenes keeps cells in a proliferative state that could lead to the loss of differentiated gene expression and function. An appealing improvement of immortalization methodology is the use of temperature-sensitive oncogenes that generate cell lines that can proliferate at a permissive temperature and "differentiate" at a nonpermissive temperature. The proliferation of such conditionally immortalized cell lines can be suppressed simply by increasing the temperature. Cell lines maintained at the nonpermissive temperature can enter into a stage in which they express differentiated properties of the cell. The potential ability of conditionally immortalized neural cell lines to accurately reflect their in vivo function has now been demonstrated on several occasions through transplantation experiments. In this report, the generation of these cell lines is described along with a discussion of their potential applications in neurobiology. PMID:8954859

Bongarzone; Foster; Byravan; Verity; Landry; Schonmann; Amur-Umarjee; Campagnoni

1996-12-01

299

Optimized protocol for derivation of human embryonic stem cell lines.  

PubMed

For the past 12 years, the biology and applications of human embryonic stem cells (hESCs) have received great attention from the scientific community. Derivatives of the first hESC line obtained by J. Thomson's group (Science 282(5391):1145-1147, 1998) have been used in clinical trials in patients with spinal cord injury, and other hESC lines have now been used to generate cells for use in treating blindness (Lancet 379(9817):713-720, 2012). In addition to the classical protocol based on mouse or human feeder layers using open culture methods (In Vitro Cellular & Developmental Biology - Animal 46(3-4):386-394, 2010; Stem Cells 23(9):1221-1227, 2005; Nature Biotechnology 24(2):185-187, 2006; Human Reproduction 21(2):503-511, 2006; Human Reproduction 20(8):2201-2206, 2005; Fertility and Sterility 83(5):1517-1529, 2005), novel hESC lines have been derived xeno-free (without using animal derived reagents) (PLoS One 5 (4):1024-1026, 2010), feeder-free (without supporting cell monolayers) (Lancet 365(9471):1601-1603, 2005), in microdrops under oil (In Vitro Cellular & Developmental Biology - Animal 46(3-4):236-41, 2010) and in suspension with ROCK inhibitor (Nature Biotechnology 28(4):361-4, 2010). Regardless of the culture system, successful hESC derivation usually requires optimization of embryo culture, the careful and timely isolation of its inner cell mass (ICM), and precise culture conditions up to the establishment of pluripotent cell growth during hESC line derivation. Herein we address the crucial steps of the hESC line derivation protocol, and provide tips to apply quality control to each step of the procedure. PMID:22614996

Camarasa, María Vicenta; Galvez, Víctor Miguel; Brison, Daniel Roy; Bachiller, Daniel

2012-09-01

300

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

PubMed

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

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

1989-07-01

301

Gene expression profiling of cell lines derived from T-cell malignancies  

Microsoft Academic Search

The expression profiles of eight cell lines derived from T-cell malignancies were compared to CD4-positive T-cells using cDNA microarray technology. Unsupervised hierarchical clustering of 4364 genes demonstrated substantial heterogeneity resulting in four distinct groups. While no genes were found to be uniformly up- or down-regulated across all cell lines, we observed 111 over-expressed genes (greater than two-fold) and 1118 down-regulated

G. Chris Fillmore; Zhaosheng Lin; Sandra D Bohling; Ryan S Robetorye; Chan-Hwan Kim; Stephen D Jenson; Kojo S. J Elenitoba-Johnson; Megan S Lim

2002-01-01

302

Susceptibility of MHC Class I Expressing Extravillous Trophoblast Cell Lines to Killing by Natural Killer Cells  

Microsoft Academic Search

Purified human first trimester extravillous trophoblast (EVT) cell lines HTR-8 and HT-116 were examined for susceptibility to natural killer (NK) cell-mediated lysis. Based upon nucleic acid sequencing of an amplified fragment of cDNA, Western blot analysis and immunostaining of fixed and live cells, it was shown that both EVT cell lines expressed HLA-G mRNA and protein within the cytoplasm when

M. Zdravkovic; G. Aboagye-Mathiesen; M.-J. Guimond; H. Hager; P. Ebbesen; P. K. Lala

1999-01-01

303

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines  

PubMed Central

Background Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of CSCs could help to develop novel therapeutic strategies specifically targeting CSCs. Methods Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the PLC/PRF/5 sphere-forming cells, and the expression levels of stem cell related proteins in the PLC/PRF/5 sphere-forming cells were assessed, comparing with the parental cells. The stem cell RT-PCR array was performed to further explore the biological properties of liver CSCs. Results The PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice, and the tumor initiating capability was not decreased when spheres were passaged. Besides, downstream proteins DTX1 and Ep300 of the CSL (CBF1 in humans, Suppressor of hairless in Drosophila and LAG1 in C. elegans) -independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability. Conclusions Nonadherent tumor spheres from hepatoma cell lines cultured in stem cell conditioned medium possess liver CSC properties, and the CSL-independent Notch signaling pathway may play a role in liver CSCs.

2011-01-01

304

Analysis of p53 in human cutaneous melanoma cell lines.  

PubMed

Mutations in the p53 tumour suppressor gene have been detected in a variety of human malignancies. Mutations have been found predominantly in conserved regions two to five. Our aim was to analyse p53 at the protein and DNA level in seven melanoma cell lines of cutaneous origin (HMB-2, DX3, LT5.1, MJM, SK23, A375P and A375M), including two parental/metastatic derivatives (A375P and A375M; DX3 and LT5.1). By immunohistochemical staining with three mouse monoclonal antibodies and a rabbit polyclonal serum, it was possible to observe differential nuclear expression of p53. The quantitation of p53 protein levels by ELISA correlated with the nuclear staining pattern. Western blotting showed an intact p53 protein in all cell lines; p53 was polymorphic in three cell lines (MJM, A375P and A375M). DNA sequencing studies showed that all cell lines had wild type p53. These results suggest that p53 is unlikely to play a significant role in the genesis of cutaneous melanoma. PMID:8152807

Montano, X; Shamsher, M; Whitehead, P; Dawson, K; Newton, J

1994-05-01

305

Mammalian Cell Lines Specifically Deficient in O-Linked Glycosylation.  

National Technical Information Service (NTIS)

Genetically modified cell lines that express a UDP-galactose 4-epimerase (GALE) capable of interconverting UDP-galactose (UDP-gal) and UDP-glucose (UDP-glc), but essentially incapable of interconverting UDP-N-acetylgalactosamine (UDP-galNAc) and UDP-N-ace...

H. M. Holden J. B. Thoden J. L. Fridovich-Kell J. M. Schulz M. Krieger

2004-01-01

306

Use of Cell Lines in the Investigation of Pharmacogenetic Loci  

PubMed Central

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.

Zhang, Wei; Dolan, M. Eileen

2009-01-01

307

Molecular cytogenetic analysis of the monoblastic cell line U937  

Microsoft Academic Search

Previous reports on the analysis of the human monoblastic cell line U937 had described several sublines containing unidentified rearrangements and marker chromosomes. In order to determine the true nature of the rearrangements, conventional banding analysis was carried out with various combinations of molecular cytogenetic techniques: comparative genomic hybridization, fluorescence in situ hybridization (FISH) with whole chromosome painting probes, and microdissection

Ji-Yun Lee; Chul-Hoon Lee; Sung-Han Shim; Han-Kyu Seo; Jee-Hong Kyhm; Sechin Cho; Youl-Hee Cho

2002-01-01

308

Curbing rampant cross-contamination and misidentification of cell lines.  

PubMed

A son's challenge started an emeritus professor of biology on a three-year odyssey to get biological researchers to correct a decades-long problem with cross-contaminated and misidentified cell lines. These errors may account for more than 15% of mammalian cultures, wasting resources and undermining the integrity of research. PMID:18816888

Nardone, Roland M

2008-09-01

309

Telomere stability genes are not mutated in osteosarcoma cell lines  

Microsoft Academic Search

Osteosarcoma (OS), the most common primary bone tumor in adolescents and young adults, is characterized by a high degree of chromosomal abnormalities. Because telomeres are important for maintaining chromosomal integrity, it is plausible that germ-line or somatic mutations in the genes responsible for stabilizing the telomere complex could contribute to OS. We performed bi-directional sequence analysis in five OS cell

Sharon A. Savage; Brian J. Stewart; Jason S. Liao; Lee J. Helman; Stephen J. Chanock

2005-01-01

310

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...

311

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...

312

Ultra-wideband time-delay line inspired by composite right\\/left-handed transmission line unit cell  

Microsoft Academic Search

This paper presents a design of ultra-wideband time-delay line inspired by the composite right\\/left-handed transmission line (CRLH TL) unit cell. A rotated version of the conventional CRLH TL unit cell is used to increase the operating bandwidth. The time-delay line is optimized using computer simulation and then fabricated on a PCB for measurement. For comparison, the time-delay lines using the

J. Zhang; S. W. Cheung; T. I. Yuk

2010-01-01

313

A human gall-bladder signet ring cell carcinoma cell line.  

PubMed

To date, very few reports of the establishment of gall-bladder cancer cell lines have appeared, although many cancer cell lines of various kinds have been established. On the other hand, no reports could be found on signet ring cell carcinoma cell lines derived from the gall-bladder and only five cell lines from the stomach. A human gall-bladder cancer cell line (FU-GBC-2) was established in tissue culture from the ascitic fluid of a 69-year-old Japanese female patient. The tumor cells growing in tissue culture exhibited the morphological characteristics of signet ring cells in phase contrast and electron microscopy. The population doubling time was 43 hours. Heterotransplantation was succeeded by inoculation into the dermis of BALB/c nude mice. An immunocytochemical study showed that most of the cultured cells were positive for carcinoembryonic antigen, CA19-9 and epithelial membrane antigen, but negative for vimentin. The modal chromosome number was 120 with a range of 100-124. Flow cytometry showed an aneuploidy pattern in the cultured cells at passage 30. Markedly amplified c-myc oncogene was observed by Southern blot analysis. This cell line may be useful in the study of the morphological and biological characteristics of signet ring cell carcinoma and gall-bladder adenocarcinoma. PMID:9211524

Nishida, T; Iwasaki, H; Johzaki, H; Tanaka, S; Watanabe, R; Kikuchi, M

1997-06-01

314

Zebrafish kidney stromal cell lines support multilineage hematopoiesis  

PubMed Central

Studies of zebrafish hematopoiesis have been largely performed using mutagenesis approaches and retrospective analyses based upon gene expression patterns in whole embryos. We previously developed transplantation assays to test the repopulation potentials of candidate hematopoietic progenitor cells. We have been impaired, however, in determining cellular differentiation potentials by a lack of short-term functional assays. To enable more precise analyses of hematopoietic progenitor cells, we have created zebrafish kidney stromal (ZKS) cell lines. Culture of adult whole kidney marrow with ZKS cells results in the maintenance and expansion of hematopoietic precursor cells. Hematopoietic growth is dependent upon ZKS cells, and we show that ZKS cells express many growth factors and ligands previously demonstrated to be important in maintaining mammalian hematopoietic cells. In the absence of exogenous growth factors, ZKS cells maintain early hematopoietic precursors and support differentiation of lymphoid and myeloid cells. With the addition of zebrafish erythropoietin, ZKS cells also support the differentiation of erythroid precursors. These conditions have enabled the ability to ascertain more precisely the points at which hematopoietic mutants are defective. The development of robust in vitro assays now provide the means to track defined, functional outcomes for prospectively isolated blood cell subsets in the zebrafish.

Stachura, David L.; Reyes, Jason R.; Bartunek, Petr; Paw, Barry H.; Zon, Leonard I.

2009-01-01

315

Deoxyribonucleic Acid Profiling Analysis of 40 Human Thyroid Cancer Cell Lines Reveals Cross-Contamination Resulting in Cell Line Redundancy and Misidentification  

PubMed Central

Context: Cell lines derived from human cancers provide critical tools to study disease mechanisms and develop novel therapies. Recent reports indicate that up to 36% of cell lines are cross- contaminated. Objective: We evaluated 40 reported thyroid cancer-derived cell lines using short tandem repeat and single nucleotide polymorphism array analysis. Results: Only 23 of 40 cell lines tested have unique genetic profiles. The following groups of cell lines are likely derivatives of the same cell line: BHP5-16, BHP17-10, BHP14-9, and NPA87; BHP2-7, BHP10-3, BHP7-13, and TPC1; KAT5, KAT10, KAT4, KAT7, KAT50, KAK1, ARO81-1, and MRO87-1; and K1 and K2. The unique cell lines include BCPAP, KTC1, TT2609-C02, FTC133, ML1, WRO82-1, 8505C, SW1736, Cal-62, T235, T238, Uhth-104, ACT-1, HTh74, KAT18, TTA1, FRO81-2, HTh7, C643, BHT101, and KTC-2. The misidentified cell lines included the DRO90-1, which matched the melanoma-derived cell line, A-375. The ARO81-1 and its derivatives matched the HT-29 colon cancer cell line, and the NPA87 and its derivatives matched the M14/MDA-MB-435S melanoma cell line. TTF-1 and Pax-8 mRNA levels were determined in the unique cell lines. Conclusions: Many of these human cell lines have been widely used in the thyroid cancer field for the past 20 yr and are not only redundant, but not of thyroid origin. These results emphasize the importance of cell line integrity, and provide the short tandem repeat profiles for a panel of thyroid cancer cell lines that can be used as a reference for comparison of cell lines from other laboratories.

Schweppe, Rebecca E.; Klopper, Joshua P.; Korch, Christopher; Pugazhenthi, Umarani; Benezra, Miriam; Knauf, Jeffrey A.; Fagin, James A.; Marlow, Laura A.; Copland, John A.; Smallridge, Robert C.; Haugen, Bryan R.

2008-01-01

316

Effects of hypoxia on human cancer cell line chemosensitivity  

PubMed Central

Background Environment inside even a small tumor is characterized by total (anoxia) or partial oxygen deprivation, (hypoxia). It has been shown that radiotherapy and some conventional chemotherapies may be less effective in hypoxia, and therefore it is important to investigate how different drugs act in different microenvironments. In this study we perform a large screening of the effects of 19 clinically used or experimental chemotherapeutic drugs on five different cell lines in conditions of normoxia, hypoxia and anoxia. Methods A panel of 19 commercially available drugs: 5-fluorouracil, acriflavine, bortezomib, cisplatin, digitoxin, digoxin, docetaxel, doxorubicin, etoposide, gemcitabine, irinotecan, melphalan, mitomycin c, rapamycin, sorafenib, thalidomide, tirapazamine, topotecan and vincristine were tested for cytotoxic activity on the cancer cell lines A2780 (ovarian), ACHN (renal), MCF-7 (breast), H69 (SCLC) and U-937 (lymphoma). Parallel aliquots of the cells were grown at different oxygen pressures and after 72 hours of drug exposure viability was measured with the fluorometric microculture cytotoxicity assay (FMCA). Results Sorafenib, irinotecan and docetaxel were in general more effective in an oxygenated environment, while cisplatin, mitomycin c and tirapazamine were more effective in a low oxygen environment. Surprisingly, hypoxia in H69 and MCF-7 cells mostly rendered higher drug sensitivity. In contrast ACHN appeared more sensitive to hypoxia, giving slower proliferating cells, and consequently, was more resistant to most drugs. Conclusions A panel of standard cytotoxic agents was tested against five different human cancer cell lines cultivated at normoxic, hypoxic and anoxic conditions. Results show that impaired chemosensitivity is not universal, in contrast different cell lines behave different and some drugs appear even less effective in normoxia than hypoxia.

2013-01-01

317

Endosialin expression in side populations in human sarcoma cell lines.  

PubMed

The Hoechst 33342 exclusion side population (SP) assay is a validated method used to identify cells with stem cell-like properties. When isolated from tumors, SP cells have been shown to have high malignant potential. SPs have been found in both carcinomas and sarcomas. The molecular profile of sarcoma SP is poorly understood. The purpose of the present study was to determine whether endosialin is a suitable therapeutic target for sarcomas. Six cell lines (HT-1080 fibrosarcoma, SJSA-1 and HOS osteosarcoma, A-673 and SK-ES-1 Ewing sarcoma) were used for the SP analysis. Flow cytometry was used to count and examine the cells. Results showed for the first time that endosialin (CD248), which was previously identified as a sarcoma marker, is expressed in sarcoma SP cells. This observation supports the hypothesis that endosialin is a promising therapeutic target for sarcomas. PMID:22740905

Rouleau, Cecile; Sancho, Jose; Campos-Rivera, Juanita; Teicher, Beverly A

2012-02-01

318

Metal mutagenesis in transgenic Chinese hamster cell lines.  

PubMed Central

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.

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

1994-01-01

319

Cytotoxicity and genotoxicity of phenazine in two human cell lines.  

PubMed

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

McGuigan, Claire F; Li, Xing-Fang

2014-06-01

320

Cytotoxic effects of Euterpe oleracea Mart. in malignant cell lines  

PubMed Central

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 ?g/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?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.

2014-01-01

321

Functional role of Rab11 in GLUT4 trafficking in cardiomyocytes  

Microsoft Academic Search

We have recently shown the co-localization of Rab11 and the glucose transporter GLUT4 in cardiac muscle and an insulin-stimulated increase of Rab11 in GLUT4-containing vesicles in this tissue. We now assessed the effect of Rab11 wt and a dominant-negative mutant (N124I) on GLUT4 trafficking in the cardiomyoblast cell line H9c2 stably overexpressing the insulin receptor (H9c2-E2) and in human primary

Mathias Uhlig; Waltraud Passlack; Jürgen Eckel

2005-01-01

322

Sphere-forming cell subpopulations with cancer stem cell properties in human hepatoma cell lines  

Microsoft Academic Search

Background  Cancer stem cells (CSCs) are regarded as the cause of tumor formation and recurrence. The isolation and identification of\\u000a CSCs could help to develop novel therapeutic strategies specifically targeting CSCs.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Human hepatoma cell lines were plated in stem cell conditioned culture system allowed for sphere forming. To evaluate the\\u000a stemness characteristics of spheres, the self-renewal, proliferation, chemoresistance, tumorigenicity of the

Lu Cao; Yanming Zhou; Beibei Zhai; Jian Liao; Wen Xu; Ruixiu Zhang; Jing Li; Yu Zhang; Lei Chen; Haihua Qian; Mengchao Wu; Zhengfeng Yin

2011-01-01

323

Singlet oxygen toxicity is cell line-dependent: A study of lipid peroxidation in leukemia cell lines  

Microsoft Academic Search

Singlet oxygen (1O2) can be quenched by water, lipids, proteins, nucleic acids and other small molecules. Poly- unsaturated fatty acids (PUFA) of cells principally quench 1O 2 by chemical mechanisms, producing lipid hy- droperoxides, while proteins physically and chemically quench 1O 2. Because cell lines can have different PUFA and protein levels, we hypothesized that 1O2 toxicity will vary between

Freya Q. Schafer; Garry R. Buettner

1998-01-01

324

Characterization of side population cells isolated from the colon cancer cell line SW480.  

PubMed

Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer. Many types of cell lines and tissues have demonstrated the presence of SP cells, including colon cancer cell lines. This study aimed to identify cancer stem cells (CSCs) in the SP of the colon cancer cell line SW480. SP cells were isolated by fluorescence-activated cell sorting (FACS), followed by serum-free medium (SFM) culture. The self-renewal, differentiated progeny, clone formation, proliferation, invasion ability, cell cycle, chemosensitivity and tumorigenic properties in SP and non-SP (NSP) cells were investigated through in vitro culture and in vivo serial transplantation. The expression profiles of ATP-binding cassette (ABC) protein transporters and stem cell?related genes were examined by RT-PCR and western blot analysis. The human colon cancer cell lines SW480, Lovo and HCT116 contain 1.1±0.10, 0.93±0.11 and 1.33±0.05% SP cells, respectively. Flow cytometry analysis revealed that SP cells could differentiate into SP and NSP cells. SP cells had a higher proliferation potency and CFE than NSP cells. Compared to NSP cells, SP cells were also more resistant to CDDP and 5-FU, and were more invasive and displayed increased tumorigenic ability. Moreover, SP cells showed higher mRNA and protein expression of ABCG2, MDR1, OCT-4, NANOG, SOX-2, CD44 and CD133. SP cells isolated from human colon cancer cell lines harbor CSC properties that may be related to the invasive potential and therapeutic resistance of colon cancer. PMID:24926880

Xiong, Binghong; Ma, Li; Hu, Xiang; Zhang, Caiquan; Cheng, Yong

2014-09-01

325

Trichothecene-induced cytotoxicity on human cell lines  

Microsoft Academic Search

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type\\u000a D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2,\\u000a HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent\\u000a cell proliferation assay assessing

Carina Nielsen; Maximilian Casteel; Andrea Didier; Richard Dietrich; Erwin Märtlbauer

2009-01-01

326

Morphogenetic behavior of simian virus 40-transformed human mammary epithelial stem cell lines on collagen gels  

Microsoft Academic Search

Summary  Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned,\\u000a epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell\\u000a lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The\\u000a myoepithelial-related cell lines give rise only to

Philip S. Rudland; Gillian E. Ollerhead; Angela M. Platt-Higgins

1991-01-01

327

Targeted genetic modification of cell lines for recombinant protein production  

PubMed Central

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.

Piskareva, Olga; Muniyappa, Mohan

2007-01-01

328

Androglobin knockdown inhibits growth of glioma cell lines  

PubMed Central

Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro.

Huang, Bo; Lu, Yi-Sheng; Li, Xia; Zhu, Zhi-Chuan; Li, Kui; Liu, Ji-Wei; Zheng, Jing; Hu, Ze-Lan

2014-01-01

329

Over-expression of secreted proteins from mammalian cell lines  

PubMed Central

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.

Dalton, Annamarie C; Barton, William A

2014-01-01

330

The interaction of normal lymphocytes and cells from lymphoid cell lines  

PubMed Central

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.

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

1973-01-01

331

Spontaneous transformation of human granulosa cell tumours into an aggressive phenotype: a metastasis model cell line  

Microsoft Academic Search

BACKGROUND: Granulosa cell tumours (GCTs) are frequently seen in menopausal women and are relatively indolent. Although the physiological properties of normal granulosa cells have been studied extensively, little is known about the molecular mechanism of GCT progression. Here, we characterise the unique behavioural properties of a granulosa tumour cell line, KGN cells, for the molecular analysis of GCT progression. METHODS:

Misa Imai; Miho Muraki; Kiyoshi Takamatsu; Hidekazu Saito; Motoharu Seiki; Yuji Takahashi

2008-01-01

332

Beta-cell differentiation from a human pancreatic cell line in vitro and in vivo.  

PubMed

Cell transplantation therapy for diabetes is limited by an inadequate supply of cells exhibiting glucose-responsive insulin secretion. To generate an unlimited supply of human beta-cells, inducibly transformed pancreatic beta-cell lines have been created by expression of dominant oncogenes. The cell lines grow indefinitely but lose differentiated function. Induction of beta-cell differentiation was achieved by stimulating the signaling pathways downstream of the transcription factor PDX-1, cell-cell contact, and the glucagon-like peptide (GLP-1) receptor. Synergistic activation of those pathways resulted in differentiation into functional beta-cells exhibiting glucose-responsive insulin secretion in vitro. Both oncogene-expressing and oncogene-deleted cells were transplanted into nude mice and found to exhibit glucose-responsive insulin secretion in vivo. The ability to grow unlimited quantities of human beta-cells is a major step toward developing a cell transplantation therapy for diabetes. PMID:11222748

de la Tour, D; Halvorsen, T; Demeterco, C; Tyrberg, B; Itkin-Ansari, P; Loy, M; Yoo, S J; Hao, E; Bossie, S; Levine, F

2001-03-01

333

Heterogeneity of a human T-lymphoblastoid cell line  

SciTech Connect

A human T-lymphoblastoid cell line (Jurkat) was cloned, and four resulting sublines were characterized in a variety of ways with the objective of gaining information on heterogeneity in cell lines. Within a few weeks of cloning, distinct cellular morphologies and growth patterns became apparent in the four sublines. Growth rate measurements made over 3 months did not show any significant differences between the sublines. Surface protein profiles obtained by radioimmunoprecipitation using antisera in conjunction with extracts from (/sup 35/S)Met and /sup 125/I-labeled cells revealed differences between the sublines. Analysis of total cell DNA showed that one of the sublines possessed only half the chromosome complement of the other sublines and the parental line. Karyotyping confirmed this result and, in addition, demonstrated that chromosome numbers fluctuated around a mean value for each subline. Karyotypic variability became apparent within 2 months of cloning and tended to increase with time in culture. G-banding analysis showed that the analyzed cell populations contained distinctive cytogenetic aberrations. Properties of the cloned sublines were monitored over a 9-month period. One of the sublines that had shown heterogeneous morphology even after 6 weeks maintained the heterogeneity throughout this time. Another subline underwent a marked change in morphology (round to irregular) and growth habit (single cells to large clumps) with increasing time in culture. Interestingly, several alterations to surface proteins accompanied these growth changes. A third subline had relatively stable morphology and chromosome number throughout the 9-month period. The modal chromosome number was hypotetraploid for three sublines and the parent line, but was diploid for another subline.

Snow, K.; Judd, W.

1987-08-01

334

Carbon nanoparticles for gene transfection in eukaryotic cell lines.  

PubMed

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed ?-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. PMID:24863237

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

2014-06-01

335

Cytotoxicity evaluation of silica nanoparticles using fish cell lines.  

PubMed

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

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

2014-05-01

336

Production and Origination of Cell Lines and Isolation of Histocompatibility Antigens from Lymphoid Cell Lines.  

National Technical Information Service (NTIS)

A method for culturing the L1210 cells so as to obtain large amounts of antigen with high levels of glycoprotein was developed. The feasibility of extraction of large quantities of H2-d antigen from cells frozen in dimethyl sulfoxide (DMSO) and liquid nit...

P. M. Lincoln J. L. Glick B. A. Maurer

1973-01-01

337

Bryostatin analogue-induced apoptosis in mantle cell lymphoma cell lines  

PubMed Central

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

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

2014-01-01

338

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

PubMed Central

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

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

2014-01-01

339

Glycoprotein profiles of human breast cells demonstrate a clear clustering of normal/benign versus malignant cell lines and basal versus luminal cell lines.  

PubMed

Gene expression profiling has defined molecular subtypes of breast cancer including those identified as luminal and basal. To determine if glycoproteins distinguish various subtypes of breast cancer, we obtained glycoprotein profiles from 14 breast cell lines. Unsupervised hierarchical cluster analysis demonstrated that the glycoprotein profiles obtained can serve as molecular signatures to classify subtypes of breast cancer, as well as to distinguish normal and benign breast cells from breast cancer cells. Statistical analyses were used to identify glycoproteins that are overexpressed in normal versus cancer breast cells, and those that are overexpressed in luminal versus basal breast cancer. Among the glycoproteins distinguishing normal breast cells from cancer cells are several proteins known to be involved in cell adhesion, including proteins previously identified as being altered in breast cancer. Basal breast cancer cell lines overexpressed a number of CD antigens, including several integrin subunits, relative to luminal breast cancer cell lines, whereas luminal breast cancer cells overexpressed carbonic anhydrase 12, clusterin, and cell adhesion molecule 1. The differential expression of glycoproteins in these breast cancer cell lines readily allows the classification of the lines into normal, benign, malignant, basal, and luminal groups. PMID:22106898

Yen, Ten-Yang; Macher, Bruce A; McDonald, Claudia A; Alleyne-Chin, Chris; Timpe, Leslie C

2012-02-01

340

Internal arsenite bioassay calibration using multiple bioreporter cell lines  

PubMed Central

Summary Bioassays with bioreporter bacteria are usually calibrated with analyte solutions of known concentrations that are analysed along with the samples of interest. This is done as bioreporter output (the intensity of light, fluorescence or colour) does not only depend on the target concentration, but also on the incubation time and physiological activity of the cells in the assay. Comparing the bioreporter output with standardized colour tables in the field seems rather difficult and error?prone. A new approach to control assay variations and improve application ease could be an internal calibration based on the use of multiple bioreporter cell lines with drastically different reporter protein outputs at a given analyte concentration. To test this concept, different Escherichia coli?based bioreporter strains expressing either cytochrome c peroxidase (CCP, or CCP mutants) or ??galactosidase upon induction with arsenite were constructed. The reporter strains differed either in the catalytic activity of the reporter protein (for CCP) or in the rates of reporter protein synthesis (for ??galactosidase), which, indeed, resulted in output signals with different intensities at the same arsenite concentration. Hence, it was possible to use combinations of these cell lines to define arsenite concentration ranges at which none, one or more cell lines gave qualitative (yes/no) visible signals that were relatively independent of incubation time or bioreporter activity. The discriminated concentration ranges would fit very well with the current permissive (e.g. World Health Organization) levels of arsenite in drinking water (10?µg?l?1).

Wackwitz, Anke; Harms, Hauke; Chatzinotas, Antonis; Breuer, Uta; Vogne, Christelle; Van Der Meer, Jan Roelof

2008-01-01

341

Processing of proSAAS in neuroendocrine cell lines.  

PubMed Central

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.

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

2002-01-01

342

Epitope tagging of endogenous genes in diverse human cell lines  

PubMed Central

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.

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

2008-01-01

343

Serial analysis of gene expression in a microglial cell line.  

PubMed

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

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

344

First continuous human pheochromocytoma cell line: KNA Biological, cytogenetic and molecular characterization of KNA cells  

Microsoft Academic Search

Pheochromocytomas are rare tumours, with an incidence of 1–2 per million which arise from chromaffin cells of the adrenal medulla. They occur sporadically or as part of dominantly inherited cancer syndromes like multiple endocrine neoplasia 2 (MEN2A and 2B) and others. Continuous cell lines, not available so far, are essential tools for studies in these tumours. A continuous cell line

R. Pfragner; A. Behmel; D. P. Smith; B. A. J. Ponder; G. Wirnsberger; I. Rinner; S. Porta; T. Henn; B. Niederle

1998-01-01

345

Characterization of human embryonic stem cell lines by the International Stem Cell Initiative  

Microsoft Academic Search

The International Stem Cell Initiative characterized 59 human embryonic stem cell lines from 17 laboratories worldwide. Despite diverse genotypes and different techniques used for derivation and maintenance, all lines exhibited similar expression patterns for several markers of human embryonic stem cells. They expressed the glycolipid antigens SSEA3 and SSEA4, the keratan sulfate antigens TRA-1-60, TRA-1-81, GCTM2 and GCT343, and the

Oluseun Adewumi; Behrouz Aflatoonian; Lars Ahrlund-Richter; Michal Amit; Gemma Beighton; Paul A Bello; Nissim Benvenisty; Lorraine S Berry; Simon Bevan; Barak Blum; Justin Brooking; Kevin G Chen; Andre B H Choo; Gary A Churchill; Marie Corbel; Ivan Damjanov; Jon S Draper; Petr Dvorak; Katarina Emanuelsson; Roland A Fleck; Angela Ford; Karin Gertow; Marina Gertsenstein; Paul J Gokhale; Rebecca S Hamilton; Ales Hampl; Lyn E Healy; Outi Hovatta; Johan Hyllner; Marta P Imreh; Joseph Itskovitz-Eldor; Jamie Jackson; Jacqueline L Johnson; Mark Jones; Kehkooi Kee; Benjamin L King; Barbara B Knowles; Majlinda Lako; Franck Lebrin; Barbara S Mallon; Daisy Manning; Yoav Mayshar; Ronald D G Mckay; Anna E Michalska; Milla Mikkola; Masha Mileikovsky; Stephen L Minger; Harry D Moore; Christine L Mummery; Andras Nagy; Norio Nakatsuji; Carmel M O'Brien; Steve K W Oh; Cia Olsson; Timo Otonkoski; Kye-Yoon Park; Robert Passier; Hema Patel; Minal Patel; Roger Pedersen; Martin F Pera; Marian S Piekarczyk; Renee A Reijo Pera; Benjamin E Reubinoff; Allan J Robins; Janet Rossant; Peter Rugg-Gunn; Thomas C Schulz; Henrik Semb; Eric S Sherrer; Henrike Siemen; Glyn N Stacey; Miodrag Stojkovic; Hirofumi Suemori; Jin Szatkiewicz; Tikva Turetsky; Timo Tuuri; Steineke van den Brink; Kristina Vintersten; Sanna Vuoristo; Dorien Ward; Thomas A Weaver; Lesley A Young; Weidong Zhang; Peter W Andrews

2007-01-01

346

Establishment and characterization of a new murine cell line (SR4987) derived from marrow stromal cells  

Microsoft Academic Search

A new murine cell line designated as SR-4987 was established by treating a long-term bone marrow culture with the supernatant\\u000a from Y-1 cells which actively produce viral C-particles (MuLV). The line showed a fibrolbast-like morphology and its mesodermal\\u000a origin was confirmed by immunocytochemical staining. Flow cytometric analysis of DNA index evidenced a tetraploid number of\\u000a chromosomes whereas cell cycle analysis

Augusto Pessina; Elisabetta Mineo; Maria Grazia Neri; Laura Gribaldo; Robert Colombi; Paolo Brambilla; Gintaras Zaleskis

1992-01-01

347

Establishment and characterization of renal cell carcinoma cell lines with multidrug resistance  

Microsoft Academic Search

Many of the discoveries of multidrug resistance (MDR) have resulted from studies using drug-resistant cultured tumor cell\\u000a lines as experimental models. To date, there has been no report on the detailed characterization of such a cell line from\\u000a renal cell carcinoma (RCC). By long-term exposure of an established RCC (RCC8701) to increasing concentrations of adriamycin,\\u000a we established a series of

Dah-Shyong Yu; Cheng-Ping Ma; Sun-Yran Chang

2000-01-01

348

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

PubMed

Culture of cell lines from fetuses or postnatal animals is an essential part of somatic cell cloning. Fetal bovine serum (FBS) is commonly used in media for propagation of these cells. Unfortunately, bovine fetuses and postnatal animals as well as FBS are all possible sources of non-cytopathic bovine viral diarrhea virus (BVDV) which is widely distributed among cattle. This study was prompted when screening of samples sent to veterinary diagnostic labs revealed that 15 of 39 fetal fibroblast cell lines used in cloning research were positive for BVDV as determined by various assays including reverse transcription-polymerase chain reaction (RT-PCR). Goals of the research were to use both virus isolation and reverse transcription-nested polymerase chain reaction (RT-nPCR) to confirm which of the cell lines were actually infected with BVDV and to assay samples of media, FBS and the earliest available passages of each cell line in an attempt to determine the source of the viral infections. Sequence analysis of amplified cDNA from all isolates was performed to provide a definitive link between possible sources of virus and infected cell lines. Only 5 of the 39 cell lines were actually infected with BVDV. Three of these five lines were not infected at the earliest cryopreserved passage, leading to the conclusion that they likely became infected after culture in media containing contaminated FBS. In fact, sequence comparison of the amplified cDNA from one lot of FBS confirmed that it was the source of infection for one of these cell lines. Since BVDV was isolated from the remaining two cell lines at the earliest available passage, the fetuses from which they were established could not be ruled out as the source of the virus. PMID:15710188

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

2005-03-01

349

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

PubMed Central

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

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

1994-01-01

350

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

PubMed

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

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

2013-08-01

351

Red Cell Apheresis with Automated In-Line Filtration  

PubMed Central

Summary Background The aim of this study was to provide data on concurrent red blood cell (RBC) and platelet (PLT) apheresis with RBC in-line leukoreduction and automated addition of saline-adenine-glucose-mannitol (SAGM) using the new version (V6.0) of Trima Accel®. Methods In this two-center paired study, each subject completed a test and a control procedure with an interval of 9 weeks between procedures. In the test arm, single RBC and PLT units were collected on the Trima Accel V6.0 (in-line leukofiltration and automated addition of SAGM). In the control arm, they were collected on Trima Accel V5.1/V5.2 (post-collection leukoreduction, manual SAGM addition). RBC percent hemolysis, potassium concentration and adenosine triphosphate over storage, hemoglobin (Hb) yield, and residual white blood cells (WBC) were determined. Results 34 subjects successfully completed both test and control procedures. Post-storage hemolysis was similar in both groups, and all values were less than 0.8% for both arms. Residual WBC counts in all RBC units were less than 1 × 106/unit. In-line processed RBC units (V6.0) have a significantly higher volume and more Hb/unit due to filtration recovery improvements. All procedures were well tolerated by the subjects. Conclusion In-line filtration and automated addition of storage solution on Trima Accel V6.0 allows collection of ready-to-use RBC units that meet EU requirements.

Matthes, Gert; Ingilizov, Marin; Dobao, Maria Luz; Marques, Susana; Callaert, Martine

2014-01-01

352

Choosing the right cell line for breast cancer research.  

PubMed

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

Holliday, Deborah L; Speirs, Valerie

2011-01-01

353

Designing of promiscuous inhibitors against pancreatic cancer cell lines  

PubMed Central

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.

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

2014-01-01

354

Designing of promiscuous inhibitors against pancreatic cancer cell lines  

NASA Astrophysics Data System (ADS)

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.

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

2014-04-01

355

New cell lines from mouse epiblast share defining features with human embryonic stem cells.  

PubMed

The application of human embryonic stem (ES) cells in medicine and biology has an inherent reliance on understanding the starting cell population. Human ES cells differ from mouse ES cells and the specific embryonic origin of both cell types is unclear. Previous work suggested that mouse ES cells could only be obtained from the embryo before implantation in the uterus. Here we show that cell lines can be derived from the epiblast, a tissue of the post-implantation embryo that generates the embryo proper. These cells, which we refer to as EpiSCs (post-implantation epiblast-derived stem cells), express transcription factors known to regulate pluripotency, maintain their genomic integrity, and robustly differentiate into the major somatic cell types as well as primordial germ cells. The EpiSC lines are distinct from mouse ES cells in their epigenetic state and the signals controlling their differentiation. Furthermore, EpiSC and human ES cells share patterns of gene expression and signalling responses that normally function in the epiblast. These results show that epiblast cells can be maintained as stable cell lines and interrogated to understand how pluripotent cells generate distinct fates during early development. PMID:17597760

Tesar, Paul J; Chenoweth, Josh G; Brook, Frances A; Davies, Timothy J; Evans, Edward P; Mack, David L; Gardner, Richard L; McKay, Ronald D G

2007-07-12

356

Can we develop ethically universal embryonic stem-cell lines?  

Microsoft Academic Search

Human embryonic stem-cell (hESC) research faces opposition from those who object to the destruction of human embryos. Over the past few years, a series of new approaches have been proposed for deriving hESC lines without injuring a living embryo. Each of these presents scientific challenges and raises ethical and political questions. Do any of these methods have the potential to

Ronald M Green

2007-01-01

357

Characterization of 5-Fluorouracil-Resistant Cholangiocarcinoma Cell Lines  

Microsoft Academic Search

Background: Although 5-fluorouracil (5-FU) is the drug of choice for the palliative treatment of cholangiocarcinoma (CCA), resistance to the drug is a therapeutic obstacle. The aim of this study was to explore the mechanisms underlying 5-FU resistance of CCA using cell lines derived from CCA associated with liver fluke infection. Methods: A stepwise exposure was used for inducing 5-FU-resistant CCA

Nisana Namwat; Piyawan Amimanan; Watcharin Loilome; Patcharee Jearanaikoon; Banchob Sripa; Vajarabhongsa Bhudhisawasdi; Wichittra Tassaneeyakul

2008-01-01

358

Multiple genetic manipulations of DT40 cell line.  

PubMed

Reverse genetics is gaining importance in the field of modern biological sciences. Gene disruption and the use of siRNAs are the favored techniques for current research. Many researchers, however, are aware that the data from siRNA experiments are frequently inconsistent and that epistatic analysis of multiple genes using siRNAs is barely feasible. In recognition of the drawbacks of the siRNA technique, many researchers, especially in the field of DNA repair, are now introducing multiple genetic disruption techniques using the chicken DT40 cell line into their research. Thus, recent publications increasingly include data utilizing DT40 cells. In this chapter, we describe the current standard methods of multiple genetic manipulation in DT40 cells. We place a particular emphasis on describing the basic concepts and theoretical background of the genetic manipulation of DT40 cells for researchers who are new to such techniques. PMID:24557895

Motegi, Akira; Takata, Minoru

2014-01-01

359

Phenotypic characterization of Ewing sarcoma cell lines with monoclonal antibodies.  

PubMed

The histogenesis of Ewing sarcoma, the second most frequent bone tumor in humans, remains controversial. Four Ewing cell lines were analyzed by immunological methods. A panel of antibodies directed to T, B, and myelomonocytic markers gave negative results. Surface antigens recognized on Ewing cells were found to be related to the neuroectoderm lineage. Ganglioside GD2, a marker of neuroectodermal tissues and tumors, was present on all lines. These were also stained by the mouse monoclonal antibody HNK-1, which detects a carbohydrate epitope present on several glycoconjugates of the nervous system, including two glycoproteins, the myelin-associated glycoprotein and the neural cell-adhesion molecule (N-CAM), and an acidic glycolipid of the peripheral nervous system. The P61 monoclonal antibody, which reacts with a peptide moiety of N-CAM, and a rabbit antiserum, raised to purified mouse N-CAM and not recognizing the HNK-1-defined epitope, were also reactive. By contrast, all antibodies specific for hematopoietic cell surface antigens were totally negative. Besides these antigenic features, Ewing sarcoma cells are characterized by a specific t(11;22)(q24;q12) translocation also observed in neuroepithelioma, a neuroectodermal tumor, suggesting a possible evolutionary related origin. The recent finding that the human N-CAM gene is located at the vicinity of the breakpoint on chromosome 11 indicates that it might be involved in genetic rearrangements occurring in this region. PMID:3760036

Lipinski, M; Braham, K; Philip, I; Wiels, J; Philip, T; Dellagi, K; Goridis, C; Lenoir, G M; Tursz, T

1986-01-01

360

The hairy cell leukemia cell line Eskol spontaneously synthesizes tumor necrosis factor- ? and nitric oxide  

Microsoft Academic Search

Tumor necrosis factor-? (TNF-?) and nitric oxide (NO) exert a wide array of immunoregulatory, partly related effects. We examined the production of these two mediators by the human hairy cell leukemia cell line Eskol. Combined cell lysate and supernatant of Eskol cells (0.5×106 cells ml?1) incubated for 18 h, contained a mean of 1.5 ng ml?1 TNF-?. This spontaneous TNF-?

Andreas Eigler; Kerstin Waller-Fontaine; Jochen Moeller; Gunther Hartmann; Ulrich T Hacker; Stefan Endres

1998-01-01

361

Cancer cell lines for drug discovery and development.  

PubMed

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. Cancer Res; 74(9); 2377-84. ©2014 AACR. PMID:24717177

Wilding, Jennifer L; Bodmer, Walter F

2014-05-01

362

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

PubMed Central

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

Fallon, Ann M.; Gerenday, Anna

2010-01-01

363

Cell surface and secreted protein profiles of human thyroid cancer cell lines reveal distinct glycoprotein patterns.  

PubMed

Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated, and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using two-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hurthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57% are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g., CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hurthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e., anaplastic). On the basis of the results obtained, a set of glycoprotein biomarker candidates for thyroid cancer is proposed. PMID:19530676

Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A

2009-08-01

364

Cell Surface and Secreted Protein Profiles of Human Thyroid Cancer Cell Lines Reveal Distinct Glycoprotein Patterns  

PubMed Central

Cell surface proteins have been shown to be effective therapeutic targets. In addition, shed forms of these proteins and secreted proteins can serve as biomarkers for diseases, including cancer. Thus, identification of cell surface and secreted proteins has been a prime area of interest in the proteomics field. Most cell surface and secreted proteins are known to be glycosylated and therefore, a proteomics strategy targeting these proteins was applied to obtain proteomic profiles from various thyroid cancer cell lines that represent the range of thyroid cancers of follicular cell origin. In this study, we oxidized the carbohydrates of secreted proteins and those on the cell surface with periodate and isolated them via covalent coupling to hydrazide resin. The glycoproteins obtained were identified from tryptic peptides and N-linked glycopeptides released from the hydrazide resin using 2-dimensional liquid chromatography-tandem mass spectrometry in combination with the gas phase fractionation. Thyroid cancer cell lines derived from papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), Hürthle cell carcinoma (XTC-1), and anaplastic thyroid cancer (ARO and DRO-1) were evaluated. An average of 150 glycoproteins were identified per cell line, of which more than 57 percent are known cell surface or secreted glycoproteins. The usefulness of the approach for identifying thyroid cancer associated biomarkers was validated by the identification of glycoproteins (e.g. CD44, galectin 3 and metalloproteinase inhibitor 1) that have been found to be useful markers for thyroid cancer. In addition to glycoproteins that are commonly expressed by all of the cell lines, we identified others that are only expressed in the more well-differentiated thyroid cancer cell lines (follicular, Hürthle cell and papillary), or by cell lines derived from undifferentiated tumors that are uniformly fatal forms of thyroid cancer (i.e. anaplastic). Based on the results obtained, a set of glycoprotein biomarker candidates for thyroid cancer is proposed.

Arcinas, Arthur; Yen, Ten-Yang; Kebebew, Electron; Macher, Bruce A.

2009-01-01

365

Cloned mice and embryonic stem cell lines generated from adult somatic cells by nuclear transfer.  

PubMed

Mice can now be cloned from cultured and noncultured adult-, fetus-, male-, or female-derived cells. Using the mouse as a model, research is moving towards a comprehensive description of clones generated by somatic cell nuclear transfer. In addition, embryonic stem (ES) cell lines can be generated from adult somatic cells via nuclear transfer (ntES cells). ntES cells contribute to an extensive variety of cell types including neurons in vitro and germ cells in vivo. Recent advances in mouse cloning are reported to illustrate its strengths and promise in the study of mammalian biology and biomedicine. PMID:12725519

Wakayama, Teruhiko

2003-01-01

366

Cell Type-specific ?2-Adrenergic Receptor Clusters Identified Using Photoactivated Localization Microscopy Are Not Lipid Raft Related, but Depend on Actin Cytoskeleton Integrity*  

PubMed Central

Recent developments in the field of optical super-resolution techniques allow both a 10-fold increase in resolution as well as an increased ability to quantify the number of labeled molecules visualized in the fluorescence measurement. By using photoactivated localization microscopy (PALM) and an experimental approach based on the systematic comparison with a nonclustering peptide as a negative control, we found that the prototypical G protein-coupled receptor ?2-adrenergic receptor is partially preassociated in nanoscale-sized clusters only in the cardiomyocytes, such as H9C2 cells, but not in other cell lines, such as HeLa and Chinese hamster ovary (CHO). The addition of the agonist for very short times or the addition of the inverse agonist did not significantly affect the organization of receptor assembly. To investigate the mechanism governing cluster formation, we altered plasma membrane properties with cholesterol removal and actin microfilament disruption. Although cholesterol is an essential component of cell membranes and it is supposed to be enriched in the lipid rafts, its sequestration and removal did not affect receptor clustering, whereas the inhibition of actin polymerization did decrease the number of clusters. Our findings are therefore consistent with a model in which ?2 receptor clustering is influenced by the actin cytoskeleton, but it does not rely on lipid raft integrity, thus ruling out the possibility that cell type-specific ?2 receptor clustering is associated with the raft.

Scarselli, Marco; Annibale, Paolo; Radenovic, Aleksandra

2012-01-01

367

Mogoltacin enhances vincristine cytotoxicity in human transitional cell carcinoma (TCC) cell line  

Microsoft Academic Search

Bladder cancer is the second common cancer of the genitourinary system throughout the world and intravesical chemotherapy is usually used to reduce tumour recurrence and progression. Human transitional cell carcinoma (TCC) is an epithelial-like adherent cell line originally established from primary bladder carcinoma.Here we report the effect of mogoltacin, a sesquiterpene coumarin from Ferula badrakema on TCC cells. Mogoltacin was

F. Behnam Rassouli; M. M. Matin; M. Iranshahi; A. R. Bahrami; V. Neshati; S. Mollazadeh; Z. Neshati

2009-01-01

368

Pulse and Trapezoidal Voltage Clamp Applied To Jurkat Cells: A T-Lymphocyte Cell Line.  

National Technical Information Service (NTIS)

The whole cell patch technique was applied to Jurkat cells, a human leukemic T-lymphocyte cell line. From a holding potential of 70 mv, a sequence of depolarizing square voltage pulses ranging from 0 to 130 mv., spaced 10 mv apart and of duration 190ms., ...

S. Yeandle W. A. Gottschalk

1993-01-01

369

The invasiveness of five medulloblastoma cell lines in collagen gels.  

PubMed

Local recurrence continues to limit survival in medulloblastoma patients, largely related to the persistence of invasive cells at the site of tumour resection and leptomeningeal dissemination. Given the relative dearth of understanding of causative mechanisms behind the invasiveness of medulloblastomas, and a general lack of validated in vitro models with which to study them, our objectives were (1) to obtain quantitative data on the invasiveness of five distinct medulloblastoma cell lines within a 3-dimensional in vitro collagen-based model; and (2) to characterize some of the mechanisms behind invasion, specifically striving to identify proteolytic processes that occur as medulloblastoma cells disrupt and thereby invade the normal tissue surrounding them, and specific inhibitors of these proteolytic enzymes. Five different medulloblastoma cell lines (UW228-1, 2 and 3; Daoy, and Madsen) were implanted onto a 3-dimensional, type I collagen gel assay to assess tumour invasion distance and mean doubling time over 5 days. Proteolytic activity was assessed against collagen types I and IV by measuring the degradation of 3H-collagen I and IV to products soluble in 100% w/v trichloroacetic acid; and general (neutral) proteolytic activity evaluated by measuring the degradation of 3H-albumin. In other experiments, cells were pre-exposed to a variety of protease inhibitors, including inhibitors of metalloproteinases and cysteine, serine and aspartic proteases, and then plated to identify any inhibition of invasion. Inter-group differences in mean invasion distance were assessed by means of Student's t-tests for non-paired subjects, with P < 0.05 set as the threshold for statistical significance. For the inhibitor studies, an inhibition index, called the inhibitory concentration 50, IC-50, was calculated by performing a regression analysis for each inhibitor tested over a range of concentrations, for each cell line. Within hours of implantation, individual cells readily detached from the surface of the cell aggregates and invaded the collagen matrix, to distances of up to 1,200 mum and at rates of up to 300-mum per day; the UW228-1 cell line clearly was less invasive than the other four cell lines. Proteolytic activity was identified against collagen type I, but not against collagen type IV or albumin; but there was no apparent correlation between invasion distance and either cell doubling time or the amount of collagen type I proteolytic activity. Both metalloproteinase inhibitors suppressed tumour invasion, as did one of two cysteine protease inhibitors; but there was no tumour suppression with either serine or aspartic protease inhibition. MMP-1 and 2, and TIMP-1 and 2 all were detectable by Western blot analysis. Medulloblastoma cell invasiveness within the 3-dimensional model used here appears to depend upon a combination of metalloproteinase and cysteine protease activity, a finding that may suggest areas for potential future clinical investigation and therapy. PMID:19847623

Ranger, Adrianna; McDonald, Warren; Moore, Emi; Delmaestro, Rolando

2010-01-01

370