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Sample records for preadipocyte cell line

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

    PubMed

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

    2010-10-01

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

  2. Effects of lysophosphatidic acid on the in vitro proliferation and differentiation of a novel porcine preadipocyte cell line.

    PubMed

    Nobusue, Hiroyuki; Kondo, Daisuke; Yamamoto, Makiko; Kano, Koichiro

    2010-12-01

    We examined the effects of lysophosphatidic acid (LPA) on in vitro proliferation and differentiation of a porcine preadipocyte cell line, DFAT-P, and a mouse preadipocyte cell line, 3T3-L1. During the proliferation and differentiation phases, DFAT-P and 3T3-L1 cells expressed only the endothelial differentiation gene (EDG)-2 receptor and not EDG-4 and EDG-7 receptors. LPA promoted the proliferation of DFAT-P cells more extensively than that of 3T3-L1 cells. After adipogenic induction, LPA inhibited glycerol-3-phosphate dehydrogenase activity and lipid droplet accumulation, and suppressed peroxisome proliferator-activated receptor γ (PPARγ) protein expression, this inhibitory effect in DFAT-P cells was twice as high as that in 3T3-L1 cells. Furthermore, treatments with low LPA concentrations significantly inhibited adipocyte differentiation in DFAT-P cells but not in 3T3-L1 cells. We conclude that LPA promotes the proliferation of porcine preadipocytes through the EDG-2 receptor but inhibits their differentiation, and these effects depend on the down-regulation of PPARγ expression via the EDG-2 receptor. Furthermore, DFAT-P cells are more sensitive to LPA than 3T3-L1 cells. These findings in a porcine model will contribute to the understanding of LPA action mechanisms on in vitro proliferation and differentiation of preadipocytes in domestic animals and/or humans.

  3. Establishment of a preadipocyte cell line derived from mature adipocytes of GFP transgenic mice and formation of adipose tissue.

    PubMed

    Nobusue, Hiroyuki; Endo, Tsuyoshi; Kano, Koichiro

    2008-06-01

    We established a preadipocyte cell line from mature adipocytes obtained from subcutaneous fat tissue of green fluorescent protein (GFP) transgenic mice. The floating top layer, containing mature adipocytes, was isolated from subcutaneous fat tissue by collagenase digestion and filtration. Fluorescence-activated cell sorting and microscopic analysis revealed that the floating cell fraction comprised a highly homogeneous adipocyte population with no adipose stromal-vascular cells. Isolated mature adipocytes dedifferentiated into fibroblast-like cells and actively proliferated in ceiling culture. In vitro studies showed that the cells could redifferentiate into mature adipocytes in an identical way to 3T3-L1 preadipocytes. No changes in the differentiation pattern were observed during the propagation of our cells. They were successfully maintained and differentiated for at least 22 passages. We named these cells dedifferentiated fat (DFAT-GFP) cells. When DFAT-GFP cells were implanted subcutaneously into C57BL/6N mice, they developed highly vascularized fat pads that morphologically resembled normal subcutaneous adipose tissue and consisted of GFP-positive cells; however, implanted 3T3-L1 cells did not have such an effect on the mice. We conclude that DFAT-GFP cells provide a model that should enable us to study the mechanisms of adipocyte differentiation and adipose tissue formation in vivo and in vitro.

  4. Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

    SciTech Connect

    Chang, Young-Chae; Cho, Hyun-Ji

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

  5. PCB126 Inhibits Adipogenesis of Human Preadipocytes

    PubMed Central

    Gadupudi, Gopi; Gourronc, Francoise A.; Ludewig, Gabriele; Robertson, Larry W.; Klingelhutz, Aloysius J.

    2014-01-01

    Emerging evidence indicates that persistent organic pollutants (POPs), including polychlorinated biphenyls (PCBs), are involved in the development of diabetes. Dysfunctional adipocytes play a significant role in initiating insulin resistance. Preadipocytes make up a large portion of adipose tissue and are necessary for the generation of functional mature adipocytes through adipogenesis. PCB126 is a dioxin-like PCB and a potent aryl hydrocarbon receptor (AhR) agonist. We hypothesized that PCB126 may be involved in the development of diabetes through disruption of adipogenesis. Using a newly developed human preadipocyte cell line called NPAD (Normal PreADipocytes), we found that exposure of preadipocytes to PCB126 resulted in significant reduction in their subsequent ability to fully differentiate into adipocytes, more so than when the cells were exposed to PCB126 during differentiation. Reduction in differentiation by PCB126 was associated with downregulation of transcript levels of a key adipocyte transcription factor, PPARγ, and late adipocyte differentiation genes. An AhR antagonist, CH223191, blocked this effect. These studies indicate that preadipocytes are particularly sensitive to the effects of PCB126 and suggest that AhR activation inhibits PPARγ transcription and subsequent adipogenesis. Our results validate the NPAD cell line as a useful model for studying the effects of POPs on adipogenesis. PMID:25304490

  6. "The preadipocyte factor" DLK1 marks adult mouse adipose tissue residing vascular cells that lack in vitro adipogenic differentiation potential.

    PubMed

    Andersen, Ditte Caroline; Jensen, Line; Schrøder, Henrik Daa; Jensen, Charlotte Harken

    2009-09-03

    Delta-like 1 (Dlk1) is expressed in 3T3-L1 preadipocytes and has frequently been referred to as "the" preadipocyte marker, yet the phenotype of DLK1(+) cells in adipose tissue remains undetermined. Herein, we demonstrate that DLK1(+) cells encompass around 1-2% of the adult mouse adipose stromal vascular fraction (SVF). Unexpectedly, the DLK1(+)SVF population was enriched for cells expressing genes generally ascribed to the vascular lineage and did not possess any adipogenic differentiation potential in vitro. Instead, DLK1(+) cells comprised an immediate ability for cobblestone formation, generation of tube-like structures on matrigel, and uptake of Acetylated Low Density-Lipoprotein, all characteristics of endothelial cells. We therefore suggest that DLK1(+)SVF cells are of a vascular origin and not them-selves committed preadipocytes as assumed hitherto.

  7. Stable SREBP-1a knockdown decreases the cell proliferation rate in human preadipocyte cells without inducing senescence

    SciTech Connect

    Alvarez, María Soledad; Fernandez-Alvarez, Ana; Cucarella, Carme; Casado, Marta

    2014-04-25

    Highlights: • SGBS cells mostly expressed SREBP-1a variant. • SREBP-1a knockdown decreased the proliferation of SGBS cells without inducing senescence. • We have identified RBBP8 and CDKN3 genes as potential SREBP-1a targets. - Abstract: Sterol regulatory element binding proteins (SREBP), encoded by the Srebf1 and Srebf2 genes, are important regulators of genes involved in cholesterol and fatty acid metabolism. Whereas SREBP-2 controls the cholesterol synthesis, SREBP-1 proteins (-1a and -1c) function as the central hubs in lipid metabolism. Despite the key function of these transcription factors to promote adipocyte differentiation, the roles of SREBP-1 proteins during the preadipocyte state remain unknown. Here, we evaluate the role of SREBP-1 in preadipocyte proliferation using RNA interference technology. Knockdown of the SREBP-1a gene decreased the proliferation rate in human SGBS preadipocyte cell strain without inducing senescence. Furthermore, our data identified retinoblastoma binding protein 8 and cyclin-dependent kinase inhibitor 3 genes as new potential SREBP-1 targets, in addition to cyclin-dependent kinase inhibitor 1A which had already been described as a gene regulated by SREBP-1a. These data suggested a new role of SREBP-1 in adipogenesis via regulation of preadipocyte proliferation.

  8. Testosterone induces cell proliferation and cell cycle gene overexpression in human visceral preadipocytes.

    PubMed

    Barbosa-Desongles, Anna; Hernández, Cristina; Simó, Rafael; Selva, David M

    2013-08-01

    Evidence from the literature suggests that testosterone plays an important role in visceral fat accumulation since both men and women with hyperandrogenism accumulate more adipose tissue in the abdominal cavity than healthy women. However, the underlying mechanisms remain to be elucidated. To shed light on this issue, we have used an in vitro approach to examine the effect of testosterone on human visceral preadipocyte proliferation. Our results showed that testosterone treatment significantly increased proliferation of human visceral preadipocytes in proliferation assays using flow cytometric analysis. We next performed a microarray gene expression analysis of human visceral preadipocytes treated with testosterone or vehicle to identify which genes were involved in the testosterone-induced increase in preadipocyte proliferation. The results showed a total of 140 genes differentially expressed between testosterone vs. vehicle. Among the top 10 upregulated genes, 5 were involved in cellular cycle and proliferation, and 3 (APOBEC3b, CCNA2, and PRC1) were significantly overexpressed by testosterone treatment when analyzed by real-time PCR. We conclude that testosterone exerts a proliferative effect on preadipocytes that may participate in the sex differences in fat distribution and that it may explain visceral fat accumulation in women with hyperandrogenism.

  9. Chronic activation of pattern recognition receptors suppresses brown adipogenesis of multipotent mesodermal stem cells and brown pre-adipocytes.

    PubMed

    Bae, Jiyoung; Chen, Jiangang; Zhao, Ling

    2015-06-01

    Brown adipose tissue (BAT) holds promise to combat obesity through energy-spending, non-shivering thermogenesis. Understanding of regulation of BAT development can lead to novel strategies to increase BAT mass and function for obesity treatment and prevention. Here, we report the effects of chronic activation of PRR on brown adipogenesis of multipotent mesodermal stem C3H10T1/2 cells and immortalized brown pre-adipocytes from the classical interscapular BAT of mice. Activation of NOD1, TLR4, or TLR2 by their respective synthetic ligand suppressed brown marker gene expression and lipid accumulation during differentiation of brown-like adipocytes of C3H10T1/2. Activation of the PRR only during the commitment was sufficient to suppress the differentiation. PRR activation suppressed PGC-1α mRNA, but induced PRDM16 mRNA at the commitment. Consistently, PRR activation suppressed the differentiation of immortalized brown pre-adipocytes. Activation of PRR induced NF-κB activation in both cells, which correlated with their abilities to suppress PPARγ transactivation, a critical event for brown adipogenesis. Taken together, our results demonstrate that chronic PRR activation suppressed brown adipogenesis of multipotent mesodermal stem cells and brown pre-adipocytes, possibly through suppression of PPARγ transactivation. The results suggest that anti- inflammatory therapies targeting PRRs may be beneficial for the BAT development.

  10. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

    SciTech Connect

    Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.; Zhang, J.; Oreffo, R.O.C. . E-mail: roco@soton.ac.uk

    2006-06-10

    RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPAR{gamma}) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPAR{gamma}-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negative siRNA). The inhibitory effect of PPAR{gamma}-siRNA was supported by testing human PPAR{gamma} mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP{sub 3}) expression, an adipocyte-specific marker. The current studies indicate that PPAR{gamma}-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells.

  11. Increased expression of inflammation-related genes in cultured preadipocytes/stromal vascular cells from obese compared with non-obese Pima Indians

    PubMed Central

    Lee, Y. H.; Rousseau, E.; Tataranni, P. A.; Baier, L. J.; Bogardus, C.; Cam, M.; Permana, P. A.

    2006-01-01

    Aims/hypothesis: The specific contributions made by the various cell types in adipose tissue to obesity, particularly obesity-related inflammation, need to be clarified. The aim of this study was to elucidate the potential role of adipocyte precursor cells (preadipocytes/stromal vascular cells [SVC]). Methods: We performed Affymetrix oligonucleotide microarray expression profiling of cultured abdominal subcutaneous preadipocytes/SVC isolated from the adipose tissue of 14 non-obese (BMI 25±4 kg/m2) and 14 obese (55±8 kg/m2) non-diabetic Pima Indian subjects. Quantitative real-time PCR (RT-PCR) was used to verify the differential expression of several genes in an independent group of subjects. Results: We identified 218 differentially expressed genes with p values less than 0.01. Microarray expression profiling revealed that the expression of inflammation-related genes was significantly upregulated in preadipocytes/SVC of obese individuals. Quantitative RT-PCR confirmed the upregulation of IL8, CTSS, ITGB2, HLA-DRA, CD53, PLA2G7 and MMP9 in preadipocytes/SVC of obese subjects. Conclusions/interpretation: The upregulation of inflammation-related genes in preadipocytes/SVC of obese subjects may increase the recruitment of immune cells into adipose tissue and may also result in changes in the extracellular matrix (tissue remodelling) to accommodate adipose tissue expansion in obesity. PMID:16034612

  12. β-Catenin Mediates Anti-adipogenic and Anticancer Effects of Arctigenin in Preadipocytes and Breast Cancer Cells.

    PubMed

    Lee, Jihye; Imm, Jee-Young; Lee, Seong-Ho

    2017-03-29

    Arctigenin is a lignan abundant in Asteraceae plants and has anti-inflammatory, antiobesity, and anticancer activities. Obesity is one of the leading causes of several types of cancers including breast cancer. The current study was performed to investigate if arctigenin suppresses differentiation of preadipocytes and proliferation of breast cancer cells and to explore potential molecular mechanisms. Treatment of arctigenin reduced lipid accumulation in differentiated 3T3-L1 adipocytes in a dose- and time-dependent manner without toxicity. Arctigenin suppressed the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein-alpha (C/EBPα), perilipin, and fatty acid binding protein 4 (FABP4) in a dose-dependent manner in differentiated 3T3-L1 cells. Both total and unphosphorylated (active) β-catenin were increased in whole cell lysates and the nuclear fraction of differentiated 3T3-L1 cells treated with 25 μM arctigenin. On the other hand, arctigenin decreased proliferation of two human breast cancer cells (MCF-7 and MDA-MB-231). Arctigenin induced apoptosis and decreased expression of total and unphosphorylated (active) β-catenin and cyclin D1 in MCF-7, but not in MDA-MB-231. These data indicate that arctigenin suppressed adipogenesis in preadipocytes and activated apoptosis in estrogen receptor (ER) positive breast cancer cells through modulating expression of β-catenin.

  13. Horse serum reduces expression of membrane-bound and soluble isoforms of the preadipocyte marker Delta-like 1 homolog (Dlk1), but is inefficient for adipogenic differentiation of mouse preadipocytes.

    PubMed

    Andersen, Ditte C; Nielsen, Charlotte; Jensen, Charlotte H; Sheikh, Søren P

    2013-05-01

    Downregulation of the preadipocyte marker Delta-like 1 homologue (Dlk1), an inhibitor of adipogenesis, has been suggested to be a prerequisite for adipogenic differentiation to occur, and low Dlk1 levels are often used to verify adipogenesis. Mouse preadipocytic cell lines such as 3T3-L1, as well as primary derived preadipocytes, are important models to study adipogenic differentiation and obesity. However, in vitro adipogenic differentiation of primary derived preadipocytes remains incomplete, and identification of factors that will improve the adipogenic differentiation process is thus of high value. In this study we show that horse serum fails to improve adipogenic differentiation of mouse preadipocytes (both 3T3-L1 cells and primary derived mouse preadipocytes) as otherwise reported for bone marrow derived adipogenic precursors. Unexpectedly, while Dlk1 levels were indeed decreased using horse serum, this did not correlate with a high degree of adipogenic differentiation. In conclusion, our novel results thus reveal that horse serum clearly is insufficient for adipogenic differentiation of mouse preadipocytes and that low levels of Dlk1 alone are a poor marker of mouse in vitro adipogenesis. We would also like to emphasize that it is very important for the field of cellular differentiation that researchers thoroughly investigate the effect of individual reagents in their protocols. Such data will increase understanding of the limitations and possibilities of individual systems.

  14. Protein kinase A-mediated cell proliferation in brown preadipocytes is independent of Erk1/2, PI{sub 3}K and mTOR

    SciTech Connect

    Wang, Yanling; Sato, Masaaki; Guo, Yuan; Bengtsson, Tore; Nedergaard, Jan

    2014-10-15

    The physiological agonist norepinephrine promotes cell proliferation of brown preadipocytes during the process of tissue recruitment. In a primary culture system, cAMP mediates these adrenergic effects. In the present study, we demonstrated that, in contrast to other systems where the mitogenic effect of cAMP requires the synergistic action of (serum) growth factors, especially insulin/IGF, the cAMP effect in brown preadipocytes was independent of serum and insulin. Protein kinase A, rather than Epac, mediated the cAMP mitogenic effect. The Erk 1/2 family of MAPK, the PI{sub 3}K system and the mTOR complexes were all activated by cAMP, but these activations were not necessary for cAMP-induced cell proliferation; a protein kinase C isoform may be involved in mediating cAMP-activated cell proliferation. We conclude that the generally acknowledged cellular mediators for induction of cell proliferation are not involved in this process in the brown preadipocyte system; this conclusion may be of relevance both for examination of mechanisms for induction of brown adipose tissue recruitment but also for understanding the mechanism behind e.g. certain endocrine neoplasias. - Highlights: • cAMP can mimick norepinephrine-induced proliferation of brown preadipocytes. • The cAMP-induced proliferation can occur in the absence of serum, of any other growth factors, and of insulin. • Erk1/2, PI{sub 3}K and mTOR are cAMP activated but not involved in induction of proliferation. • A Protein Kinase C member may be in the signalling cascade. • This pathway analysis may also be of importance for certain endocrine hyper- and neoplasias.

  15. Preadipocyte proliferation is affected by norepinephrine

    SciTech Connect

    Jones, D.D.; Hausman, G.J.; Martin, R.J.

    1986-03-01

    Beta-adrenergic stimulators are known to alter body composition profiles in experimental animals. The most prominent in vivo adaptations to beta-agonists are a decreased adipose tissue and concurrent increased muscle mass. Our lab has previously reported that norepinephrine - a beta/sub 1/-agonist - compromised preadipocyte growth in a primary cell culture system. Accordingly, this study continued to examine the effect of norepinephrine (NE) on in vitro preadipocyte proliferation. Stromalvascular cells, obtained from the inguinal pad of 4-5 week old Sprague-Dawley rats, were grown in culture for two weeks. Cells were treated with NE (1 ..mu..M) during the proliferative growth phase and labelled with /sup 3/H-thymidine on day 2 of culture. Following exponential growth, all cells were placed on a differentiation promoting media. Adipocytes (including differentiated preadipocytes) and stromal cells were separated using a density gradient, and /sup 3/H-thymidine was determined for both cell types. NE exposure for 2 or 4 days significantly reduced preadipocyte /sup 3/H-thymidine uptake, indicating an inhibition of mitotic growth. Future experiments will examine possible mechanism(s) involved in the catecholamine induced suppression of preadipocyte proliferation.

  16. Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress.

    PubMed

    Hatia, S; Septembre-Malaterre, A; Le Sage, F; Badiou-Bénéteau, A; Baret, P; Payet, B; Lefebvre d'hellencourt, C; Gonthier, M P

    2014-04-01

    Obesity has been associated with a marked risk of metabolic diseases and requires therapeutic strategies. Changes in redox status with increased oxidative stress in adipose tissue have been linked with obesity-related disorders. Thus, the biological effect of antioxidants such as polyphenols is of high interest. We aimed to measure antioxidant capacities of 28 polyphenols representative of main dietary phenolic acids, flavonoids, stilbenes and curcuminoids. Then, 14 molecules were selected for the evaluation of their effect on 3T3-L1 preadipocytes and human red blood cells exposed to oxidative stress. Analysis of reducing and free radical-scavenging capacities of compounds revealed antioxidant properties related to their structure, with higher activities for flavonoids such as quercetin and epicatechin. Their effects on preadipocytes' viability also depended on their structure, dose and time of exposure. Interestingly, most of the compounds exhibited a protective effect on preadipocytes exposed to oxidative stress, by reversing H₂O₂-induced anti-proliferative action and reactive oxygen species production. Polyphenols also exerted an anti-inflammatory effect on preadipocytes exposed to H₂O₂ by reducing IL-6 secretion. Importantly, such antioxidant and anti-inflammatory effects were observed in co-exposition (polyphenol and prooxidant during 24 h) or pretreatment (polyphenol during 24 h, then prooxidant for 24 h) conditions. Moreover, compounds protected erythrocytes from AAPH radical-induced lysis. Finally, these results led to demonstrate that antioxidant and anti-inflammatory properties of polyphenols may depend on structure, dose, time of exposure and cell conditioning with oxidative stress. Such findings should be considered for a better understanding of polyphenols' benefits in strategies aiming to prevent obesity-related diseases.

  17. Dehydroepiandrosterone exerts antiglucocorticoid action on human preadipocyte proliferation, differentiation, and glucose uptake

    PubMed Central

    McNelis, Joanne C.; Manolopoulos, Konstantinos N.; Gathercole, Laura L.; Bujalska, Iwona J.; Stewart, Paul M.; Tomlinson, Jeremy W.

    2013-01-01

    Glucocorticoids increase adipocyte proliferation and differentiation, a process underpinned by the local reactivation of inactive cortisone to active cortisol within adipocytes catalyzed by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). The adrenal sex steroid precursor dehydroepiandrosterone (DHEA) has been shown to inhibit 11β-HSD1 in murine adipocytes; however, rodent adrenals do not produce DHEA physiologically. Here, we aimed to determine the effects and underlying mechanisms of the potential antiglucocorticoid action of DHEA and its sulfate ester DHEAS in human preadipocytes. Utilizing a human subcutaneous preadipocyte cell line, Chub-S7, we examined the metabolism and effects of DHEA in human adipocytes, including adipocyte proliferation, differentiation, 11β-HSD1 expression, and activity and glucose uptake. DHEA, but not DHEAS, significantly inhibited preadipocyte proliferation via cell cycle arrest in the G1 phase independent of sex steroid and glucocorticoid receptor activation. 11β-HSD1 oxoreductase activity in differentiated adipocytes was inhibited by DHEA. DHEA coincubated with cortisone significantly inhibited preadipocyte differentiation, which was assessed by the expression of markers of early (LPL) and terminal (G3PDH) adipocyte differentiation. Coincubation with cortisol, negating the requirement for 11β-HSD1 oxoreductase activity, diminished the inhibitory effect of DHEA. Further consistent with glucocorticoid-opposing effects of DHEA, insulin-independent glucose uptake was significantly enhanced by DHEA treatment. DHEA increases basal glucose uptake and inhibits human preadipocyte proliferation and differentiation, thereby exerting an antiglucocorticoid action. DHEA inhibition of the amplification of glucocorticoid action mediated by 11β-HSD1 contributes to the inhibitory effect of DHEA on human preadipocyte differentiation. PMID:24022868

  18. Beta-mecaptoethanol suppresses inflammation and induces adipogenic differentiation in 3T3-F442A murine preadipocytes.

    PubMed

    Guo, Wen; Li, Yahui; Liang, Wentao; Wong, Siu; Apovian, Caroline; Kirkland, James L; Corkey, Barbara E

    2012-01-01

    Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is "metabolically healthy". Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.

  19. Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes

    SciTech Connect

    Holmstroem, Therese E.; Mattsson, Charlotte L.; Faelting, Johanna M.; Nedergaard, Jan

    2008-11-15

    Stimulation by both adrenergic and non-adrenergic pathways can induce proliferation of brown pre-adipocytes. To understand the signalling pathways involved in non-adrenergic stimulation of cell proliferation, we examined Erk1/2 activation. In primary cultures of mouse brown pre-adipocytes, both EGF (epidermal growth factor) and PDGF (platelet-derived growth factor) induced Erk1/2 activation. EGF-stimulated Erk1/2 activation involved Src tyrosine kinases, but not PKC or PI3K, whereas in PDGF-induced Erk1/2 activation, PI3K, PKC (probably the atypical {zeta} isoform) and Src were involved sequentially. Both EGF and PDGF induced PI3K-dependent Akt activation that was not involved in Erk1/2 activation. By comparing effects of signalling inhibitors (wortmannin, SH-6, TPA, Goe6983, PP2, PD98059) on EGF- and PDGF-induced Erk1/2 activation and cell proliferation ({sup 3}H-thymidine incorporation), we conclude that while the signal transduction pathways initiated by these growth factors are clearly markedly different, their effects on cell proliferation can be fully explained through their stimulation of Erk1/2 activation; thus Erk1/2 is a common, essential step for stimulation of proliferation in these cells.

  20. Modulation of early human preadipocyte differentiation by glucocorticoids.

    PubMed

    Tomlinson, Julianna J; Boudreau, Adèle; Wu, Dongmei; Atlas, Ella; Haché, Robert J G

    2006-11-01

    Glucocorticoids provide an adipogenic stimulus that is most obvious in the truncal obesity of patients with Cushing's syndrome. Glucocorticoid treatment also strongly potentiates the differentiation of human preadipocytes in culture. However, the molecular basis of these stimulatory effects remains to be defined. In this study, we provide a detailed analysis of the specific contribution of glucocorticoid treatment to the differentiation of primary human preadipocytes cultured in chemically defined medium. Contrary to previous descriptions of glucocorticoids being required throughout the course of differentiation, our results show that glucocorticoid treatment is stimulatory only during the first 48 h of differentiation. Furthermore, stimulation by glucocorticoids and the peroxisome proliferator activator receptor-gamma agonist troglitazone is mediated sequentially. Several details of the early events in the differentiation of human preadipocytes and the contribution of steroid to these events differ from the responses observed previously in murine preadipocyte models. First, glucocorticoid treatment stimulated the early accumulation of CCAAT enhancer binding protein-beta (C/EBPbeta) in primary human preadipocytes. Second, induction of C/EBPalpha in primary human preadipocytes was noted within 4 h of adipogenic stimulus, whereas C/EBPalpha induction is not detected until 24-48 h in the murine 3T3 L1 preadipocyte model. Remarkably, by contrast to human primary preadipocytes, which do not undergo postconfluent mitosis, 3T3 L1 murine preadipocytes stimulated to differentiate under chemically defined conditions required glucocorticoids to survive the clonal expansion that precedes terminal differentiation, revealing a novel signal imparted by glucocorticoids in this immortalized murine cell system.

  1. Differentiation of preadipocytes and mature adipocytes requires PSMB8

    PubMed Central

    Arimochi, Hideki; Sasaki, Yuki; Kitamura, Akiko; Yasutomo, Koji

    2016-01-01

    The differentiation of adipocytes is tightly regulated by a variety of intrinsic molecules and also by extrinsic molecules produced by adjacent cells. Dysfunction of adipocyte differentiation causes lipodystrophy, which impairs glucose and lipid homeostasis. Although dysfunction of immunoproteasomes causes partial lipodystrophy, the detailed molecular mechanisms remain to be determined. Here, we demonstrate that Psmb8, a catalytic subunit for immunoproteasomes, directly regulates the differentiation of preadipocytes and additionally the differentiation of preadipocytes to mature adipocytes. Psmb8−/− mice exhibited slower weight gain than wild-type mice, and this was accompanied by reduced adipose tissue volume and smaller size of mature adipocytes compared with controls. Blockade of Psmb8 activity in 3T3-L1 cells disturbed the differentiation to mature adipocytes. Psmb8−/− mice had fewer preadipocyte precursors, fewer preadipocytes and a reduced ability to differentiate preadipocytes toward mature adipocytes. Our data demonstrate that Psmb8-mediated immunoproteasome activity is a direct regulator of the differentiation of preadipocytes and their ultimate maturation. PMID:27225296

  2. Metformin induces glucose uptake in human preadipocyte-derived adipocytes from various fat depots.

    PubMed

    Fischer, M; Timper, K; Radimerski, T; Dembinski, K; Frey, D M; Zulewski, H; Keller, U; Müller, B; Christ-Crain, M; Grisouard, J

    2010-04-01

    To evaluate the effect of metformin on basal and insulin-induced glucose uptake in subcutaneous and visceral preadipocyte-derived adipocytes from obese and non-obese patients, preadipocytes were obtained from subcutaneous and visceral fat depots during abdominal surgery. Differentiation efficiency was evaluated by measurement of intracellular triglyceride accumulation. Preadipocyte-derived adipocytes were treated with metformin (1 mM) for 24 h with or without the addition of insulin (100 nM) for 20 min and glucose uptake was measured. In cells from each donor, intracellular triglyceride accumulation was more abundant in subcutaneous preadipocyte-derived adipocytes than in visceral preadipocyte-derived adipocytes (p < 0.001). Insulin stimulated glucose uptake in subcutaneous preadipocyte-derived adipocytes from both non-obese and obese patients (p < 0.001 vs. basal). In visceral preadipocyte-derived adipocytes, insulin did not increase basal glucose uptake. In subcutaneous preadipocyte-derived adipocytes from non-obese and obese patients, metformin alone increased glucose uptake to 2.7 +/- 0.2 (p < 0.001) and 2.1 +/- 0.1 fold (p < 0.001) respectively. Metformin increased glucose uptake in visceral preadipocyte-derived adipocytes from non-obese (1.7 +/- 0.1 fold vs. basal, p < 0.001) and obese (2.0 +/- 0.2 fold vs. basal, p < 0.001) patients. Combined treatment with metformin and insulin increased glucose uptake in subcutaneous preadipocyte-derived adipocytes from both non-obese and obese patients (p < 0.001 vs. insulin alone). In preadipocyte-derived adipocytes glucose uptake is induced by metformin independent of the fat depot origin of the preadipocytes (subcutaneous or visceral) and the obesity state of the patients (non-obese or obese). In adipocytes, metformin seems to induce glucose uptake independent of insulin suggesting an alternative mechanism of action of this drug.

  3. Cell line provenance.

    PubMed

    Freshney, R Ian

    2002-07-01

    Cultured cell lines have become an extremely valuable resource, both in academic research and in industrial biotechnology. However, their value is frequently compromised by misidentification and undetected microbial contamination. As detailed elsewhere in this volume, the technology, both simple and sophisticated, is available to remedy the problems of misidentification and contamination, given the will to apply it. Combined with proper records of the origin and history of the cell line, assays for authentication and contamination contribute to the provenance of the cell line. Detailed records should start from the initiation or receipt of the cell line, and should incorporate data on the donor as well as the tissue from which the cell line was derived, should continue with details of maintenance, and include any accidental as well as deliberate deviations from normal maintenance. Records should also contain details of authentication and regular checks for contamination. With this information, preferably stored in a database, and suitable backed up, the provenance of the cell line so created makes the cell line a much more valuable resource, fit for validation in industrial applications and more likely to provide reproducible experimental results when disseminated for research in other laboratories.

  4. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans

    SciTech Connect

    Calvo, J.C.; Rodbard, D.; Katki, A.; Chernick, S.; Yanagishita, M. )

    1991-06-15

    The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with (35S)sulfate and (3H) glucosamine for 24 h and then extracted and analyzed. There was a 1.68 {plus minus} 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of {approximately} 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of {approximately} 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 {plus minus} 0.2-fold in media and 3.2 {plus minus} 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.

  5. Adipose tissue macrophages impair preadipocyte differentiation in humans

    PubMed Central

    Liu, Li Fen; Craig, Colleen M.; Tolentino, Lorna L.; Choi, Okmi; Morton, John; Rivas, Homero; Cushman, Samuel W.; Engleman, Edgar G.; McLaughlin, Tracey

    2017-01-01

    Aim The physiologic mechanisms underlying the relationship between obesity and insulin resistance are not fully understood. Impaired adipocyte differentiation and localized inflammation characterize adipose tissue from obese, insulin-resistant humans. The directionality of this relationship is not known, however. The aim of the current study was to investigate whether adipose tissue inflammation is causally-related to impaired adipocyte differentiation. Methods Abdominal subcutaneous(SAT) and visceral(VAT) adipose tissue was obtained from 20 human participants undergoing bariatric surgery. Preadipocytes were isolated, and cultured in the presence or absence of CD14+ macrophages obtained from the same adipose tissue sample. Adipocyte differentiation was quantified after 14 days via immunofluorescence, Oil-Red O, and adipogenic gene expression. Cytokine secretion by mature adipocytes cultured with or without CD14+macrophages was quantified. Results Adipocyte differentiation was significantly lower in VAT than SAT by all measures (p<0.001). With macrophage removal, SAT preadipocyte differentiation increased significantly as measured by immunofluorescence and gene expression, whereas VAT preadipocyte differentiation was unchanged. Adipocyte-secreted proinflammatory cytokines were higher and adiponectin lower in media from VAT vs SAT: macrophage removal reduced inflammatory cytokine and increased adiponectin secretion from both SAT and VAT adipocytes. Differentiation of preadipocytes from SAT but not VAT correlated inversely with systemic insulin resistance. Conclusions The current results reveal that proinflammatory immune cells in human SAT are causally-related to impaired preadipocyte differentiation, which in turn is associated with systemic insulin resistance. In VAT, preadipocyte differentiation is poor even in the absence of tissue macrophages, pointing to inherent differences in fat storage potential between the two depots. PMID:28151993

  6. Advanced oxidation protein products inhibit differentiation and activate inflammation in 3T3-L1 preadipocytes.

    PubMed

    Zhou, Qiu Gen; Peng, Xin; Hu, Li Li; Xie, Di; Zhou, Min; Hou, Fan Fan

    2010-10-01

    Accumulation of advanced oxidation protein products (AOPPs) is prevalent in metabolic syndromes, a condition with impaired preadipocytes differentiation. In the present study, we tested the hypothesis that AOPPs disturb preadipocyte differentiation. Exposure of 3T3-L1 preadipocytes to increased levels of AOPPs inhibited accumulation of intracellular triglyceride and decreased the expression of the essential markers of matured adipocytes, such as adipocyte fatty-acid-binding protein (aP2), CAAT/enhancer-binding protein (C/EBP)-alpha, and peroxisome proliferator-activated receptor (PPAR)-gamma, in response to standard adipogenic induction. Inhibitory effects of AOPPs on preadipocytes differentiation was time sensitive, which occurred at the early stage of differentiation. In the presence of AOPPs, induction of preadipocytes differentiation resulted in upregulated expression of C/EBP homologous protein (CHOP) and CUG-Triplet repeat-binding protein (CUGBP), two important inhibitors of preadipocytes differentiation. In addition, treatment with AOPPs increased abundance of C/EBP-beta-liver enriched inhibitory protein (C/EBP-beta-LIP), a truncated C/EBP-beta isoform without adipogenic activity. Moreover, AOPPs-treated preadipocytes expressed a macrophage marker F4/80 and overexpressed tumor necrosis factor-alpha and interleukin-6 via nuclear factor-kappaB (NF-kappaB)-dependent pathway. However, blocking inflammation with NF-kappaB inhibitor failed to improve AOPPs-induced inhibition of preadipocytes differentiation. These data suggest that accumulation of AOPPs may inhibit differentiation of preadipocytes and activate inflammation in these cells. This information might have implication for understanding the impairment of preadipocytes differentiation and fat inflammation seen in metabolic syndrome.

  7. Metformin-suppressed differentiation of human visceral preadipocytes: Involvement of microRNAs.

    PubMed

    Fujita, Koji; Iwama, Hisakazu; Oura, Kyoko; Tadokoro, Tomoko; Hirose, Kayo; Watanabe, Miwako; Sakamoto, Teppei; Katsura, Akiko; Mimura, Shima; Nomura, Takako; Tani, Joji; Miyoshi, Hisaaki; Morishita, Asahiro; Yoneyama, Hirohito; Okano, Keiichi; Suzuki, Yasuyuki; Himoto, Takashi; Masaki, Tsutomu

    2016-10-01

    Visceral adipose tissue contributes to the pathophysiology of metabolic syndrome. Metformin has been reported to suppress lipogenesis in a murine preadipocyte cell line. However, the effect of metformin on the differentiation of human visceral adipose tissue remains unknown. MicroRNAs (miRNAs or miRs) have been suggested as therapeutic targets because of their involvement in the differentiation and maturation of fatty cells. The aim of this study was to determine whether metformin suppresses the differentiation of human preadipocytes and to identify miRNAs associated with the regulation of lipid metabolism. Human visceral preadipocytes (HPrAD-vis) were preincubated in growth media and then cultured with differentiation media containing metformin for 1 or 2 weeks. Adipogenic differentiation of the cells was assessed by Oil Red O staining, and soluble adiponectin in the culture media was measured using an enzyme-linked immunosorbent assay. Cell proliferation was assessed using a WST-8 assay, and the gene and protein expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT‑enhancer-binding protein α (C/EBPα) was determined by RT-qPCR and western blot analysis, respectively. miRNAs were profiled using human miRNA Oligo chips after total RNA was extracted and labeled. Oil Red O staining showed that metformin suppressed the accumulation of lipid droplets in HPrAD-vis cells. The adiponectin concentration in the culture media was also decreased in metformin-treated cells. The WST-8 assay revealed no effect on proliferation or growth inhibition following metformin treatment, although metformin suppressed the expression of PPARγ and C/EBPα. miRNA profiling further revealed differences between the metformin-treated group and control HPrAD-vis cells. Thus, the findings of the present study demonstrated that metformin suppressed the differentiation of human preadipocytes in vitro and altered the miRNA profile of these cells. Thus, the mi

  8. Pioglitazone promotes preadipocyte proliferation by downregulating p16{sup Ink4a}

    SciTech Connect

    Hasan, Arif U.; Ohmori, Koji; Hashimoto, Takeshi; Kamitori, Kazuyo; Hirata, Yuko; Ishihara, Yasuhiro; Okamoto, Naoko; Noma, Takahisa; Kosaka, Hiroaki; Tokuda, Masaaki; Kohno, Masakazu

    2011-07-29

    Highlights: {yields} Mechanisms for preadipocyte hyperplasia by pioglitazone, a PPAR{gamma} agonist, are shown. {yields} Pioglitazone promotes cell-cycle of 3T3-L1 preadipocytes and increases their number. {yields} Pioglitazone downregulates a cyclin dependent kinase inhibitor, p16{sup Ink4a}. {yields} PPAR{gamma} transrepresses p16{sup Ink4a} gene in preadipocytes, which pioglitazone enhances. -- Abstract: Pioglitazone, a synthetic ligand of peroxisome proliferator-activated receptor (PPAR){gamma}, causes preadipocyte proliferation through a mechanism which still remains elusive. Here, to address the mechanism, we investigated the effects of PPAR{gamma} and pioglitazone on the kinetics of cyclin-dependent kinase inhibitors, especially with p16{sup Ink4a} (p16) centered, by employing 3T3-L1 preadipocytes. Pioglitazone promoted preadipocyte proliferation by increasing S and G{sub 2}/M cell-cycle entry, which was accompanied by decreased p16 mRNA expression. PPAR{gamma} overexpression along with the luciferase reporter assay confirmed that PPAR{gamma} was crucial for the downregulation of p16 mRNA transcription, and that the action was augmented by pioglitazone. Thus, pioglitazone exerted cell-cycle dependent promoting effect on preadipocyte proliferation, of which mechanisms include p16-downregulation through PPAR{gamma}.

  9. Heat Shock Protein Augmentation of Angelica gigas Nakai Root Hot Water Extract on Adipogenic Differentiation in Murine 3T3-L1 Preadipocytes.

    PubMed

    Lumbera, Wenchie Marie L; Dela Cruz, Joseph; Yang, Seung-Hak; Hwang, Seong Gu

    2016-03-01

    There is a high association of heat shock on the alteration of energy and lipid metabolism. The alterations associated with thermal stress are composed of gene expression changes and adaptation through biochemical responses. Previous study showed that Angelica gigas Nakai (AGN) root extract promoted adipogenic differentiation in murine 3T3-L1 preadipocytes under the normal temperature condition. However, its effect in heat shocked 3T3-L1 cells has not been established. In this study, we investigated the effect of AGN root hot water extract in the adipogenic differentiation of murine 3T3-L1 preadipocytes following heat shock and its possible mechanism of action. Thermal stress procedure was executed within the same stage of preadipocyte confluence (G0) through incubation at 42°C for one hour and then allowed to recover at normal incubation temperature of 37°C for another hour before AGN treatment for both cell viability assay and Oil Red O. Cell viability assay showed that AGN was able to dose dependently (0 to 400 μg/mL) increase cell proliferation under normal incubation temperature and also was able to prevent cytotoxicity due to heat shock accompanied by cell proliferation. Confluent preadipocytes were subjected into heat shock procedure, recovery and then AGN treatment prior to stimulation with the differentiation solution. Heat shocked preadipocytes exhibited reduced differentiation as supported by decreased amount of lipid accumulation in Oil Red O staining and triglyceride measurement. However, those heat shocked preadipocytes that then were given AGN extract showed a dose dependent increase in lipid accumulation as shown by both evaluation procedures. In line with these results, real-time polymerase chain reaction (RT-PCR) and Western blot analysis showed that AGN increased adipogenic differentiation by upregulating heat shock protection related genes and proteins together with the adipogenic markers. These findings imply the potential of AGN in heat

  10. Obestatin changes proliferation, differentiation and apoptosis of porcine preadipocytes.

    PubMed

    Tang, Shengqiu; Dong, Xiaoying; Zhang, Wei

    2014-02-01

    Obestatin, originally identified and purified from rat stomach extracts, was reported to bind to orphan G protein-coupled receptor, GPR39, and inhibit appetite and gastric motility. This study was conducted to investigate the effects of porcine obestatin on proliferation, differentiation and apoptosis of porcine preadipocytes isolated from subcutaneous fat of piglets. At indicated times of culture, morphology of preadipocytes and accumulated lipid droplets within the cells were identified by invert microscope. After treating with obestatin (0, 0.1, 1, 10 and 100nM), cell proliferation was measured by MTT method and protein expression of CCAAT/enhancer binding protein-α (C/EBPα), peroxisome proliferator-activated receptor-γ (PPARγ), Caspase-7 and Caspase-9 was determined by Western Blot, mRNA expression of GPR39 and Caspase-3 was analyzed by RT-PCR, and the activity of Caspase-3 was measured by spectrophotometric method. The results showed that obestatin had no effect on GPR39 expression, while promotes the optical density (OD) value of cells, enhanced protein expression of PPARγ and C/EBPa, decreased mRNA expression and activity of Caspase-3, and inhibited protein expression of Caspase-7 and Caspase-9 in a dose-dependent manner. These results suggested that obestatin enhances proliferation and differentiation of preadipocytes promoting PPARγ and C/EBPa expression, and inhibiting preadipocyte apoptosis by decreasing expression of Caspase-3, Caspase-7 and Caspase-9.

  11. CLO: The cell line ontology

    PubMed Central

    2014-01-01

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

  12. Inhibition of dipeptidyl peptidase 8/9 impairs preadipocyte differentiation

    PubMed Central

    Han, Ruijun; Wang, Xinying; Bachovchin, William; Zukowska, Zofia; Osborn, John W.

    2015-01-01

    Adipocytes are the primary cells in adipose tissue, and adipocyte dysfunction causes lipodystrophy, obesity and diabetes. The dipeptidyl peptidase (DPP) 4 family includes four enzymes, DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). DPP4 family inhibitors have been used for the treatment of type 2 diabetes patients, but their role in adipocyte formation are poorly understood. Here we demonstrate that the DPP8/9 selective inhibitor 1G244 blocks adipogenesis in preadipocyte 3T3-L1 and 3T3-F422A, while DPP4 and FAP inhibitors have no effect. In addition, knockdown of DPP8 or DPP9 significantly impairs adipocyte differentiation in preadipocytes. We further uncovered that blocking the expression or activities of DPP8 and DPP9 attenuates PPARγ2 induction during preadipocyte differentiation. Addition of PPARγ agonist thiazolidinediones (TZDs), or ectopic expression of PPARγ2, is able to rescue the adipogenic defect caused by DPP8/9 inhibition in preadipocytes. These results indicate the importance of DPP8 and DPP9 on adipogenesis. PMID:26242871

  13. [Comparative analysis of Ca(2+)-signalling in brown preadipocytes of ground squirrel Spermophillus undulatus and mouse].

    PubMed

    Dolgacheva, L P; Konakov, M V; Agafonova, T A; Rybina, V V; Zinchenko, V P; Bronnikov, G E

    2007-01-01

    Analysis of the slow Ca(2+)-responses of brown preadipocytes of ground squirrel Spermophillus undulatus and mouse was carried out. The mouse brown preadipocytes demonstrated low but prominent responses to noradrenalin with the maximum at 3 and 10 microM being the less effective. The ground squirrel brown preadipocytes practically did not practically respond to 10 nM-10 microM, whereas 30-600 microM noradrenalin was able to raise intracellular [Ca2+]i up to 600 nM with 300 microM agonist being the most effective. Stimulation of the plasma membrane Ca(2+)-channels with thimerosal showed considerable reduction of the calcium entry system in the cell precursors of both species comparing with their mature adipocytes. Intracellular calcium stores liberated in preadipocytes of both species by tapsigargin and ionomycin in Ca(2+)-free medium were insignificant, and capacitative Ca(2+)-entry in response to the cellular Ca(2+)-stores depletion was completely absent in Ca(2+)-containing medium. The Ca(2+)-responses of the ground squirrel brown preadipocytes were independent on physiological state of the animals and annual seasons. Preadipocytes of both species showed the same dose-response curves for the Ca(2+)-raise under thimerosal, and the mouse had two-fold higher kinetic constants for the Ca2+ ions entry. The ground squirrel brown adipocytes responded to ionomycin with approximately 25% higher increase in [Ca2+]i and the entry of the ions had 7-10-fold higher kinetic constants for this process. Kinetic constants for the [Ca2+]i raise in mouse preadipocytes were independent of ionomycin concentration, whereas in the ground squirrel brown preadipocytes the constant linearly increased with the ionophore concentration. It is suggested that the found difference in the function of Ca(2+)-signalling in preadipocytes of two species, which becomes apparent in the presence of ionomycin, might be responsible for the observed difference in the noradrenalin induced cellular Ca(2

  14. Dexamethasone and Acetate Modulate Cytoplasmic Leptin in Bovine Preadipocytes

    PubMed Central

    Yonekura, Shinichi; Hirota, Shohei; Tokutake, Yukako; Rose, Michael T.; Katoh, Kazuo; Aso, Hisashi

    2014-01-01

    Hormonal and nutrient signals regulate leptin synthesis and secretion. In rodents, leptin is stored in cytosolic pools of adipocytes. However, not much information is available regarding the regulation of intracellular leptin in ruminants. Recently, we demonstrated that leptin mRNA was expressed in bovine intramuscular preadipocyte cells (BIP cells) and that a cytoplasmic leptin pool may be present in preadipocytes. In the present study, we investigated the expression of cytoplasmic leptin protein in BIP cells during differentiation as well as the effects of various factors added to the differentiation medium on its expression in BIP cells. Leptin mRNA expression was observed only at 6 and 8 days after adipogenic induction, whereas the cytoplasmic leptin concentration was the highest on day 0 and decreased gradually thereafter. Cytoplasmic leptin was detected at 6 and 8 days after adipogenic induction, but not at 4 days after adipogenic induction. The cytoplasmic leptin concentration was reduced in BIP cells at 4 days after treatment with dexamethasone, whereas cytoplasmic leptin was not observed at 8 days after treatment. In contrast, acetate significantly enhanced the cytoplasmic leptin concentration in BIP cells at 8 days after treatment, although acetate alone did not induce adipocyte differentiation in BIP cells. These results suggest that dexamethasone and acetate modulate the cytoplasmic leptin concentration in bovine preadipocytes. PMID:25049989

  15. Pediatric brain tumor cell lines.

    PubMed

    Xu, Jingying; Margol, Ashley; Asgharzadeh, Shahab; Erdreich-Epstein, Anat

    2015-02-01

    Pediatric brain tumors as a group, including medulloblastomas, gliomas, and atypical teratoid rhabdoid tumors (ATRT) are the most common solid tumors in children and the leading cause of death from childhood cancer. Brain tumor-derived cell lines are critical for studying the biology of pediatric brain tumors and can be useful for initial screening of new therapies. Use of appropriate brain tumor cell lines for experiments is important, as results may differ depending on tumor properties, and can thus affect the conclusions and applicability of the model. Despite reports in the literature of over 60 pediatric brain tumor cell lines, the majority of published papers utilize only a small number of these cell lines. Here we list the approximately 60 currently-published pediatric brain tumor cell lines and summarize some of their central features as a resource for scientists seeking pediatric brain tumor cell lines for their research.

  16. AMPKα, C/EBPβ, CPT1β, GPR43, PPARγ, and SCD Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid.

    PubMed

    Choi, S H; Park, S K; Johnson, B J; Chung, K Y; Choi, C W; Kim, K H; Kim, W Y; Smith, B

    2015-03-01

    We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco's Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta (CPT1β) gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism.

  17. AMPKα, C/EBPβ, CPT1β, GPR43, PPARγ, and SCD Gene Expression in Single- and Co-cultured Bovine Satellite Cells and Intramuscular Preadipocytes Treated with Palmitic, Stearic, Oleic, and Linoleic Acid

    PubMed Central

    Choi, S. H.; Park, S. K.; Johnson, B. J.; Chung, K. Y.; Choi, C. W.; Kim, K. H.; Kim, W. Y.; Smith, B.

    2015-01-01

    We previously demonstrated that bovine subcutaneous preadipocytes promote adipogenic gene expression in muscle satellite cells in a co-culture system. Herein we hypothesize that saturated fatty acids would promote adipogenic/lipogenic gene expression, whereas mono- and polyunsaturated fatty acids would have the opposite effect. Bovine semimembranosus satellite cells (BSC) and intramuscular preadipocytes (IPA) were isolated from crossbred steers and cultured with 10% fetal bovine serum (FBS)/Dulbecco’s Modified Eagle Medium (DMEM) and 1% antibiotics during the 3-d proliferation period. After proliferation, cells were treated for 3 d with 3% horse serum/DMEM (BSC) or 5% FBS/DMEM (IPA) with antibiotics. Media also contained 10 μg/mL insulin and 10 μg/mL pioglitazone. Subsequently, differentiating BSC and IPA were cultured in their respective media with 40 μM palmitic, stearic, oleic, or linoleic acid for 4 d. Finally, BSC and IPA were single- or co-cultured for an additional 2 h. All fatty acid treatments increased (p = 0.001) carnitine palmitoyltransferase-1 beta (CPT1β) gene expression, but the increase in CPT1β gene expression was especially pronounced in IPA incubated with palmitic and stearic acid (6- to 17- fold increases). Oleic and linoleic acid decreased (p = 0.001) stearoyl-CoA desaturase (SCD) gene expression over 80% in both BSC and IPA. Conversely, palmitic and stearic acid increased SCD gene expression three fold in co-cultured in IPA, and stearic acid increased AMPKα gene expression in single- and co-cultured BSC and IPA. Consistent with our hypothesis, saturated fatty acids, especially stearic acid, promoted adipogenic and lipogenic gene expression, whereas unsaturated fatty acids decreased expression of those genes associated with fatty acid metabolism. PMID:25656188

  18. Xanthohumol influences preadipocyte differentiation: implication of antiproliferative and apoptotic effects.

    PubMed

    Mendes, Vanda; Monteiro, Rosário; Pestana, Diogo; Teixeira, Diana; Calhau, Conceição; Azevedo, Isabel

    2008-12-24

    There is interest in the research of natural compounds that may interfere with the adipocyte life cycle, due to the growing prevalence of obesity and related complications. We aimed at studying the effect of xanthohumol (XN), a Humulus lupulus L. prenylflavonoid, on adipocytes measuring differentiation, proliferation, and apoptosis in 3T3-L1 cells. XN reduced differentiation, as revealed by decreased lipid content and peroxisome proliferator-activated receptor gamma expression, an effect more pronounced when cells were treated before or during differentiation induction. XN also decreased proliferation, as measured by sulforhodamine staining (IC(50) between 26 and 12 microM for 24, 48, and 72 h), and preadipocyte Ki67 expression. Apoptosis was increased in preadipocytes and adipocytes. NF-kappaB activity was stimulated by XN in preadipocytes. Results suggest that XN may reduce adipocyte number, contributing to adipocyte hypertrophy. Taking into consideration the consequences of adipocyte hypertrophy, XN does not seem to improve the metabolic profile associated with obesity.

  19. miR-429 Inhibits Differentiation and Promotes Proliferation in Porcine Preadipocytes

    PubMed Central

    Peng, Ying; Chen, Fen-Fen; Ge, Jing; Zhu, Jia-Yu; Shi, Xin-E; Li, Xiao; Yu, Tai-Yong; Chu, Gui-Yan; Yang, Gong-She

    2016-01-01

    MicroRNAs (miRNAs) are crucial regulatory molecules for adipogenesis. They contribute to the controlling of proliferation and differentiation of preadipocytes. Previous studies revealed an important role of miR-429 in cell invasion, migration, and apoptosis. Our previous work has shown that the expression of miR-429 in subcutaneous fat can be observed in newly born (3-day-old) Rongchang piglets rather than their adult counterparts (180-day-old). This expression pattern suggests that miR-429 might be functionally related to postnatal adipogenesis. However, we currently lack a mechanistic understanding of miR-429 within the context of preadipocyte differentiation. In this study, we investigated the function of miR-429 in porcine subcutaneous and intramuscular preadipocyte proliferation and differentiation. In our porcine preadipocyte differentiation model, miR-429 expression decreased remarkably upon adipogenic induction. Overexpression of miR-429 notably down-regulated the expression of adipogenic marker genes: PPARγ, aP2, FAS and impaired the triglyceride accumulation, while the expression of lipolytic gene ATGL was not affected. In addition, we observed that miR-429 significantly promoted the proliferation of porcine preadipocytes. We also found that miR-429 could directly bind to the 3′-UTRs of KLF9 and p27, which have been well documented to promote preadipocyte differentiation and repress cell cycle progression. Taken together, our data support a novel role of miR-429 in regulating porcine preadipocyte differentiation and proliferation, and KLF9 and p27 are potent targets of miR-429 during these processes. PMID:27941616

  20. Effects of proposed adipogenic factors in fetal swine sera upon preadipocyte development

    SciTech Connect

    Ramsay, T.G.; Hausman, G.J.; Martin, R.J.

    1986-03-01

    Genetic obesity has been detected in fetal pigs which suggests primary factors that cause the obesity develop prenatally. Growth hormone and thyroid hormones have been implicated as regulatory factors in fetal serum for preadipocyte differentiation. This experiment examined effects of growth hormone (GH) and thyroxine (T4) addition upon preadipocyte proliferation and differentiation when supplemented to deficient fetal pig sea. Hormones were added to decapitated fetal pig (Decap) sera to concentrations present in intact littermate (Reference) sera. Primary stromal-vascular cell cultures were prepared from rat inguinal adipose tissue. Cells were incubated with 5% decap or reference sera and hormones in media 199 during: days 1 to 5 for a /sup 3/H-thymidine incorporation assay; days 1 to 15 for assay of ..cap alpha..-glycerol phosphate dehydrogenase; days 5 to 14 for a complete differentiation assay. Decap sera promoted less proliferation and enzyme differentiation than reference sera with no effect of GH addition. GH reduced detection of lipid accumulating cells on percol density gradients by 81%. T4 addition stimulated preadipocyte multiplication and produced a 30% increase in completely differentiated preadipocytes. These results indicate thyroid hormones are important components of fetal sera for regulation of preadipocyte development, whereas GH may only affect cellular metabolism.

  1. Thyroid cell lines in research on goitrogenesis.

    PubMed

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

    1991-12-01

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

  2. Differences in type II, IV, V and VI adenylyl cyclase isoform expression between rat preadipocytes and adipocytes.

    PubMed

    Serazin-Leroy, V; Morot, M; de Mazancourt, P; Giudicelli, Y

    2001-11-26

    Adenylyl cyclase catalytic activity is low in preadipocyte membranes when compared to adipocytes. Under conditions promoting inhibition of adipocyte adenylyl cyclase activity by Gpp(NH)p, a stable GTP analog, a paradoxical increase in preadipocyte adenylyl cyclase activity was obtained. In order to explain this contradiction, expression of types II, IV, V and VI adenylyl cyclase isoforms was compared in adipocytes and undifferentiated preadipocytes both by western blots and by a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Type II, IV, V and VI mRNAs and proteins were present in both adipocytes and preadipocytes. However, in undifferentiated preadipocytes, expression of type II mRNA and protein were significantly higher whereas expression of type IV, V and VI adenylyl cyclase mRNAs and proteins were significantly weaker than in adipocytes. In late differentiated preadipocytes, the adenylyl cyclase subtype mRNA expression pattern was intermediary between the undifferentiated and the full differentiation states except for type IV which remained weakly expressed. Moreover, one of the representative regulators of G-protein signaling (RGS protein), RGS4, was less expressed in undifferentiated preadipocyte membranes and cytosol extracts, which contrasts with adipocytes where RGS4 is clearly expressed. Thus, the preferential expression of type II adenylyl cyclase (G(betagamma) subunit-stimulated) in preadipocytes might explain why Gpp(NH)p elicits stimulation of adenylyl cyclase under conditions designed to promote inhibition. Conversely, the preferential expression of type V and VI adenylyl cyclases and the slightly higher expression of type IV adenylyl cyclase in adipocytes could contribute to explain the elevated total catalytic activity observed in mature fat cells compared to their precursor cells.

  3. Chapter 6. available lepidopteran insect cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  4. Non-transactivational, dual pathways for LPA-induced Erk1/2 activation in primary cultures of brown pre-adipocytes

    SciTech Connect

    Holmstroem, Therese E.; Mattsson, Charlotte L.; Wang, Yanling; Iakovleva, Irina; Petrovic, Natasa; Nedergaard, Jan

    2010-10-01

    In many cell types, G-protein-coupled receptor (GPCR)-induced Erk1/2 MAP kinase activation is mediated via receptor tyrosine kinase (RTK) transactivation, in particular via the epidermal growth factor (EGF) receptor. Lysophosphatidic acid (LPA), acting via GPCRs, is a mitogen and MAP kinase activator in many systems, and LPA can regulate adipocyte proliferation. The mechanism by which LPA activates the Erk1/2 MAP kinase is generally accepted to be via EGF receptor transactivation. In primary cultures of brown pre-adipocytes, EGF can induce Erk1/2 activation, which is obligatory and determinant for EGF-induced proliferation of these cells. Therefore, we have here examined whether LPA, via EGF transactivation, can activate Erk1/2 in brown pre-adipocytes. We found that LPA could induce Erk1/2 activation. However, the LPA-induced Erk1/2 activation was independent of transactivation of EGF receptors (or PDGF receptors) in these cells (whereas in transformed HIB-1B brown adipocytes, the LPA-induced Erk1/2 activation indeed proceeded via EGF receptor transactivation). In the brown pre-adipocytes, LPA instead induced Erk1/2 activation via two distinct non-transactivational pathways, one G{sub i}-protein dependent, involving PKC and Src activation, the other, a PTX-insensitive pathway, involving PI3K (but not Akt) activation. Earlier studies showing LPA-induced Erk1/2 activation being fully dependent on RTK transactivation have all been performed in cell lines and transfected cells. The present study implies that in non-transformed systems, RTK transactivation may not be involved in the mediation of GPCR-induced Erk1/2 MAP kinase activation.

  5. Gga-miR-21 inhibits chicken pre-adipocyte proliferation in part by down-regulating Kruppel-like factor 5.

    PubMed

    Wang, Weishi; Cheng, Min; Qiao, Shupei; Wang, Yuxiang; Li, Hui; Wang, Ning

    2017-01-01

    Gga-miR-21 is abundantly expressed in chicken pre-adipocytes, but its role is unclear. The present study investigated the role of gga-miR-21 in chicken pre-adipocyte proliferation. Cell proliferation assay and gene expression analysis of proliferating cell nuclear antigen (PCNA) showed that the gga-miR-21 mimic inhibited pre-adipocyte proliferation. In contrast, the gga-miR-21 inhibitor enhanced pre-adipocyte proliferation. The subsequent investigation identified Kruppel-like factor 5 (KLF5) mRNA as a target of gga-miR-21. The gga-miR-21 mimic inhibited KLF5 3(')UTR reporter activity and decreased endogenous KLF5 expression in primary pre-adipocytes. KLF5 knockdown using RNAi had a similar effect to that of the gga-miR-21 mimic on cell proliferation. The promoting effect of the gga-miR-21 inhibitor on pre-adipocyte proliferation was partially attenuated by KLF5 knockdown. Taken together, our results demonstrated that miR-21 inhibits chicken pre-adipocyte proliferation, at least in part, by targeting KLF5.

  6. In vitro adipogenic differentiation of preadipocytes varies with differentiation stimulus, culture dimensionality, and scaffold composition.

    PubMed

    Stacey, D Heath; Hanson, Summer E; Lahvis, Garet; Gutowski, Karol A; Masters, Kristyn S

    2009-11-01

    Despite the rapidly growing body of work on stem cell-based adipose tissue engineering, there remains much to be learned about the role of the scaffold and culture environments in directing the adipogenic differentiation of cells. The present study examined how various culture environments and differentiation stimuli (traditional differentiation medium [DM] and coculture with mature adipocytes) impacted the adipogenic differentiation of human preadipocytes, with studies progressing from two-dimensions (2D) to three-dimensions (3D) in vitro. Assays for adipogenic markers (leptin, adiponectin, and glycerol) and Oil Red O staining were used to assess differentiation. After 16 days of 2D culture, adipogenesis was substantially greater when preadipocytes were cocultured with adipocytes rather than treated with DM. In a 3D in vitro environment, the production of adipogenic markers was significantly elevated relative to 2D conditions, and the coculture condition continued to stimulate greater adipogenesis. Alterations in 3D scaffold physical properties had only a minimal effect on the function of mature adipocytes, but significantly impacted the ability of preadipocytes to undergo adipogenic differentiation in vitro. These alterations in scaffold environment and in medium conditions, particularly the application of adipocyte/preadipocyte coculture methods in lieu of traditional DM, may provide further means for optimizing adipogenic outcomes in vitro and in vivo.

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

    PubMed Central

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

    2013-01-01

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

  8. Vaspin promotes 3T3-L1 preadipocyte differentiation

    PubMed Central

    Liu, Ping; Wu, Jine; Zhou, Xin; Wang, Liping; Han, Wenqi; Lv, Ying; Sun, Chaofeng

    2015-01-01

    Vaspin, a novel adipocyte factor secreted from visceral adipose tissues, is associated with obesity and insulin resistance and can regulate glucose and lipid metabolism, increase insulin sensitivity, and suppress inflammation; however, the underlying mechanisms remain unknown. Proliferation and maladaptive differentiation are important pathological mechanisms underlying obesity. This study aimed to evaluate the effects of vaspin on the proliferation and differentiation of preadipocyte 3T3-L1 cells and to explore the likely mechanisms responsible for 3T3-L1 differentiation. Vaspin was added to cultured 3T3-L1 cells, and the differentiation of adipocytes was evaluated using Oil Red O staining. The AKT signaling pathway and specific differentiation factors related to the differentiation of preadipocyte 3T3-L1 cells, peroxisome proliferator-activated γ and the CCAAT/enhancer-binding protein (C/EBP) family, were evaluated using reverse transcription polymerase chain reaction (RT-PCR) and western blot analyses during the early phase of differentiation. Additionally, adiponectin mRNA, interleukin-6 mRNA (IL-6 mRNA), and glucose transporter-4 (GLUT4) protein levels were measured in the differentiated adipocytes. The results indicated that vaspin promotes the intracellular accumulation of lipids and increases differentiation-related factors, including peroxisome proliferator-activated receptor γ, C/EBPα, and free fatty acid-binding protein 4 (FABP4), in a dose-dependent manner. Additionally, vaspin (200 ng/mL) increased the mRNA and protein levels of C/EBPβ, peroxisome proliferator-activated γ, C/EBPα, and FABP4. Moreover, compared with the control, significantly smaller eight-day differentiated adipocytes were observed, and these cells exhibited decreased IL-6 mRNA and increased GLUT4 mRNA levels; these results also indicated the potential of vaspin to promote the insulin-mediated AKT signaling pathway during the early phase of differentiation. In conclusion

  9. ClC-3 deficiency protects preadipocytes against apoptosis induced by palmitate in vitro and in type 2 diabetes mice.

    PubMed

    Huang, Yun-Ying; Huang, Xiong-Qin; Zhao, Li-Yan; Sun, Fang-Yun; Chen, Wen-Liang; Du, Jie-Yi; Yuan, Feng; Li, Jie; Huang, Xue-Lian; Liu, Jie; Lv, Xiao-Fei; Guan, Yong-Yuan; Chen, Jian-Wen; Wang, Guan-Lei

    2014-11-01

    Palmitate, a common saturated free fatty acid (FFA), has been demonstrated to induce preadipocyte apoptosis in the absence of adipogenic stimuli, suggesting that preadipocytes may be prone to apoptosis under adipogenic insufficient conditions, like type 2 diabetes mellitus (T2DM). ClC-3, encoding Cl(-) channel or Cl(-)/H(+) antiporter, is critical for cell fate choices of proliferation versus apoptosis under diseased conditions. However, it is unknown whether ClC-3 is related with preadipocyte apoptosis induced by palmitate or T2DM. Palmitate, but not oleate, induced apoptosis and increase in ClC-3 protein expression and endoplasmic reticulum (ER) stress in 3T3-L1 preadipocyte. ClC-3 specific siRNA attenuated palmitate-induced apoptosis and increased protein levels of Grp78, ATF4, CHOP and phosphorylation of JNK1/2, whereas had no effects on increased phospho-PERK and phospho-eIF2α protein expression. Moreover, the enhanced apoptosis was shown in preadipocytes from high-sucrose/fat, low-dose STZ induced T2DM mouse model with hyperglycemia, hyperlipidemia (elevated serum TG and FFA levels) and insulin resistance. ClC-3 knockout significantly attenuated preadipocyte apoptosis and the above metabolic disorders in T2DM mice. These data demonstrated that ClC-3 deficiency prevent preadipocytes against palmitate-induced apoptosis via suppressing ER stress, and also suggested that ClC-3 may play a role in regulating cellular apoptosis and disorders of glucose and lipid metabolism during T2DM.

  10. LINE-1 Cultured Cell Retrotransposition Assay.

    PubMed

    Kopera, Huira C; Larson, Peter A; Moldovan, John B; Richardson, Sandra R; Liu, Ying; Moran, John V

    2016-01-01

    The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells.

  11. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes.

    PubMed

    Xue, Ruidan; Lynes, Matthew D; Dreyfuss, Jonathan M; Shamsi, Farnaz; Schulz, Tim J; Zhang, Hongbin; Huang, Tian Lian; Townsend, Kristy L; Li, Yiming; Takahashi, Hirokazu; Weiner, Lauren S; White, Andrew P; Lynes, Maureen S; Rubin, Lee L; Goodyear, Laurie J; Cypess, Aaron M; Tseng, Yu-Hua

    2015-07-01

    Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes.

  12. Co-expression of FBN1 with mesenchyme-specific genes in mouse cell lines: implications for phenotypic variability in Marfan syndrome

    PubMed Central

    Summers, Kim M; Raza, Sobia; van Nimwegen, Erik; Freeman, Thomas C; Hume, David A

    2010-01-01

    Mutations in the human FBN1 gene cause Marfan syndrome, a complex disease affecting connective tissues but with a highly variable phenotype. To identify genes that might participate in epistatic interactions with FBN1, and could therefore explain the observed phenotypic variability, we have looked for genes that are co-expressed with Fbn1 in the mouse. Microarray expression data derived from a range of primary mouse cells and cell lines were analysed using the network analysis tool BioLayout Express3D. A cluster of 205 genes, including Fbn1, were selectively expressed by mouse cell lines of different mesenchymal lineages and by mouse primary mesenchymal cells (preadipocytes, myoblasts, fibroblasts, osteoblasts). Promoter analysis of this gene set identified several candidate transcriptional regulators. Genes within this co-expressed cluster are candidate genetic modifiers for Marfan syndrome and for other connective tissue diseases. PMID:20551991

  13. Phenamil enhances the adipogenic differentiation of hen preadipocytes.

    PubMed

    Regassa, Alemu; Park, Kye Won; Kim, Woo Kyun

    2016-10-01

    A study was conducted to examine the effect of phenamil on adipogenic differentiation and expression of key adipogenic transcripts in hen preadipocytes. Preadipocytes were isolated from 20-week old Single Comb White Leghorn hens (Gallas gallus, Lohman strain). The experiment lasted for 48 h and had six treatments. Non-treated control (C) cells, cells treated with dexamethasone, 3-isobutyl-1-methylxanthine, insulin, and oleic acid (DMIOA) (T1), DMIOA + 15 μM phenamil (T2), DMIOA + 30 μM phenamil (T3), 15 μM phenamil alone (T4), and 30 μM phenamil alone (T5). Neutral lipid accumulation and the mRNA expression of key adipogenic transcripts were measured in all treatments and compared. Lipid accumulation was detected in T1, T2, and T3 only. Expression of peroxisome proliferator receptor-activator gamma 2 (PPARγ2), the core enhancer binding protein α (C/EBPα), C/EBPβ, fatty acid binding protein 4 (FABP4), and lipoprotein lipase (LPL) as well as ETS variant 4 (ETV4) and 5 was higher (P < 0.05) in T2, T3, T4, and T5 compared to C. Expression of these transcripts was higher (P < 0.05) in T2 and T3 compared to T4 and T5. The core enhancer binding protein α, C/EBPβ, and FABP4 were highly expressed (P < 0.05) in T1 compared to C. However, the expression of PPARγ2, LPL, and ETV4 and ETV5 was not significantly different. Expression of C/EBPα, C/EBPβ, and FABP4 was higher (P < 0.05) in T2 and T3 compared to T1. Expression of sterol regulatory element binding protein 1 (SREBP1) and leptin receptor (LEPR) was not significantly different among the treatments. In conclusion, phenamil enhances DMIOA-induced adipogenic differentiation of hen preadipocytes but does not induce adipogenesis by itself.

  14. Stimulation of glycerol kinase in grass carp preadipocytes by EPA.

    PubMed

    Lei, Caixia; Tian, Jingjing; Ji, Hong

    2017-01-05

    This study was conducted to assess the effect of eicosapentaenoic acid (EPA) on grass carp preadipocyte glycerol kinase (GyK) expression, as well as to explore the mechanism. Here, we cloned partial sequence of grass carp GyK gene and analyzed its tissue distribution. The result showed that GyK gene expressed most in the liver, followed by adipose tissue and the kidney. Besides, 400 μM oleic acid (18:1n-9, OA) was used to establish a hypertrophic preadipocyte model. GyK gene expression and enzyme activity were significantly enhanced after model cells were treated with 100 μM eicosapentaenoic acid (20:5n-3, EPA) for 6, 12, and 24 h. Meanwhile, peroxisome proliferative-activated receptor (PPAR)γ, adipose triglyceride lipase (ATGL), and the two isoforms of grass carp HSL gene were first identified by Sun et al (2016), and they defined the two isoforms as HSLa and HSLb. Therefore, maybe HSLa and HSLb are appropriate.. The content of triglyceride was dramatically increased by EPA treatment for 24 h. Further, a competitive ATGL antagonist, HY-15859, attenuated the increase in GyK induced by EPA at 12 h. Surprisingly, the enhanced lipolysis and PPARγ gene expression induced by serum deprivation were paralleled by an increase in GyK gene expression, whereas a stabilization in GyK enzyme activity. Other fatty acids, including docosahexaenoic acid, alpha-linolenic acid, linoleic acid, and OA also promoted GyK gene expression. Moreover, an irreversible PPARγ antagonist, GW9662, was used to investigate the role of PPARγ in GyK induction. Data showed that GW9662 abolished the induction of GyK by EPA at 12 h. Together, these data suggested that EPA elevated grass carp preadipocytes GyK expression. ATGL and PPARγ contributed to the induction of GyK. PPARγ may be a key regulator in response to GyK expression induced by EPA.

  15. Antidepressant phenelzine alters differentiation of cultured human and mouse preadipocytes.

    PubMed

    Chiche, Françoise; Le Guillou, Morwenna; Chétrite, Gérard; Lasnier, Françoise; Dugail, Isabelle; Carpéné, Christian; Moldes, Marthe; Fève, Bruno

    2009-05-01

    Change in body weight is a frequent side effect of antidepressants and is considered to be mediated by central effects on food intake and energy expenditure. The antidepressant phenelzine (Nardil) potently inhibits both monoamine oxidase and semicarbazide-sensitive amine oxidase activities, two enzymes that are highly expressed in adipose tissue, raising the possibility that it could directly alter adipocyte biology. Treatment with this compound is rather associated with weight gain. The aim of this work was to examine the effects of phenelzine on differentiation and metabolism of cultured human and mouse preadipocytes and to characterize the mechanisms involved in these effects. In all preadipocyte models, phenelzine induced a time- and dose-dependent reduction in differentiation and triglyceride accumulation. Modulation of lipolysis or glucose transport was not involved in phenelzine action. This effect was supported by the reduced expression in the key adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and CCAAT/enhancer binding protein-alpha, which was observed only at the highest drug concentrations (30-100 microM). The PPAR-gamma agonists thiazolidinediones did not reverse phenelzine effects. By contrast, the reduction in both cell triglycerides and sterol regulatory element-binding protein-1c (SREBP-1c) was detectable at lower phenelzine concentrations (1-10 microM). Phenelzine effect on triglyceride content was prevented by providing free fatty acids to the cells and was partially reversed by overexpression of a dominant-positive form of SREBP-1c, showing the privileged targeting of the lipogenic pathway. When considered together, these findings demonstrate that an antidepressant directly and potently inhibits adipocyte lipid storage and differentiation, which could contribute to psychotropic drug side effects on energy homeostasis.

  16. Adipogenesis of bovine perimuscular preadipocytes

    SciTech Connect

    Taniguchi, Masaaki; Le Luo Guan; Zhang Bing; Dodson, Michael V.; Okine, Erasmus; Moore, Stephen S.

    2008-02-01

    In this study, non-transformed progeny adipofibroblasts, derived from mature adipocyte dedifferentiation, was used as a novel in vitro model to study adipogenic gene expression in cattle. Adipofibroblasts from dedifferentiated mature perimuscular fat (PMF) tissue were cultured with differentiation stimulants until the cells exhibited morphological differentiation. Treated cells were harvested from day 2 to 16 for RNA extraction, whereas control cells were cultured without addition of stimulants. Results from time course gene expression assays by quantitative real-time PCR revealed that peroxisome proliferator-activated receptor gamma (PPAR-{gamma}), sterol regulatory element binding protein 1 (SREBP-1) and their six down-stream genes were co-expressed at day 2 post-differentiation induction. When compared to other adipogenesis culture systems, the adipogenic gene expression of bovine PMF adipofibroblasts culture was different, especially to the rodent model. Collectively, these results demonstrated PPAR-{gamma} and SREBP-1 cooperatively play a key role to regulate the re-differentiation of bovine adipofibroblasts, during early conversion stages in vitro.

  17. Downregulated Expression of the Secreted Glycoprotein Follistatin-like 1 (Fstl1) is a Robust Hallmark of Preadipocyte to Adipocyte Conversion

    PubMed Central

    Wu, Yu; Zhou, Shengli; Smas, Cynthia M.

    2010-01-01

    Obesity is a public health crisis in The United States. Targeting preadipocyte to adipocyte conversion may be an effective approach to regulate adipose mass. Using differential screening we identified Fstl1, a secreted glycoprotein with roles in immunomodulation, cell growth, cardioprotection, and vascularization, as a “preadipokine”. Fstl1 is highly expressed in 3T3-L1 preadipocytes and dramatically downregulated early in their differentiation to adipocytes. Northern blot analysis of murine tissues reveals white adipose tissue (WAT), lung and heart as primary sites of Fstl1 transcript expression. In WAT, Fstl1 transcript is restricted to the preadipocyte-containing stromal-vascular cell population. Time course studies in multiple adipogenesis models reveal downregulation of Fstl1 is a hallmark of white and brown adipocyte conversion. By Western blot, we show culture media of 3T3-L1 preadipocytes contains high levels of Fstl1 protein that rapidly decline in adipocyte conversion. Moreover, we observe a correlation between preadipocyte phenotype and Fstl1 expression in that TNFα-mediated dedifferentiation of 3T3-L1 adipocytes is accompanied by re-expression of Fstl1 transcript and protein. Treatment of 3T3-L1 preadipocytes with a panel of 18 hormones and other agents revealed the demethylating agent 5-aza-cytidine decreases Fstl1 transcript and protein levels by ~90%. Furthermore, of 10 additional preadipocyte-expressed genes analyzed we find Pref-1, Col1A1, Sca-1/Ly6a, Lox and Thbs2, are also downregulated by 5-aza-cytidine. Using luciferase reporter constructs containing 791 or 3922 bp of the Fstl1 5’-flanking region, we determine negative transcriptional regulation by Kruppel-like factor 15. Together, our data suggest downregulation of Fstl1 expression may be an important feature of preadipocyte to adipocyte conversion. PMID:20043993

  18. The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes.

    PubMed

    Pavlikova, Nela; Weiszenstein, Martin; Pala, Jan; Halada, Petr; Seda, Ondrej; Elkalaf, Moustafa; Trnka, Jan; Kovar, Jan; Polak, Jan

    2015-12-01

    Experiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.

  19. Effects of Rosmarinus officinalis extract on human primary omental preadipocytes and adipocytes.

    PubMed

    Stefanon, Bruno; Pomari, Elena; Colitti, Monica

    2015-07-01

    The prevalence of obesity is increasing all over the world. Although it has been shown that natural substances influence fat metabolism, little is known about the effect on cellular and molecular mechanisms in human. In this in vitro study, the activity of Rosmarinus officinalis (RO) standardized extract in modulating human primary visceral preadipocytes differentiation, lipolysis, and apoptosis was investigated. Moreover, gene expression of key adipogenesis modulators and microRNAs-seq were evaluated. Preadipocytes treated with RO extract significantly reduced triglyceride incorporation during maturation in a dose-dependent manner without affecting cell viability. In addition, RO extract stimulated lipolytic activity in differentiating preadipocytes and mature adipocytes in treated cells compared to controls. Differentiating preadipocytes incubated in the presence of RO extract showed a decreased expression of cell cycle genes such as cyclin D1, cyclin-dependent kinase 4, cyclin-dependent kinase inhibitor 1A (p21, Cip1) and an increased expression of GATA binding protein 3, wingless-type MMTV integration site family, member 3A mRNA levels. Recent studies have demonstrated that some phytochemicals alter the expression of specific genes and microRNAs that play a fundamental role in the pathogenesis of obesity and related diseases. Interestingly, genes modulated in RO-treated cells were found to be validated miRNAs targets, such as let-7f-1, miR-17, and miR-143. The results indicated that RO extract modulates human adipocyte differentiation and significantly interferes with adipogenesis and lipid metabolism, supporting its interest as dietary supplement.

  20. Cell line fingerprinting using retroelement insertion polymorphism.

    PubMed

    Ustyugova, Svetlana V; Amosova, Anna L; Lebedev, Yuri B; Sverdlov, Eugene D

    2005-04-01

    Human cell lines are an indispensable tool for functional studies of living entities in their numerous manifestations starting with integral complex systems such as signal pathways and networks, regulation of gene ensembles, epigenetic factors, and finishing with pathological changes and impact of artificially introduced elements, such as various transgenes, on the behavior of the cell. Therefore, it is highly desirable to have reliable cell line identification techniques to make sure that the cell lines to be used in experiments are exactly what is expected. To this end, we developed a set of informative markers based on insertion polymorphism of human retroelements (REs). The set includes 47 pairs of PCR primers corresponding to introns of the human genes with dimorphic LINE1 (L1) and Alu insertions. Using locus-specific PCR assays, we have genotyped 10 human cell lines of various origins. For each of these cell lines, characteristic fingerprints were obtained. An estimated probability that two different cell lines possess the same marker genotype is about 10-18. Therefore, the proposed set of markers provides a reliable tool for cell line identification.

  1. Analysis of the transcriptome of differentiating and non-differentiating preadipocytes from rats and humans by next generation sequencing.

    PubMed

    Birzele, Fabian; Fässler, Sybille; Neubauer, Heike; Hildebrandt, Tobias; Hamilton, Bradford S

    2012-10-01

    Alongside cell lines such as 3T3-L1 cells, primary cell culture models of adipogenesis have helped in developing an understanding of the process of adipocyte recruitment and maintenance, which may lead to therapeutic advances to treat the growing epidemic of obesity. Recently, it has been demonstrated that fat cell progenitors (DFAT) established through ceiling culture of adipocytes retain an enhanced ability to undergo adipocyte differentiation compared to preadipocytes isolated from the stromal vascular fraction of adipose tissue. Clonal expansion of rat DFAT cells identified differentiation capable and incapable cell strains. To understand the mechanisms underlying these differences, comparison of their transcriptomes by next generation sequencing was performed. Two hundred seventy-eight genes with a significant fold change of 1.4 were detected as being consistently deregulated between differentiating and non-differentiating strains. Bioinformatic network analyses identified components of the extra-cellular matrix and PPARγ as important genes in this process, suggesting crosstalk between ECM and transcription factors influences differentiation. Analyses of the transcriptomes of human DFAT cells in early and late passage (non-differentiating) confirmed the importance of these pathways in maintaining an adipogenic potential.

  2. Glyphosate Inhibits PPAR Gamma Induction and Differentiation of Preadipocytes and is able to Induce Oxidative Stress.

    PubMed

    Martini, Claudia N; Gabrielli, Matías; Brandani, Javier N; Vila, María Del C

    2016-08-01

    Glyphosate-based herbicides (GF) are extensively used for weed control. Thus, it is important to investigate their putative toxic effects. We have reported that GF at subagriculture concentrations inhibits proliferation and differentiation to adipocytes of 3T3-L1 fibroblasts. In this investigation, we evaluated the effect of GF on genes upregulated during adipogenesis. GF was able to inhibit the induction of PPAR gamma, the master gene in adipogenesis but not C/EBP beta, which precedes PPAR gamma activation. GF also inhibited differentiation and proliferation of another model of preadipocyte: mouse embryonic fibroblasts. In exponentially growing 3T3-L1 cells, GF increased lipid peroxidation and the activity of the antioxidant enzyme, superoxide dismutase. We also found that proliferation was inhibited with lower concentrations of GF when time of exposure was extended. Thus, GF was able to inhibit proliferation and differentiation of preadipocytes and to induce oxidative stress, which is indicative of its ability to alter cellular physiology.

  3. Differentiation of Pre-Adipocytes in Modelled Microgravity

    NASA Astrophysics Data System (ADS)

    Coinu, R.; Postiglione, I.; Meloni, M. A.; Galleri, G.; Pippia, P.; Palumbo, G.

    2008-06-01

    It has been demonstrated that microgravity affects biological and biochemical functions of cells including: morphology, cytoskeleton and embryogenesis [1]; proliferation, reduction of DNA, protein synthesis and glucose transport [2]; signalling, reduction of EGF-dependant c-fos and c-jun expression [3]; gene expression, reduction of IL2 expression and release by activated T-cells [4]. Moreover it has be found that peroxisome proliferators activated receptor γ (PPARγ2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4, are highly expressed in response to modelled microgravity [5]. These findings prompted us to investigate the effects of microgravity on cellular differentiation rate using a well characterized model. Such model consists in murine pre-adipocyte cells (3T3-L1) properly stimulated with insulin, dexamethazone and isobuthylmethyl-xantine (DMI protocol). The adipogenic program is completed within a short time. The entire process requires coordinated and temporarily beated molecular events. Early events. Growth arrest at confluence; Clonal expansion (this process involves synchronous entry of cells into S phase of the cell cycle, leading to one or two rounds of mitosis); Early expression of C/EBPβ and C/EBPδ. Late events. Expression of PPARγ and C/EBPα Assumption of rounded morphology and accumulation of lipid droplets.

  4. Standards for Cell Line Authentication and Beyond

    PubMed Central

    Cole, Kenneth D.; Plant, Anne L.

    2016-01-01

    Different genomic technologies have been applied to cell line authentication, but only one method (short tandem repeat [STR] profiling) has been the subject of a comprehensive and definitive standard (ASN-0002). Here we discuss the power of this document and why standards such as this are so critical for establishing the consensus technical criteria and practices that can enable progress in the fields of research that use cell lines. We also examine other methods that could be used for authentication and discuss how a combination of methods could be used in a holistic fashion to assess various critical aspects of the quality of cell lines. PMID:27300367

  5. Embryonic stem cell lines of nonhuman primates.

    PubMed

    Nakatsuji, Norio; Suemori, Hirofumi

    2002-06-26

    Human embryonic stem (ES) cell lines have opened great potential and expectation for cell therapy and regenerative medicine. Monkey and human ES cell lines, which are very similar to each other, have been established from monkey blastocysts and surplus human blastocysts from fertility clinics. Nonhuman primate ES cell lines provide important research tools for basic and applicative research. Firstly, they provide wider aspects of investigation of the regulative mechanisms of stem cells and cell differentiation among primate species. Secondly, their usage does not need clearance or permission from the regulative rules in many countries that are associated with the ethical aspects of human ES cells, although human and nonhuman embryos and fetuses are very similar to each other. Lastly and most importantly, they are indispensable for animal models of cell therapy to test effectiveness, safety, and immunological reaction of the allogenic transplantation in a setting similar to the treatment of human diseases. So far, ES cell lines have been established from rhesus monkey (Macaca mulatta), common marmoset (Callithrix jacchus), and cynomolgus monkey (Macaca fascicularis), using blastocysts produced naturally or by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). These cell lines seem to have very similar characteristics. They express alkaline phosphatase activity and stage-specific embryonic antigen (SSEA)-4 and, in most cases, SSEA-3. Their pluripotency was confirmed by the formation of embryoid bodies and differentiation into various cell types in culture and also by the formation of teratomas that contained many types of differentiated tissues including derivatives of three germ layers after transplantation into the severe combined immunodeficiency (SCID) mice. The noneffectiveness of the leukemia inhibitory factor (LIF) signal makes culture of primate and human ES cell lines prone to undergo spontaneous differentiation and thus it is

  6. Thyroid cancer cell lines: an overview

    PubMed Central

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

    2012-01-01

    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

  7. Comparative study of gene expression during the differentiation of white and brown preadipocytes

    NASA Astrophysics Data System (ADS)

    Boeuf, Stéphane

    2002-08-01

    Introduction Mammals have two types of adipose tissue: the lipid storing white adipose tissue and the brown adipose tissue characterised by its capacity for non-shivering thermogenesis. White and brown adipocytes have the same origin in mesodermal stem cells. Yet nothing is known so far about the commitment of precursor cells to the white and brown adipose lineage. Several experimental approaches indicate that they originate from the differentiation of two distinct types of precursor cells, white and brown preadipocytes. Based on this hypothesis, the aim of this study was to analyse the gene expression of white and brown preadipocytes in a systematic approach. Experimental approach The white and brown preadipocytes to compare were obtained from primary cell cultures of preadipocytes from the Djungarian dwarf hamster. Representational difference analysis was used to isolate genes potentially differentially expressed between the two cell types. The thus obtained cDNA libraries were spotted on microarrays for a large scale gene expression analysis in cultured preadipocytes and adipocytes and in tissue samples. Results 4 genes with higher expression in white preadipocytes (3 members of the complement system and a fatty acid desaturase) and 8 with higher expression in brown preadipocytes were identified. From the latter 3 coded for structural proteins (fibronectin, metargidin and a actinin 4), 3 for proteins involved in transcriptional regulation (necdin, vigilin and the small nuclear ribonucleoprotein polypeptide A) and 2 are of unknown function. Cluster analysis was applied to the gene expression data in order to characterise them and led to the identification of four major typical expression profiles: genes up-regulated during differentiation, genes down-regulated during differentiation, genes higher expressed in white preadipocytes and genes higher expressed in brown preadipocytes. Conclusion This study shows that white and brown preadipocytes can be distinguished

  8. Bisphenol A and Bisphenol S Induce Distinct Transcriptional Profiles in Differentiating Human Primary Preadipocytes

    PubMed Central

    Boucher, Jonathan G.; Gagné, Rémi; Rowan-Carroll, Andrea; Boudreau, Adèle; Yauk, Carole L.; Atlas, Ella

    2016-01-01

    Bisphenol S (BPS) is increasingly used as a replacement plasticizer for bisphenol A (BPA) but its effects on human health have not been thoroughly examined. Recent evidence indicates that both BPA and BPS induce adipogenesis, although the mechanisms leading to this effect are unclear. In an effort to identify common and distinct mechanisms of action in inducing adipogenesis, transcriptional profiles of differentiating human preadipocytes exposed to BPA or BPS were compared. Human subcutaneous primary preadipocytes were differentiated in the presence of either 25 μM BPA or BPS for 2 and 4 days. Poly-A RNA-sequencing was used to identify differentially expressed genes (DEGs). Functional analysis of DEGs was undertaken in Ingenuity Pathway Analysis. BPA-treatment resulted in 472 and 176 DEGs on days 2 and 4, respectively, affecting pathways such as liver X receptor (LXR)/retinoid X receptor (RXR) activation, hepatic fibrosis and cholestasis. BPS-treatment resulted in 195 and 51 DEGs on days 2 and 4, respectively, revealing enrichment of genes associated with adipogenesis and lipid metabolism including the adipogenesis pathway and cholesterol biosynthesis. Interestingly, the transcription repressor N-CoR was identified as a negative upstream regulator in both BPA- and BPS-treated cells. This study presents the first comparison of BPA- and BPS-induced transcriptional profiles in human differentiating preadipocytes. While we previously showed that BPA and BPS both induce adipogenesis, the results from this study show that BPS affects adipose specific transcriptional changes earlier than BPA, and alters the expression of genes specifically related to adipogenesis and lipid metabolism. The findings provide insight into potential BPS and BPA-mediated mechanisms of action in inducing adipogenesis in human primary preadipocytes. PMID:27685785

  9. Hypoxia-mimetic effects in the secretome of human preadipocytes and adipocytes.

    PubMed

    Rosenow, Anja; Noben, Jean-Paul; Bouwman, Freek G; Mariman, Edwin C M; Renes, Johan

    2013-12-01

    White adipose tissue (WAT) regulates energy metabolism by secretion of proteins with endocrine and paracrine effects. Dysregulation of the secretome of obesity-associated enlarged WAT may lead to obesity-related disorders. This can be caused by hypoxia as a result of poorly vascularized WAT. The effect of hypoxia on the secretome of human (pre)adipocytes is largely unknown. Therefore, we investigated the effect of CoCl2, a hypoxia mimetic, on the secretome of human SGBS (pre)adipocytes by a proteomics approach combined with bioinformatic analysis. In addition, regulation of protein secretion was examined by protein turnover experiments. As such, secretome changes were particularly associated with protein down-regulation and extracellular matrix protein dysregulation. The observed up-regulation of collagens in adipocytes may be essential for cell survival while down-regulation of collagens in preadipocytes may indicate a disturbed differentiation process. These CoCl2-induced changes reflect WAT dysfunction that ultimately may lead to obesity-associated complications. In addition, 9 novel adipocyte secreted proteins were identified from which 6 were regulated by CoCl2. Mass spectrometry data have been deposited to the ProteomeXchange with identifier PXD000162.

  10. 18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes

    PubMed Central

    Kirkwood, Jay S.; Miranda, Cristobal L.; Bobe, Gerd; Maier, Claudia S.; Stevens, Jan F.

    2016-01-01

    The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in

  11. Capsaicin Induces “Brite” Phenotype in Differentiating 3T3-L1 Preadipocytes

    PubMed Central

    Baboota, Ritesh K.; Singh, Dhirendra P.; Sarma, Siddhartha M.; Kaur, Jaspreet; Sandhir, Rajat; Boparai, Ravneet K.; Kondepudi, Kanthi K.; Bishnoi, Mahendra

    2014-01-01

    Objective Targeting the energy storing white adipose tissue (WAT) by pharmacological and dietary means in order to promote its conversion to energy expending “brite” cell type holds promise as an anti-obesity approach. Present study was designed to investigate/revisit the effect of capsaicin on adipogenic differentiation with special reference to induction of “brite” phenotype during differentiation of 3T3-L1 preadipocytes. Methods Multiple techniques such as Ca2+ influx assay, Oil Red-O staining, nutrigenomic analysis in preadipocytes and matured adipocytes have been employed to understand the effect of capsaicin at different doses. In addition to in-vitro experiments, in-vivo studies were carried out in high-fat diet (HFD) fed rats treated with resiniferatoxin (RTX) (a TRPV1 agonist) and in mice administered capsaicin. Results TRPV1 channels are expressed in preadipocytes but not in adipocytes. In preadipocytes, both capsaicin and RTX stimulate Ca2+ influx in dose-dependent manner. This stimulation may be prevented by capsazepine, a TRPV1 antagonist. At lower doses, capsaicin inhibits lipid accumulation and stimulates TRPV1 gene expression, while at higher doses it enhances accumulation of lipids and suppresses expression of its receptor. In doses of 0.1–100 µM, capsaicin promotes expression of major pro-adipogenic factor PPARγ and some of its downstream targets. In concentrations of 1 µM, capsaicin up-regulates anti-adipogenic genes. Low-dose capsaicin treatment of 3T3-L1 preadipocytes differentiating into adipocytes results in increased expression of brown fat cell marker genes. In white adipose of mice, capsaicin administration leads to increase in browning-specific genes. Global TRPV1 ablation (i.p. by RTX administration) leads to increase in locomotor activity with no change in body weight. Conclusion Our findings suggest the dual modulatory role of capsaicin in adipogenesis. Capsaicin inhibits adipogenesis in 3T3-L1 via TRPV1 activation and

  12. Virus Discovery Using Tick Cell Lines

    PubMed Central

    Bell-Sakyi, Lesley; Attoui, Houssam

    2016-01-01

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

  13. Epigenetic programming of Dnmt3a mediated by AP2α is required for granting preadipocyte the ability to differentiate

    PubMed Central

    Guo, Wei; Chen, Jiangnan; Yang, Ying; Zhu, Jianbei; Wu, Jiarui

    2016-01-01

    Adipogenesis has an important role in regulating energy homeostasis in mammals. 3T3-L1 preadipocytes have been widely used as an in vitro model for analyzing the molecular mechanism of adipogenesis. Previous reports indicated that the stage of contact inhibition (CI), through which the proliferating cells exit from the cell cycle, was required for granting preadipocyte the ability to differentiate. While this kind of the granting mechanism remains elusive. In the present study, we showed that DNA (cytosine-5) methyltransferase 3a (Dnmt3a) was upregulated at both the mRNA and protein level during the CI stage, and resulted in increasing promoter methylation of adipogenic genes. We further identified that the expression of Activator protein 2α (AP2α), a member of the transcription factor activator protein 2 (AP2) family, was highly correlated with the expression of Dnmt3a during the CI stage. In addition, we showed that AP2α transcriptionally upregulated Dnmt3a by directly binding to its proximal promoter region. Importantly, treatment of 3T3-L1 preadipocytes with AP2α-specific siRNAs inhibited the preadipocyte differentiation in a stage-dependent manner, supporting the conclusion that AP2α has an important role during the CI stage. Furthermore, overexpression of Dnmt3a partially rescued the impairment of adipogenesis induced by AP2α knockdown. Collectively, our findings reveal that AP2α is an essential regulator for granting preadipocyte the ability to differentiate through the upregulation of Dnmt3a expression during the CI stage. PMID:27906176

  14. Epigenetic programming of Dnmt3a mediated by AP2α is required for granting preadipocyte the ability to differentiate.

    PubMed

    Guo, Wei; Chen, Jiangnan; Yang, Ying; Zhu, Jianbei; Wu, Jiarui

    2016-12-01

    Adipogenesis has an important role in regulating energy homeostasis in mammals. 3T3-L1 preadipocytes have been widely used as an in vitro model for analyzing the molecular mechanism of adipogenesis. Previous reports indicated that the stage of contact inhibition (CI), through which the proliferating cells exit from the cell cycle, was required for granting preadipocyte the ability to differentiate. While this kind of the granting mechanism remains elusive. In the present study, we showed that DNA (cytosine-5) methyltransferase 3a (Dnmt3a) was upregulated at both the mRNA and protein level during the CI stage, and resulted in increasing promoter methylation of adipogenic genes. We further identified that the expression of Activator protein 2α (AP2α), a member of the transcription factor activator protein 2 (AP2) family, was highly correlated with the expression of Dnmt3a during the CI stage. In addition, we showed that AP2α transcriptionally upregulated Dnmt3a by directly binding to its proximal promoter region. Importantly, treatment of 3T3-L1 preadipocytes with AP2α-specific siRNAs inhibited the preadipocyte differentiation in a stage-dependent manner, supporting the conclusion that AP2α has an important role during the CI stage. Furthermore, overexpression of Dnmt3a partially rescued the impairment of adipogenesis induced by AP2α knockdown. Collectively, our findings reveal that AP2α is an essential regulator for granting preadipocyte the ability to differentiate through the upregulation of Dnmt3a expression during the CI stage.

  15. C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene.

    PubMed

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli; Sun, Wenxing; Gao, Ying; Zhang, Lifan; Chen, Jie

    2016-03-25

    Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes.

  16. Refractory lining for electrochemical cell

    DOEpatents

    Blander, Milton; Cook, Glenn M.

    1987-01-01

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

  17. Flavonoid inhibition of aromatase enzyme activity in human preadipocytes.

    PubMed

    Campbell, D R; Kurzer, M S

    1993-09-01

    Eleven flavonoid compounds were compared with aminoglutethimide (AG), a pharmaceutical aromatase inhibitor, for their abilities to inhibit aromatase enzyme activity in a human preadipocyte cell culture system. Flavonoids exerting no effect on aromatase activity were catechin, daidzein, equol, genistein, beta-naphthoflavone (BNF), quercetin and rutin. The synthetic flavonoid, alpha-naphthoflavone (ANF), was the most potent aromatase inhibitor, with an I50 value of 0.5 microM. Three naturally-occurring flavonoids, chrysin, flavone, and genistein 4'-methyl ether (Biochanin A) showed I50 values of 4.6, 68, and 113 microM, respectively, while AG showed an I50 value of 7.4 microM. Kinetic analyses showed that both AG and the flavonoids acted as competitive inhibitors of aromatase. The Ki values, indicating the effectiveness of inhibition, were 0.2, 2.4, 2.4, 22, and 49 microM, for ANF, AG, chrysin, flavone, and Biochanin A, respectively. Chrysin, the most potent of the naturally-occurring flavonoids, was similar in potency and effectiveness to AG, a pharmaceutical aromatase inhibitor used clinically in cases of estrogen-dependent carcinoma. These data suggest that flavonoid inhibition of peripheral aromatase activity may contribute to the observed cancer-preventive hormonal effects of plant-based diets.

  18. Effects of teicoplanin on cell number of cultured cell lines

    PubMed Central

    Kashkolinejad-Koohi, Tahere; Saadat, Iraj

    2015-01-01

    Teicoplanin is a glycopeptide antibiotic with a wide variation in human serum half-life. It is also a valuable alternative of vancomycin. There is however no study on its effect on cultured cells. The aim of the present study was to test the effect of teicoplanin on cultured cell lines CHO, Jurkat E6.1 and MCF-7. The cultured cells were exposed to teicoplanin at final concentrations of 0–11000 μg/ml for 24 hours. To determine cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was performed. At low concentrations of teicoplanin the numbers of cultured cells (due to cell proliferation) were increased in the three cell lines examined. The maximum cell proliferation rates were observed at concentrations of 1000, 400, and 200 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. Cell toxicity was observed at final concentrations over 2000, 6000, and 400 μg/ml of teicoplanin for CHO, MCF-7 and Jurkat cell lines, respectively. A dose-dependent manner of cell toxicity was observed. Our present findings indicated that teicoplanin at clinically used concentrations induced cell proliferation. It should therefore be used cautiously, particularly in children, pregnant women and patients with cancer. PMID:27486356

  19. Radiation sensitivity of Merkel cell carcinoma cell lines

    SciTech Connect

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

    1995-07-30

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

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

    SciTech Connect

    Felthaus, O.; Ettl, T.; Gosau, M.; Driemel, O.; Brockhoff, G.; Reck, A.; Zeitler, K.; Hautmann, M.; Reichert, T.E.; Schmalz, G.; Morsczeck, C.

    2011-04-01

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

  1. Investigating citrullinated proteins in tumour cell lines

    PubMed Central

    2013-01-01

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

  2. The proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°Celsius compared to euthermic conditions in pigs.

    PubMed

    Bohan, Amy E; Purvis, Katelyn N; Bartosh, Julia L; Brandebourg, Terry D

    2014-01-01

    Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

  3. The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro.

    PubMed

    Jiao, Yang; Zhang, Jingying; Lu, Lunjie; Xu, Jiaying; Qin, Liqiang

    2016-02-19

    The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO) is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05). Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT4) expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling.

  4. Dimethylfumarate suppresses adipogenic differentiation in 3T3-L1 preadipocytes through inhibition of STAT3 activity.

    PubMed

    Kang, Hyeon-Ji; Seo, Hyun-Ae; Go, Younghoon; Oh, Chang Joo; Jeoung, Nam Ho; Park, Keun-Gyu; Lee, In-Kyu

    2013-01-01

    The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBPβ, PPARγ, C/EBPα, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBPβ, C/EBPα, PPARγ, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation.

  5. Pancreastatin producing cell line from human pancreatic islet cell tumor.

    PubMed

    Funakoshi, A; Tateishi, K; Tsuru, M; Jimi, A; Wakasugi, H; Ikeda, Y; Kono, A

    1990-04-30

    It has been characterized that cell line QGP-1 derived from human non-functioning pancreatic islet cell tumor produces human pancreastatin. Exponentially growing cultures produced 5.7 fmol of pancreastatin/10(6) cells/hr. Human pancreastatin immunoreactivities in plasma and tumor after xenografting with QGP-1 into nude mouse were 92.7 fmol/ml and 160.2 pmol/g wet weight, respectively. Immunocytochemical study revealed both chromogranin A and pancreastatin immunoreactive cells in the tumor. Gel filtrations of culture medium and tumor extract identified heterogenous molecular forms of PST-LI which eluted as large and smaller molecular species. These results suggest that plasma pancreastatin levels may be useful as a tumor marker of endocrine tumor of the pancreas, and the pancreastatin producing cell line may be useful for studies of the mechanism of secretions and processing of chromogranin A and pancreastatin.

  6. Effect of glutamate analogues on brain tumor cell lines.

    PubMed

    Campbell, G L; Bartel, R; Freidman, H S; Bigner, D D

    1985-10-01

    Glutamate analogues have been used in many different experimental approaches in neurobiology. A small number of these analogues have been classified as gliotoxic. We have examined the effect of seven glutamate analogues (five gliotoxic and two neurotoxic) on the growth and viability of four human glioma cell lines, one human medulloblastoma cell line, and one human sarcoma cell line. Aminoadipic acid and homocysteic acid predominantly affected the growth of two glioma cell lines in the presence of 4 mM glutamine. Phosphonobutyric acid predominantly affected the other two glioma cell lines and the medulloblastoma cell line in the presence of 4 mM glutamine. In medium containing no glutamine, all three analogues had marked effects on all the cell lines except the sarcoma cell line. These effects were dose dependent. We postulate that these results can in part be explained on the basis of metabolic compartmentalization.

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

    PubMed Central

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

    2016-01-01

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

  8. On the Ontology Based Representation of Cell Lines

    PubMed Central

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

    2012-01-01

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

  9. EXAFS studies of prostate cancer cell lines

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

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

  10. The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation.

    PubMed

    Zhang, Meizi; Zhang, Ying; Ma, Jun; Guo, Feima; Cao, Qian; Zhang, Yu; Zhou, Bin; Chai, Jijie; Zhao, Wenqing; Zhao, Renbin

    2015-01-01

    FTO (fat mass and obesity associated gene) was genetically identified to be associated with body mass index (BMI), presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N6-methyladenosine (m6A) is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m6A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ.

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

    PubMed Central

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

    2016-01-01

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

  12. In vitro suppression of the lipogenic pathway by the nonnucleoside reverse transcriptase inhibitor efavirenz in 3T3 and human preadipocytes or adipocytes.

    PubMed

    El Hadri, Khadija; Glorian, Martine; Monsempes, Christelle; Dieudonné, Marie-Noëlle; Pecquery, René; Giudicelli, Yves; Andreani, Marise; Dugail, Isabelle; Fève, Bruno

    2004-04-09

    A serious metabolic syndrome combining insulin-resistance, dyslipidemia, central adiposity, and peripheral lipoatrophy has arisen in HIV-infected patients receiving highly active antiretroviral therapy. The aim of this work was to examine the effects of the nonnucleoside reverse transcriptase inhibitor (NNRTI) efavirenz on adipocyte differentiation and metabolism. When induced to differentiate in the presence of efavirenz (5-50 microm), 3T3-F442A preadipocytes failed to accumulate cytoplasmic triacylglycerol droplets. This phenomenon was rapidly reversible and was also readily detectable in the 3T3-L1 preadipose cell line and in primary cultures of human preadipocytes. When applied to mature 3T3-F442A adipocytes, efavirenz induced a delayed and moderate reduction in cell triglyceride content. Measurement of [(3)H]deoxyglucose uptake, basal and agonist-stimulated lipolysis, and cell viability indicated that these pathways are not involved in efavirenz effects on triacylglycerol accumulation. By contrast, we found that the NNRTI induced a dramatic dose- and time-dependent decrease in gene and protein expression of the lipogenic transcription factor sterol regulatory element-binding protein-1c (SREBP-1c). Adipose conversion was only altered at the highest efavirenz concentrations, as suggested by the mild reduction in peroxisome proliferator-activated receptor-gamma and CCAAT/enhancer-binding protein-alpha. CCAAT/enhancer-binding protein-beta remained unchanged. The inhibition of SREBP-1c expression was accompanied by a sharp reduction in the expression of SREBP-1c target genes and in the adipocyte lipogenic activity in efavirenz-treated cells. Finally, the inhibitory effect of efavirenz on cell triglyceride accumulation was prevented by directly providing free fatty acids to the cells and was reversed by overexpression of a dominant positive form of SREBP-1c, reinforcing the implication of this transcription factor in the antilipogenic effect of the drug. When

  13. Growth hormone (GH) differentially regulates NF-kB activity in preadipocytes and macrophages: implications for GH's role in adipose tissue homeostasis in obesity.

    PubMed

    Kumar, P Anil; Chitra, P Swathi; Lu, Chunxia; Sobhanaditya, J; Menon, Ram

    2014-06-01

    Adipose tissue remodeling in obesity involves macrophage infiltration and chronic inflammation. NF-kB-mediated chronic inflammation of the adipose tissue is directly implicated in obesity-associated insulin resistance. We have investigated the effect of growth hormone (GH) on NF-kB activity in preadipocytes (3T3-F442A) and macrophages (J774A.1). Our studies indicate that whereas GH increases NF-kB activity in preadipocytes, it decreases NF-kB activity in macrophages. This differential response of NF-kB activity to GH correlates with the GH-dependent expression of a cadre of NF-kB-activated cytokines in these two cell types. Activation of NF-kB by GH in preadipocytes heightens inflammatory response by stimulating production of multiple cytokines including TNF-α, IL-6, and MCP-1, the mediators of both local and systemic insulin resistance and chemokines that recruit macrophages. Our studies also suggest differential regulation of miR132 and SIRT1 expression as a mechanism underlying the observed variance in GH-dependent NF-kB activity and altered cytokine profile in preadipocytes and macrophages. These findings further our understanding of the complex actions of GH on adipocytes and insulin sensitivity.

  14. DNA profiling and characterization of animal cell lines.

    PubMed

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

    2014-01-01

    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.

  15. Isoniazid suppresses antioxidant response element activities and impairs adipogenesis in mouse and human preadipocytes

    SciTech Connect

    Chen, Yanyan; Xue, Peng; Hou, Yongyong; Zhang, Hao; Zheng, Hongzhi; Zhou, Tong; Qu, Weidong; Teng, Weiping; Zhang, Qiang; Andersen, Melvin E.; Pi, Jingbo

    2013-12-15

    Transcriptional signaling through the antioxidant response element (ARE), orchestrated by the Nuclear factor E2-related factor 2 (Nrf2), is a major cellular defense mechanism against oxidative or electrophilic stress. Here, we reported that isoniazid (INH), a widely used antitubercular drug, displays a substantial inhibitory property against ARE activities in diverse mouse and human cells. In 3T3-L1 preadipocytes, INH concentration-dependently suppressed the ARE-luciferase reporter activity and mRNA expression of various ARE-dependent antioxidant genes under basal and oxidative stressed conditions. In keeping with our previous findings that Nrf2-ARE plays a critical role in adipogenesis by regulating expression of CCAAT/enhancer-binding protein β (C/EBPβ) and peroxisome proliferator-activated receptor γ (PPARγ), suppression of ARE signaling by INH hampered adipogenic differentiation of 3T3-L1 cells and human adipose-derived stem cells (ADSCs). Following adipogenesis induced by hormonal cocktails, INH-treated 3T3-L1 cells and ADSCs displayed significantly reduced levels of lipid accumulation and attenuated expression of C/EBPα and PPARγ. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by INH occurred in the early stage of terminal adipogenic differentiation, where reduced expression of C/EBPβ and C/EBPδ was observed. To our knowledge, the present study is the first to demonstrate that INH suppresses ARE signaling and interrupts with the transcriptional network of adipogenesis, leading to impaired adipogenic differentiation. The inhibition of ARE signaling may be a potential underlying mechanism by which INH attenuates cellular antioxidant response contributing to various complications. - Highlights: • Isoniazid suppresses ARE-mediated transcriptional activity. • Isoniazid inhibits adipogenesis in preadipocytes. • Isoniazid suppresses adipogenic gene expression during adipogenesis.

  16. Neuronal cell lines as model dorsal root ganglion neurons

    PubMed Central

    Yin, Kathleen; Baillie, Gregory J

    2016-01-01

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

  17. 77 FR 5489 - Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-03

    ... National Institute of Standards and Technology Identification of Human Cell Lines Project AGENCY: National... tandem repeat (STR) profiling up to 1500 human cell line samples as part of the Identification of Human... its intent to unambiguously identify by short tandem repeat (STR) profiling up to 1500 human cell...

  18. Immunoglobulin expression and synthesis by human haemic cell lines.

    PubMed Central

    Gordon, J; Hough, D; Karpas, A; Smith, J L

    1977-01-01

    Twenty-six human cell lines derived from a variety of lymphoid and non-lymphoid malignancies, were investigated for their immunological markers, with special reference to the class of immunoglobulin expressed. Twenty-five of the lines stained positively for surface immunoglobulin and IgD together with IgM proved to be the major immunoglobulin classes on these cells. Six of the lines were chosen for a study of their immunoglobulin synthesis patterns over an 18-h period and the immunoglobulin produced was analysed on SDS-polyacrylamide gel electrophoresis. Patterns obtained from the cell lines were similar to that from normal lymph node lymphocytes and differed markedly to plasma cells. Two of the cell lines had abnormal immunoglobulin synthesis patterns characterized as free light chains in one case. The cell lines are evaluated for their usefulness as models of immunoglobulin synthesis and analogues of normal and neoplastic states. PMID:608682

  19. Effect of blueberry polyphenols on 3T3-F442A preadipocyte differentiation.

    PubMed

    Moghe, Shiwani S; Juma, Shanil; Imrhan, Victorine; Vijayagopal, Parakat

    2012-05-01

    Today obesity is an epidemic, and its prevalence has increased significantly over the last few decades. To avoid excessive accumulation of fat, optimum energy intake along with regular exercise is mandatory. Polyphenols present in green tea, grape seeds, orange, and grapefruit combat adipogenesis at the molecular level and also induce lipolysis. However, very little is known regarding the role of blueberry polyphenols on adipocyte differentiation. Hence we tested the dose-dependent effects of blueberry polyphenols on mouse 3T3-F442A preadipocyte differentiation and lipolysis. 3T3-F442A preadipocytes were incubated with three doses of blueberry polyphenols (150, 200, and 250 μg/mL [BB-150, BB-200, and BB-250, respectively]), and intracellular lipid content, cell proliferation, and lipolysis were assayed. Blueberry polyphenols suppressed adipocyte differentiation determined by Oil Red-O staining and AdipoRed assay. Intracellular lipid content in control (11,385.51±1,169.6 relative fluorescence units) was significantly higher (P<.05) than with the three doses of blueberry polyphenols (8336.86±503.57, 4235.67±323.17, and 3027.97±346.61, respectively). This corresponds to a reduction of 27%, 63%, and 74%, respectively. Cell proliferation was observed to be significantly higher in the control (0.744±0.035 optical density units) than with BB-150 (0.517±0.031), BB-200 (0.491±0.023), and BB-250 (0.455±0.012). However, when tested for lipolysis, there was no significant difference observed among the groups. We conclude that blueberry polyphenols may play an effective role in inhibiting adipogenesis and cell proliferation.

  20. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators.

    PubMed

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone; Dai, Jie; Rogowska-Wrzesinska, Adelina; Mandrup, Susanne; Jensen, Ole N

    2017-03-01

    Adipocytes (fat cells) are important endocrine and metabolic cells critical for systemic insulin sensitivity. Both adipose excess and insufficiency are associated with adverse metabolic function. Adipogenesis is the process whereby preadipocyte precursor cells differentiate into lipid-laden mature adipocytes. This process is driven by a network of transcriptional regulators (TRs). We hypothesized that protein PTMs, in particular phosphorylation, play a major role in activating and propagating signals within TR networks upon induction of adipogenesis by extracellular stimulus. We applied MS-based quantitative proteomics and phosphoproteomics to monitor the alteration of nuclear proteins during the early stages (4 h) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation levels of proteins involved in gene expression, cell organization, and oxidation-reduction pathways. Furthermore, proteins acting as negative modulators involved in negative regulation of gene expression, insulin stimulated glucose uptake, and cytoskeletal organization showed a decrease in their nuclear abundance and/or phosphorylation levels during the first 4 h of adipogenesis. Among 288 identified TRs, 49 were regulated within 4 h of adipogenic stimulation including several known and many novel potential adipogenic regulators. We created a kinase-substrate database for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase family and the mitogen-activated protein kinase family including P38 and c-Jun N-terminal kinases, suggesting that these kinases act as orchestrators of early adipogenesis.

  1. Continuous human cell lines and method of making same

    SciTech Connect

    Stampfer, M.R.

    1989-02-28

    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. No Drawings

  2. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, M.R.

    1985-07-01

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

  3. Continuous human cell lines and method of making same

    DOEpatents

    Stampfer, Martha R.

    1989-01-01

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

  4. Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Lee, Youngyi; Bae, Eun Ju

    2013-11-01

    Adipocytes are the key player in adipose tissue inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and differentiation of preadipocytes. Fistein, a polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-diabetic effects. In this study, we aimed to investigate the effect of fisetin on adipocyte proliferation and differentiation in 3T3-L1 preadipocyte cell line and its mechanism of action. We found that fisetin inhibits adipocyte differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor γ. Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the differentiation medium. We also found that fisetin inhibition of adipocyte differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of preadipocyte proliferation at early stage of differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the inhibition of adipocyte differentiation by fisetin may be at least in part mediated by cell cycle arrest during adipogenesis.

  5. Susceptibilities of 14 cell lines to bluetongue virus infection.

    PubMed Central

    Wechsler, S J; McHolland, L E

    1988-01-01

    The effect of bluetongue virus (BTV) infection was investigated in 14 cell lines. The cell lines included the following vertebrate cells: baby hamster kidney, African green monkey kidney (Vero), rabbit kidney, bovine kidney, canine kidney, bovine turbinate, bovine endothelium (CPAE), bighorn sheep tongue, equine dermis, gekko lung, rainbow trout gonad, and mouse fibroblast (L929); they also included the following invertebrate lines: mosquito and biting midge. Comparisons between the cell lines were made on the basis of time to observed cytopathic effects, titer in 50% tissue culture infectious doses, and titer in plaque-forming units. The CPAE cell line produced the highest BTV 50% tissue culture infectious dose of all cell lines tested. The Vero and L929 cells gave the most discrete plaques in plaque assays. Of the 14 cell lines tested, the CPAE cells were the most susceptible to both cell culture-adapted and animal source BTV. Bovine endothelial cells demonstrate significant potential as a cell culture system for BTV investigations. PMID:2853175

  6. Re-characterization of established human retinoblastoma cell lines.

    PubMed

    Busch, Maike; Philippeit, Claudia; Weise, Andreas; Dünker, Nicole

    2015-03-01

    Retinoblastoma (RB) is the most common malignant intraocular childhood tumor. Forty years after their first description, in the present study, we re-characterized seven established retinoblastoma cell lines with regard to their RB1 mutation status, morphology, growth pattern, endogenous apoptosis levels, colony formation efficiency in soft agar and invasiveness and dissemination capacity in chick chorioallantoic membrane (CAM) assays. All RB cell lines predominantly resemble small epithelioid cells with little cytoplasm and large nucleus, which mainly grow in cell clusters, but sometimes form chain-like structures with incident loops or three-dimensional aggregates. We observed different growth rates for the different retinoblastoma cells investigated. RBL-30, RBL-13 and RBL 383 cells grew very slowly, whereas Y-79 cells grew fastest under our culture conditions. Apoptosis rates likewise differed with highest cell death levels in RB 383 and RB 355 and lowest in WERI-Rb1 and RBL-15. Contradicting former reports, six of the seven RB cell lines analyzed were able to form colonies in soft agarose after single cell seeding within 3 weeks of incubation. Upon inoculation of four out of seven RB cell lines on the dorsal CAM, GFP-positive cells were detectable in the ventral CAM and two RB cell lines caused tumor development, indicating their intravasation and dissemination potential. All RB cell lines exhibited the potential to extravasate from the capillary system after intravenous CAM injection. Our study provides valuable new details for future therapy-related retinoblastoma basic research in vitro.

  7. Identification of cell lines permissive for human coronavirus NL63.

    PubMed

    Schildgen, Oliver; Jebbink, Maarten F; de Vries, Michel; Pyrc, Krzysztov; Dijkman, Ronald; Simon, Arne; Müller, Andreas; Kupfer, Bernd; van der Hoek, Lia

    2006-12-01

    Six cell lines routinely used in laboratories were tested for permissiveness to the infection with the newly identified human coronavirus NL63. Two monkey epithelial cell lines, LLC-MK2 and Vero-B4, showed a cytopathic effect (CPE) and clear viral replication, whereas no CPE or replication was observed in human lung fibroblasts MRC-5s. In Rhabdomyosarcoma cells, Madin-Darby-Canine-kidney cells and in an undefined monkey kidney cell line some replication was observed but massive exponential rise in virus yield lacked The results will lead to an improved routine diagnostic algorithm for the detection of the human coronavirus NL63.

  8. Trichloroethylene toxicity in a human hepatoma cell line

    SciTech Connect

    Thevenin, E.; McMillian, J.

    1994-12-31

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

  9. TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation

    PubMed Central

    Funcke, Jan-Bernd; Zoller, Verena; El Hay, Muad Abd; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2015-01-01

    Upon obesity, adipose tissue is excessively expanded and characterized by pathologic processes like hypoxia, fibrosis, and inflammation. Death ligands belonging to the TNF superfamily such as TNF-α are important contributors to these derangements and exert a pronounced influence on the metabolic and cellular homeostasis of adipose tissue. Here, we sought to identify the effect of the death ligand TNF-related apoptosis-inducing ligand (TRAIL) on the adipose tissue precursor cell pool and therefore investigated its influence on preadipocyte proliferation. Treatment of human preadipocytes with TRAIL resulted in a time- and dose-dependent increase in proliferation (EC50 3.4 ng/ml) comparable to IGF-1. Although no apoptosis was observed, TRAIL triggered a rapid cleavage of caspase-8 and -3. Neither inhibition of caspase activity by zVAD.fmk (20 µM) nor ablation of caspase-8 expression by lentivirus-delivered small hairpin RNA (shRNA) abolished the proliferative response. TRAIL triggered a delayed and sustained activation of ERK1/2, leaving Akt, p38, JNK, and NF-κB unaffected. Importantly, inhibition of ERK1/2 activation by PD0325901 (300 nM) or AZD6244 (5 or 10 µM) completely abolished the proliferative response. We thus reveal a hitherto unknown function of TRAIL in regulating adipose tissue homeostasis by promoting the proliferation of tissue-resident precursor cells.—Funcke, J.-B., Zoller, V., Abd El Hay, M., Debatin, K.-M., Wabitsch, M., Fischer-Posovszky, P. TNF-related apoptosis-inducing ligand promotes human preadipocyte proliferation via ERK1/2 activation. PMID:25857555

  10. Motoneuron differentiation of immortalized human spinal cord cell lines.

    PubMed

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

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

    SciTech Connect

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

    2012-01-15

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

  12. Germ line, stem cells, and epigenetic reprogramming.

    PubMed

    Surani, M A; Durcova-Hills, G; Hajkova, P; Hayashi, K; Tee, W W

    2008-01-01

    The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells.

  13. Establishment of a Human Thymic Myoid Cell Line

    PubMed Central

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

    1999-01-01

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

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

    PubMed

    Hacker, David L; Balasubramanian, Sowmya

    2016-06-01

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

  15. Quantitative methods to characterize morphological properties of cell lines.

    PubMed

    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

    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.

  16. Growth of Murine Cytomegalovirus in Various Cell Lines

    PubMed Central

    Kim, Kwang Soo; Carp, Richard I.

    1971-01-01

    Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV. PMID:4327583

  17. The mechanics of hyaluronic acid/adipic acid dihydrazide hydrogel: towards developing a vessel for delivery of preadipocytes to native tissues.

    PubMed

    Shoham, Naama; Sasson, Aviad Levi; Lin, Feng-Huei; Benayahu, Dafna; Haj-Ali, Rami; Gefen, Amit

    2013-12-01

    Promising treatment approaches in repairing tissue defects include implementation of regenerative medicine strategies, particularly delivery of preadipocytes to sites where adipose tissue damage needs to be repaired or where fat needs to be generated. In this study, we suggest that the injectable hyaluronic acid/adipic acid dihydrazide (HA/ADH) hydrogel may be an adipose-tissue-like material in terms of biological compatibility as well as mechanical behavior. First, we show that the hydrogel enables and supports growth, proliferation and differentiation of 3T3-L1 preadipocytes. Second, given that adipose tissue is a weight-bearing biological structure, we investigate the large deformation mechanical behavior of the hydrogel with and without embedded preadipocytes, by performing confined and unconfined compression tests and then calibrating a strain energy density (SED) function to the results. Four test groups were examined: (1) Hydrogel specimens right after the preparation without cells, (2) and (3) 3-days-cultured hydrogel specimens with and without cells, respectively, and (4) 6-days-cultured hydrogel specimens with cells. A one-term Ogden SED was found to adequately describe the hyperelastic behavior of the hydrogel specimens in all experimental groups. Importantly, we found that the mechanical properties of the hydrogel, when subjected to compression, are in good agreement with those of native adipose tissue, with the better fit occurring 3-6 days after preparation of the hydrogel. Third, computational finite element studies of the mechanical (stress-strain) behavior of the HA/ADH hydrogel when containing mature adipocytes indicated that the stiffnesses of the constructs were mildly affected by the presence of the adipocytes. Hence, we conclude that injectable HA/ADH hydrogel may serve as a vessel for protecting preadipocytes during, and at a short-term after delivery to native tissues, e.g. in research towards regenerative medicine in tissue reconstructions.

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

    NASA Astrophysics Data System (ADS)

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

    AIM To investigate the radiosensitivity of hepatoma cell lines and human normal liver cell lines METHODS Accelerated carbon ions by heavy ion research facility in Lanzhou HIRFL have high LET We employed it to study the radiosensitivity of hepatoma cell lines SMMC-7721 and human normal liver cell lines L02 using premature chromosome condensation technique PCC Cell survive was documented by a colony assay Chromatid breaks were measured by counting the number of chromatid breaks and isochromatid breaks immediately after prematurely chromosome condensed by Calyculin-A RESULTS The survival curve of the two cell lines presented a good linear relationship and the survival fraction of L02 is higher than that of SMMC-7721 Additionally the two types of G 2 phase chromosome breaks chromatid breaks and isochromatid breaks of L02 are lower than that of SMMC-7721 CONCLUSION Human normal liver cell line have high radioresistance than that of hepatoma cell line It imply that it is less damage to normal organs when radiotherapy to hepatoma

  19. Regulation of germ line stem cell homeostasis

    PubMed Central

    Garcia, T.X.; Hofmann, M.C.

    2015-01-01

    Mammalian spermatogenesis is a complex process in which spermatogonial stem cells of the testis (SSCs) develop to ultimately form spermatozoa. In the seminiferous epithelium, SSCs self-renew to maintain the pool of stem cells throughout life, or they differentiate to generate a large number of germ cells. A balance between SSC self-renewal and differentiation is therefore essential to maintain normal spermatogenesis and fertility. Stem cell homeostasis is tightly regulated by signals from the surrounding microenvironment, or SSC niche. By physically supporting the SSCs and providing them with these extrinsic molecules, the Sertoli cell is the main component of the niche. Earlier studies have demonstrated that GDNF and CYP26B1, produced by Sertoli cells, are crucial for self-renewal of the SSC pool and maintenance of the undifferentiated state. Down-regulating the production of these molecules is therefore equally important to allow germ cell differentiation. We propose that NOTCH signaling in Sertoli cells is a crucial regulator of germ cell fate by counteracting these stimulatory factors to maintain stem cell homeostasis. Dysregulation of this essential niche component can lead by itself to sterility or facilitate testicular cancer development.

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

    PubMed

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

    2013-10-01

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

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

    PubMed

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

    2014-09-01

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

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

    PubMed

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

    2014-01-01

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

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

    PubMed

    Quisbert-Valenzuela, Edwin O; Calaf, Gloria M

    2016-06-01

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

  4. Diacylglycerol production induced by growth hormone in Ob1771 preadipocytes arises from phosphatidylcholine breakdown

    SciTech Connect

    Catalioto, R.M.; Ailhaud, G.; Negrel, R. )

    1990-12-31

    Growth Hormone has recently been shown to stimulate the formation of diacylglycerol in Ob1771 mouse preadipocyte cells without increasing inositol lipid turnover. Addition of growth hormone to Ob1771 cells prelabelled with ({sup 3}H)glycerol or ({sup 3}H)choline led to a rapid, transient and stoechiometric formation of labelled diacylglycerol and phosphocholine, respectively. In contrast, no change was observed in the level of choline and phosphatidic acid whereas the release of water-soluble metabolites in ({sup 3}H)ethanolamine prelabelled cells exposed to growth hormone was hardly detectable. Stimulation by growth hormone of cells prelabelled with (2-palmitoyl 9, 10 ({sup 3}H))phosphatidylcholine also induced the production of labelled diacyglycerol. Pertussis toxin abolished both diacylglycerol and phosphocholine formation induced by growth hormone. It is concluded that growth hormone mediates diacylglycerol production in Ob1771 cells by means of phosphatidylcholine breakdown involving a phospholipase C which is likely coupled to the growth hormone receptor via a pertussis toxin-sensitive G-protein.

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

    PubMed

    Klingemann, Hans; Boissel, Laurent; Toneguzzo, Frances

    2016-01-01

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

  6. Different anti-adipogenic effects of bio-compounds on primary visceral pre-adipocytes and adipocytes

    PubMed Central

    Colitti, Monica; Stefanon, Bruno

    2016-01-01

    Several natural compounds exhibit strong capacity for decreasing triglyceride accumulation, enhancing lipolysis and inducing apoptosis. The present study reports the anti-adipogenic effects of Silybum marianum (SL), Citrus aurantium (CA), Taraxacum officinale (TO), resveratrol (RE), Curcuma longa (CU), caffeine (CF), oleuropein (OL) and docosahexaenoic acid (DHA) in reducing differentiation and increasing lipolysis and apoptosis. Analyses were performed on human primary visceral pre-adipocytes after 10 (P10) and 20 (P20) days of treatment during differentiation and on mature adipocytes after 7 days of treatment (A7). The percentage of apoptosis induced by TO extract in P10 and P20 cells was significantly higher than that induced by all other compounds and in CTRL cells. Triglyceride accumulation was significantly lower in cells treated with DHA, CF, RE in comparison to cells treated with OL and in CTRL cells. Treatments with CF, DHA and OL significantly incremented lipolysis in P20 cells in comparison to other compounds and in CTRL cells. On the contrary, the treatment of A7 cells with OL, CA and TO compounds significantly increased cell lipolysis. The addition of CF in differentiating P20 pre-adipocytes significantly increased the expression of genes involved in inhibition of adipogenesis, such as GATA2, GATA3, WNT1, WNT3A, SFRP5, and DLK1. Genes involved in promoting adipogenesis such as CCND1, CEBPB and SREBF1 were significantly down-regulated by the treatment. The screening of bioactive compounds for anti-adipogenic effects showed that in differentiating cells TO extract was the most effective in inducing apoptosis and CF and DHA extracts were more efficient in inhibition of differentiation and in induction of cell lipolysis. PMID:27540349

  7. Investigation of the selenium metabolism in cancer cell lines.

    PubMed

    Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan; Andresen, Lars; Skov, Søren; Gammelgaard, Bente

    2011-02-01

    The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 μM were incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size exclusion chromatography and ICP-MS detection. The selenium compounds exhibited large differences in their ability to induce cell death in the three cell lines and the susceptibilities of the cell lines were different. Full recovery of selenium in the cellular fractions was observed for all Se compounds except MeSeA. Speciation analysis showed that MeSeA was completely transformed during the incubations, while metabolic conversion of the other Se compounds was limited. Production of volatile dimethyl diselenide was observed for MeSeA and MeSeCys. MeSeA, MeSeCys and selenite showed noticeable protein binding. Correlations between cell death induction and the Se compounds transformations could not be demonstrated.

  8. Induction of apoptosis by opium in some tumor cell lines.

    PubMed

    Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

    2016-09-30

    The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

  9. Strategies for selecting recombinant CHO cell lines for cGMP manufacturing: improving the efficiency of cell line generation.

    PubMed

    Porter, Alison J; Racher, Andrew J; Preziosi, Richard; Dickson, Alan J

    2010-01-01

    Transfectants with a wide range of cellular phenotypes are obtained during the process of cell line generation. For the successful manufacture of a therapeutic protein, a means is required to identify a cell line with desirable growth and productivity characteristics from this phenotypically wide-ranging transfectant population. This identification process is on the critical path for first-in-human studies. We have stringently examined a typical selection strategy used to isolate cell lines suitable for cGMP manufacturing. One-hundred and seventy-five transfectants were evaluated as they progressed through the different assessment stages of the selection strategy. High producing cell lines, suitable for cGMP manufacturing, were identified. However, our analyses showed that the frequency of isolation of the highest producing cell lines was low and that ranking positions were not consistent between each assessment stage, suggesting that there is potential to improve upon the strategy. Attempts to increase the frequency of isolation of the 10 highest producing cell lines, by in silico analysis of alternative selection strategies, were unsuccessful. We identified alternative strategies with similar predictive capabilities to the typical selection strategy. One alternate strategy required fewer cell lines to be progressed at the assessment stages but the stochastic nature of the models means that cell line numbers are likely to change between programs. In summary, our studies illuminate the potential for improvement to this and future selection strategies, based around use of assessments that are more informative or that reduce variance, paving the way to improved efficiency of generation of manufacturing cell lines.

  10. Insulin sensitization of human preadipocytes through glucocorticoid hormone induction of forkhead transcription factors.

    PubMed

    Tomlinson, Julianna J; Boudreau, Adèle; Wu, Dongmei; Abdou Salem, Houssein; Carrigan, Amanda; Gagnon, AnneMarie; Mears, Alan J; Sorisky, Alexander; Atlas, Ella; Haché, Robert J G

    2010-01-01

    Glucocorticoids are synthesized locally in adipose tissue and contribute to metabolic disease through the facilitation of adipose tissue expansion. Here we report that exposure of human primary preadipocytes to glucocorticoids increases their sensitivity to insulin and enhances their subsequent response to stimuli that promote differentiation. This effect was observed in primary human preadipocytes but not in immortalized 3T3-L1 murine preadipocytes or in fully differentiated primary human adipocytes. Stimulation of insulin signaling was mediated through induction of insulin receptor (IR), IR substrate protein 1 (IRS1), IRS2, and the p85 regulatory subunit of phosphoinositide-3-3-kinase, which led to enhanced insulin-mediated activation of Akt. Although induction of IRS2 was direct, induction of IR and IRS1 by glucocorticoids occurred subsequent to primary induction of the forkhead family transcription factors FoxO1A and FoxO3A. These results reveal a new role for glucocorticoids in preparing preadipocytes for differentiation.

  11. Effects of ethanol on an intestinal epithelial cell line

    SciTech Connect

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

    1990-02-01

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

  12. Co-cultivation of human aortic smooth muscle cells with epicardial adipocytes affects their proliferation rate.

    PubMed

    Ždychová, J; Čejková, S; Králová Lesná, I; Králová, A; Malušková, J; Janoušek, L; Kazdová, L

    2014-01-01

    The abnormal proliferation of vascular smooth muscle cells (VSMC) is thought to play a role in the pathogenesis of atherosclerosis. Adipocytes produce several bioactive paracrine substances that can affect the growth and migration of VSMCs. Our study focuses on the direct effect of the bioactive substances in conditioned media (CM) that was obtained by incubation with primary adipocyte-derived cell lines, including cell lines derived from both preadipocytes and from more mature cells, on the proliferation rate of human aortic smooth muscle cells (HAoSMCs). We used a Luminex assay to measure the adipokine content of the CM and showed that there was a higher concentration of monocyte chemoattractant protein-1 in renal preadipocyte-CM compared with the HAoSMC control (p<0.5). The addition of both renal preadipocyte- and epicardial adipocyte- CM resulted in the elevated production of vascular endothelial growth factor compared with the control HASoSMC CM (p<0.001). The adiponectin content in renal adipocyte-CM was increased compared to all the remaining adipocyte-CM (p<0.01). Moreover, the results showed a higher proliferation rate of HAoSMCs after co-culture with epicardial adipocyte-CM compared to the HAoSMC control (p<0.05). These results suggest that bioactive substances produced by adipocytes have a stimulatory effect on the proliferation of VSMCs.

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

    PubMed Central

    2014-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  15. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines.

    PubMed

    Harenza, Jo Lynne; Diamond, Maura A; Adams, Rebecca N; Song, Michael M; Davidson, Heather L; Hart, Lori S; Dent, Maiah H; Fortina, Paolo; Reynolds, C Patrick; Maris, John M

    2017-03-28

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma.

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

    PubMed

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

    2002-01-01

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

  17. Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines

    PubMed Central

    Harenza, Jo Lynne; Diamond, Maura A.; Adams, Rebecca N.; Song, Michael M.; Davidson, Heather L.; Hart, Lori S.; Dent, Maiah H.; Fortina, Paolo; Reynolds, C. Patrick; Maris, John M.

    2017-01-01

    Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma. PMID:28350380

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

    PubMed

    Sadowska, Anna; Krętowski, Rafał; Szynaka, Beata; Cechowska-Pasko, Marzanna; Car, Halina

    2013-10-01

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

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

    PubMed Central

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

    1996-01-01

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

  20. Myelination in coculture of established neuronal and Schwann cell lines.

    PubMed

    Sango, Kazunori; Kawakami, Emiko; Yanagisawa, Hiroko; Takaku, Shizuka; Tsukamoto, Masami; Utsunomiya, Kazunori; Watabe, Kazuhiko

    2012-06-01

    Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

  1. Experimental Adaptation of Rotaviruses to Tumor Cell Lines

    PubMed Central

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

    2016-01-01

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

  2. Sclerostin Antibody Administration Converts Bone Lining Cells Into Active Osteoblasts.

    PubMed

    Kim, Sang Wan; Lu, Yanhui; Williams, Elizabeth A; Lai, Forest; Lee, Ji Yeon; Enishi, Tetsuya; Balani, Deepak H; Ominsky, Michael S; Ke, Hua Zhu; Kronenberg, Henry M; Wein, Marc N

    2016-11-14

    Sclerostin antibody (Scl-Ab) increases osteoblast activity, in part through increasing modeling-based bone formation on previously quiescent surfaces. Histomorphometric studies have suggested that this might occur through conversion of bone lining cells into active osteoblasts. However, direct data demonstrating Scl-Ab-induced conversion of lining cells into active osteoblasts are lacking. Here, we used in vivo lineage tracing to determine if Scl-Ab promotes the conversion of lining cells into osteoblasts on periosteal and endocortical bone surfaces in mice. Two independent, tamoxifen-inducible lineage-tracing strategies were used to label mature osteoblasts and their progeny using the DMP1 and osteocalcin promoters. After a prolonged "chase" period, the majority of labeled cells on bone surfaces assumed a thin, quiescent morphology. Then, mice were treated with either vehicle or Scl-Ab (25 mg/kg) twice over the course of the subsequent week. After euthanization, marked cells were enumerated, their thickness quantified, and proliferation and apoptosis examined. Scl-Ab led to a significant increase in the average thickness of labeled cells on periosteal and endocortical bone surfaces, consistent with osteoblast activation. Scl-Ab did not induce proliferation of labeled cells, and Scl-Ab did not regulate apoptosis of labeled cells. Therefore, direct reactivation of quiescent bone lining cells contributes to the acute increase in osteoblast numbers after Scl-Ab treatment in mice. © 2017 American Society for Bone and Mineral Research.

  3. Establishment and characterization of 10 cell lines derived from patients with adult T-cell leukemia.

    PubMed Central

    Hoshino, H; Esumi, H; Miwa, M; Shimoyama, M; Minato, K; Tobinai, K; Hirose, M; Watanabe, S; Inada, N; Kinoshita, K; Kamihira, S; Ichimaru, M; Sugimura, T

    1983-01-01

    By using human T-cell growth factor (TCGF), 10 cell lines were established from tissue samples of 10 patients with adult T-cell leukemia (ATL). Three cell lines were adapted to growth in medium lacking TCGF. The surface markers of all cell lines were characteristic of inducer/helper T cells, i.e., OKT3+, OKT4+, OKT6-, OKT8-, OKIa1+, and human Lyt2+ and Lyt3+, except that one cell line was OKT3-. The expression of the viral antigen was examined during establishment of 8 of the 10 cell lines. The viral antigen was not expressed in leukemic cells before cultivation. In 5 lines, the viral antigen was detected by immunofluorescent staining after a short period of cultivation. However, 3 cell lines, ATL-6A, ATL-9Y, and ATL-1K did not express the viral antigen during short-term culture: the ATL-6A and ATL-9Y cell lines became positive for the viral antigen after 5 and 2 months of cultivation, respectively; the ATL-1K cell line remained antigen-negative throughout a culture period of 13 months. Southern blot hybridization assay showed that all of the cell lines, including the viral antigen-negative ATL-1K cell line, contained the viral genome. Thus, the retrovirus was associated with all 10 cell lines established from ATL patients, but there was a heterogeneity in the expression time of the retroviral antigen in leukemic cells maintained in vitro. Our findings suggested that the expression of the viral antigen was not required for maintenance of the leukemic state in vivo and for growth of leukemic cells in vitro. Images PMID:6193528

  4. Cytoglobin: a potential marker for adipogenic differentiation in preadipocytes in vitro.

    PubMed

    Doğan, Ayşegül; Demirci, Selami; Kıratlı, Binnur; Şahin, Fikrettin

    2017-02-01

    Obesity, mainly characterized by the excess fat storage, is a global health problem resulting in serious morbidity and mortality. Identification of molecular mechanisms in adipogenic differentiation pathway might lead to development of new strategies for diagnosis, prevention and therapy of obesity and associated diseases. Discovery of new genes and proteins in the differentiation pathway could help to understand the key specific regulators of the adipogenesis. Cytoglobin (Cygb), identified as a new globin family member protein, is expressed in various tissues. Although its interaction with oxygen and nitric oxide indicates the potential role in antioxidant pathways, the exact role remains unclear. In the current study, expression level of Cygb was determined in proliferating and differentiating 3T3-F442A cells by gene expression and protein expression analysis. Results revealed that Cygb expression up-regulated in differentiated cells in parallel with adipogenic differentiation markers; PPARγ, CEBPα and FABP4 expressions. Besides, Cygb overexpression in preadipocytes contributed to the adipogenic differentiation as verified by detection of higher lipid droplets and increased PPARγ, CEBPα and FABP4 expressions with respect to control cells. These findings will shed light on the unknown roles of Cygb in adipogenesis and obesity.

  5. Disabled-2 Determines Commitment of a Pre-adipocyte Population in Juvenile Mice

    PubMed Central

    Tao, Wensi; Moore, Robert; Meng, Yue; Yeasky, Toni M.; Smith, Elizabeth R.; Xu, Xiang-Xi

    2016-01-01

    Disabled-2 (Dab2) is a widely expressed clathrin binding endocytic adaptor protein and known for the endocytosis of the low-density lipoprotein (LDL) family receptors. Dab2 also modulates endosomal Ras/MAPK (Erk1/2) activity by regulating the disassembly of Grb2/Sos1 complexes associated with clathrin-coated vesicles. We found that the most prominent phenotype of Dab2 knockout mice was their striking lean body composition under a high fat and high caloric diet, although the weight of the mutant mice was indistinguishable from wild-type littermates on a regular chow. The remarkable difference in resistance to high caloric diet-induced weight gain of the dab2-deleted mice was presented only in juvenile but not in mature mice. Investigation using Dab2-deficient embryonic fibroblasts and mesenchymal stromal cells indicated that Dab2 promoted adipogenic differentiation by modulation of MAPK (Erk1/2) activity, which otherwise suppresses adipogenesis through the phosphorylation of PPARγ. The results suggest that Dab2 is required for the excessive calorie-induced differentiation of an adipocyte progenitor cell population that is present in juvenile but depleted in mature animals. The finding provides evidence for a limited pre-adipocyte population in juvenile mammals and the requirement of Dab2 in the regulation of Ras/MAPK signal in the commitment of the precursor cells to adipose tissues. PMID:27779214

  6. Three-dimensional cultured glioma cell lines

    NASA Technical Reports Server (NTRS)

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

    1991-01-01

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

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

    PubMed

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

    2007-12-15

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

  8. Solid Oxide Fuel Cell Systems PVL Line

    SciTech Connect

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

    2012-05-01

    In July 2010, Stark State College (SSC), received Grant DE-EE0003229 from the U.S. Department of Energy (DOE), Golden Field Office, for the development of the electrical and control systems, and mechanical commissioning of a unique 20kW scale high-pressure, high temperature, natural gas fueled Stack Block Test System (SBTS). SSC worked closely with subcontractor, Rolls-Royce Fuel Cell Systems (US) Inc. (RRFCS) over a 13 month period to successfully complete the project activities. This system will be utilized by RRFCS for pre-commercial technology development and training of SSC student interns. In the longer term, when RRFCS is producing commercial products, SSC will utilize the equipment for workforce training. In addition to DOE Hydrogen, Fuel Cells, and Infrastructure Technologies program funding, RRFCS internal funds, funds from the state of Ohio, and funding from the DOE Solid State Energy Conversion Alliance (SECA) program have been utilized to design, develop and commission this equipment. Construction of the SBTS (mechanical components) was performed under a Grant from the State of Ohio through Ohio's Third Frontier program (Grant TECH 08-053). This Ohio program supported development of a system that uses natural gas as a fuel. Funding was provided under the Department of Energy (DOE) Solid-state Energy Conversion Alliance (SECA) program for modifications required to test on coal synthesis gas. The subject DOE program provided funding for the electrical build, control system development and mechanical commissioning. Performance testing, which includes electrical commissioning, was subsequently performed under the DOE SECA program. Rolls-Royce Fuel Cell Systems is developing a megawatt-scale solid oxide fuel cell (SOFC) stationary power generation system. This system, based on RRFCS proprietary technology, is fueled with natural gas, and operates at elevated pressure. A critical success factor for development of the full scale system is the capability to

  9. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    SciTech Connect

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  10. Canine mammary tumour cell lines established in vitro.

    PubMed

    Hellmén, E

    1993-01-01

    Mammary tumours are the most common tumours in the female dog. The tumours have a complex histology and exist in epithelial, mixed and mesenchymal forms. To study the biology of canine mammary tumours, five cell lines have been established and characterized. The results indicate that canine mammary tumours might be derived from mammary stem cells and that the tumour growth is independent of oestrogens. The established canine mammary tumour cell lines will be valuable tools in further studies of the histogenesis and pathogenesis of these tumours.

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

    PubMed Central

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

    2014-01-01

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

  12. Design of tunable microwave transmission lines using metamaterial cells

    NASA Astrophysics Data System (ADS)

    Bensafieddine, D.; Djerfaf, F.; Chouireb, F.; Vincent, D.

    2017-04-01

    In this paper, frequency tunable transmission lines are designed using metasurface split ring resonator unit cell. We prove that the tuning principle in metasurface transmission lines is based on the variation of the resonance frequency of the permeability. The frequency-tuning arises by changing the values of two gaps in the inner and outer rings of unit cell ( g1 and g2). The branches of a disconnected gaps type conductor of each unit cell can be joined by switches (PIN diodes, MEMs, etc.). According to switch states ON or OFF, the unit cell has four different commutable behaviors which are 00, 01, 11, and 10. The results show that the resonance frequency of our metasurface transmission line is strongly shifted by about 2.5 GHz between the cases (01) and (11).

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

    PubMed

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

    2014-09-09

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

  14. Susceptibility of nonprimate cell lines to hepatitis A virus infection.

    PubMed Central

    Dotzauer, A; Feinstone, S M; Kaplan, G

    1994-01-01

    Hepatitis A virus (HAV) has been adapted to grow in primate cell cultures. We investigated replication of HAV in nonprimate cells by inoculating 20 cell lines from different species with the tissue culture-adapted HM175 strain. Slot blot hybridization and immunofluorescence analysis revealed that HAV replicated in GPE, SP 1K, and IB-RS-2 D10 cells of guinea pig, dolphin, and pig origin, respectively. Studies in IB-RS-2 D10 cells were discontinued because cultures were contaminated with classical swine fever virus. A growth curve showed that HAV grew poorly in GPE cells and intermediately in SP 1K cells compared with growth in FRhK-4 cells. Therefore, the cell surface receptor(s) and other host factor(s) required for HAV replication are present in nonprimate as well as primate cells. Images PMID:8057483

  15. Non-targeted radiation effects in vertebrate cell lines

    NASA Astrophysics Data System (ADS)

    Ryan, Lorna

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

  16. Induction of experimental autoimmune uveoretinitis by T-cell lines.

    PubMed Central

    Rozenszajn, L A; Muellenberg-Coulombre, C; Gery, I; el-Saied, M; Kuwabara, T; Mochizuki, M; Lando, Z; Nussenblatt, R B

    1986-01-01

    Experimental autoimmune uveoretinitis was induced in genetically susceptible Lewis rats by passive transfer of T-lymphocyte cell lines from long-term cultures primed against soluble retinal antigen (S-Ag). A continuous T-cell line was established from non-adherent lymph node cells of S-Ag-immunized Lewis rats. The lymphoid cells were propagated in vitro by serially restimulating them with S-Ag in the presence of irradiated syngeneic spleen cells and expanding them in IL-2-containing media. The cell lines exhibited markers specific for T lymphocytes and the majority had the helper phenotype. When naïve rats were inoculated intravenously with anti S-Ag T-cell lines re-exposed to the antigen prior to injection, they developed uveoretinitis with both clinical and histological characteristics in half the time required by S-Ag to induce the disease by active immunization. The rats exhibited a delayed hypersensitivity skin reaction towards S-Ag. Images Figure 2 Figure 3 PMID:3485569

  17. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

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

  18. Butyrate-Induced Apoptosis in Prostate Cancer Cell Lines

    DTIC Science & Technology

    2001-09-01

    butyrate-induced apoptosis was independent of cell cycle phase. 14. SUBJECT TERMS 15. NUMBER OF PAGES prostate cancer, histone deacetylase inhibitors, bone...of cells plated) HDI histone deacetylase inhibitor SBHA suberoylbishydroxamate PKC protein kinase C activator SDS-PAGE SDS polyacrylamide gel...cancer cell lines 1. Summary of goals and findings Histone deacetylase inhibitors (HDI) such as butyrate and suberoylbishydroxamate (SBHA) have

  19. Consecutive Positive Feedback Loops Create a Bistable Switch that Controls Preadipocyte-to-Adipocyte Conversion

    PubMed Central

    Park, Byung Ouk; Ahrends, Robert; Teruel, Mary N.

    2016-01-01

    SUMMARY Adipogenesis, or the conversion of proliferating preadipocytes into nondividing adipocytes, is an important part of the vertebrate weight-maintenance program. It is not yet understood how and when an irreversible transition occurs into a distinct state capable of accumulating lipid. Here, we use single-cell fluorescence imaging to show that an all-or-none switch is induced before lipid accumulation occurs. Conversion begins by glucocorticoid and cAMP signals raising C/EBPβ levels above a critical threshold, triggering three consecutive positive feedback loops: from PPARγ to C/EBPα, then to C/EBPβ, and last to the insulin receptor. Experiments and modeling show that these feedbacks create a robust, irreversible transition to a terminally differentiated state by rejecting short- and low-amplitude stimuli. After the differentiation switch is triggered, insulin controls fat accumulation in a graded fashion. Altogether, our study introduces a regulatory motif that locks cells in a differentiated state by engaging a sequence of positive feedback loops. PMID:23063366

  20. Up-regulated miR-145 Expression Inhibits Porcine Preadipocytes Differentiation by Targeting IRS1

    PubMed Central

    Guo, Yunxue; Chen, Yaosheng; Zhang, Yun; Zhang, Yue; Chen, Luxi; Mo, Delin

    2012-01-01

    Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering. PMID:23197937

  1. Up-regulated miR-145 expression inhibits porcine preadipocytes differentiation by targeting IRS1.

    PubMed

    Guo, Yunxue; Chen, Yaosheng; Zhang, Yun; Zhang, Yue; Chen, Luxi; Mo, Delin

    2012-01-01

    Generally, most miRNAs that were up-regulated during differentiation promoted adipogenesis, but our research indicated that up-regulation of miR-145 in porcine preadipocytes did not promote but inhibit adipogenesis. In this study, miR-145 was significantly up-regulated during porcine dedifferentiated fat (DFAT) cells differentiation. In miR-145 overexpressed DFAT cells, adipogenesis was inhibited and triglycerides accumulation was decreased after hormone stimulation (P<0.05). Furthermore, up-regulation of miR-145 expression repressed induction of mRNA levels of adipogenic markers, such as CCAAT/enhancer-binding protein α (C/EBPα), and peroxisome proliferator-activated receptor γ2 (PPARγ2). These effects caused by miR-145 overexpression were mediated by Insulin receptor substrate 1 (IRS1) as a mechanism. These data suggested that induced miR-145 expression during differentiation could inhibit adipogenesis by targeting IRS1, and miR-145 may be novel agent for adipose tissue engineering.

  2. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations.

    PubMed

    Meeth, Katrina; Wang, Jake Xiao; Micevic, Goran; Damsky, William; Bosenberg, Marcus W

    2016-09-01

    The remarkable success of immune therapies emphasizes the need for immune-competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here, we describe the Yale University Mouse Melanoma (YUMM) lines, a comprehensive system of mouse melanoma cell lines that are syngeneic to C57BL/6, have well-defined human-relevant driver mutations, and are genomically stable. This will be a useful tool for the study of tumor immunology and genotype-specific cancer biology.

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

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

  4. Electrophysiological characterization of Nsc-34 cell line using Microelectrode Array.

    PubMed

    Sabitha, K R; Sanjay, D; Savita, B; Raju, T R; Laxmi, T R

    2016-11-15

    Neurons communicate with each other through intricate network to evolve higher brain functions. The electrical activity of the neurons plays a crucial role in shaping the connectivity. With motor neurons being vulnerable to neurodegenerative diseases, understanding the electrophysiological properties of motor neurons is the need of the hour, in order to comprehend the impairment of connectivity in these diseases. NSC-34 cell line serves as an excellent model to study the properties of motor neurons as they express Choline acetyltransferase (ChAT). Although NSC-34 cell lines have been used to study the effect of various toxicological, neurotrophic and neuroprotective agents, the electrical activity of these cells has not been elucidated. In the current study, we have characterized the electrophysiological properties of NSC-34 cell lines using Micro-Electrode Array (MEA) as a tool. Based on the spike waveform, firing frequency, auto- and cross-correlogram analysis, we demonstrate that NSC-34 cell culture has >2 distinct types of neuronal population: principal excitatory neurons, putative interneurons and unclassified neurons. The presence of interneurons in the NSC-34 culture was characterized by increased expression of GAD-67 markers. Thus, finding an understanding of the electrophysiological properties of different population of neurons in NSC-34 cell line, will have multiple applications in the treatment of neurological disorders.

  5. Active form Notch4 promotes the proliferation and differentiation of 3T3-L1 preadipocytes

    SciTech Connect

    Lai, Peng-Yeh; Tsai, Chong-Bin; Tseng, Min-Jen

    2013-01-18

    Highlights: ► Notch4IC modulates the ERK pathway and cell cycle to promote 3T3-L1 proliferation. ► Notch4IC facilitates 3T3-L1 differentiation by up-regulating proadipogenic genes. ► Notch4IC promotes proliferation during the early stage of 3T3-L1 adipogenesis. ► Notch4IC enhances differentiation during subsequent stages of 3T3-L1 adipogenesis. -- Abstract: Adipose tissue is composed of adipocytes, which differentiate from precursor cells in a process called adipogenesis. Many signal molecules are involved in the transcriptional control of adipogenesis, including the Notch pathway. Previous adipogenic studies of Notch have focused on Notch1 and HES1; however, the role of other Notch receptors in adipogenesis remains unclear. Q-RT-PCR analyses showed that the augmentation of Notch4 expression during the differentiation of 3T3-L1 preadipocytes was comparable to that of Notch1. To elucidate the role of Notch4 in adipogenesis, the human active form Notch4 (N4IC) was transiently transfected into 3T3-L1 cells. The expression of HES1, Hey1, C/EBPδ and PPARγ was up-regulated, and the expression of Pref-1, an adipogenic inhibitor, was down-regulated. To further characterize the effect of N4IC in adipogenesis, stable cells expressing human N4IC were established. The expression of N4IC promoted proliferation and enhanced differentiation of 3T3-L1 cells compared with those of control cells. These data suggest that N4IC promoted proliferation through modulating the ERK pathway and the cell cycle during the early stage of 3T3-L1 adipogenesis and facilitated differentiation through up-regulating adipogenic genes such as C/EBPα, PPARγ, aP2, LPL and HSL during the middle and late stages of 3T3-L1 adipogenesis.

  6. Caffeine augments Alprazolam induced cytotoxicity in human cell lines.

    PubMed

    Saha, Biswarup; Mukherjee, Ananda; Samanta, Saheli; Saha, Piyali; Ghosh, Anup Kumar; Santra, Chitta Ranjan; Karmakar, Parimal

    2009-09-01

    Combined effects of alprazolam (Alp), a member of benzodiazepine group of drugs and caffeine on human cell lines, HeLa and THP1 were investigated in this study. Alp mediated cytotoxicity was enhanced while caffeine was present. The cell death was confirmed by observing morphological changes, LDH assay and membrane anisotropic study. Also such combined effects induced elevated level of ROS and depletion of GSH. The mechanism of cell death induced by simultaneous treatment of Alp and caffeine was associated with the calcium-mediated activation of mu-calpain, release of lysosomal protease cathepsin B, activation of PARP and cleavage of caspase 3. Our results indicate that, Alp alone induces apoptosis in human cells but in the presence of caffeine it augments necrosis in a well-regulated pathway. Thus our observations strongly suggest that, alprazolam and caffeine together produce severe cytotoxicity in human cell lines.

  7. A new cell line from a human chondrosarcoma.

    PubMed Central

    de Man, J. C.; Snoep, M. P.; Huiskens-van der Meij, J. W.; Warnaar, S. O.; Schaberg, A.

    1977-01-01

    Morphological and growth characteristics are described of a rapidly growing cell line with epithelioid and giant-cell characteristics derived from a chondrosarcoma in a male patient 65 years of age. This cell line is of considerable interest because in these cells cross-reacting antigens with known animal oncorna-viruses are present. Biochemically, the cells contain particles with a density of 1-16 with "cores" of density 1'23 associated with a reverse-transcriptase-like enzyme and with 70S RNA. Occasionally, virus-like particles were demonstrated by electron microscope in material derived from the culture medium. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 8 Fig. 9 PMID:857824

  8. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines

    PubMed Central

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-01-01

    Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells. PMID:27916824

  9. Graphene Oxide Nanoribbons Induce Autophagic Vacuoles in Neuroblastoma Cell Lines.

    PubMed

    Mari, Emanuela; Mardente, Stefania; Morgante, Emanuela; Tafani, Marco; Lococo, Emanuela; Fico, Flavia; Valentini, Federica; Zicari, Alessandra

    2016-11-29

    Since graphene nanoparticles are attracting increasing interest in relation to medical applications, it is important to understand their potential effects on humans. In the present study, we prepared graphene oxide (GO) nanoribbons by oxidative unzipping of single-wall carbon nanotubes (SWCNTs) and analyzed their toxicity in two human neuroblastoma cell lines. Neuroblastoma is the most common solid neoplasia in children. The hallmark of these tumors is the high number of different clinical variables, ranging from highly metastatic, rapid progression and resistance to therapy to spontaneous regression or change into benign ganglioneuromas. Patients with neuroblastoma are grouped into different risk groups that are characterized by different prognosis and different clinical behavior. Relapse and mortality in high risk patients is very high in spite of new advances in chemotherapy. Cell lines, obtained from neuroblastomas have different genotypic and phenotypic features. The cell lines SK-N-BE(2) and SH-SY5Y have different genetic mutations and tumorigenicity. Cells were exposed to low doses of GO for different times in order to investigate whether GO was a good vehicle for biological molecules delivering individualized therapy. Cytotoxicity in both cell lines was studied by measuring cellular oxidative stress (ROS), mitochondria membrane potential, expression of lysosomial proteins and cell growth. GO uptake and cytoplasmic distribution of particles were studied by Transmission Electron Microscopy (TEM) for up to 72 h. The results show that GO at low concentrations increased ROS production and induced autophagy in both neuroblastoma cell lines within a few hours of exposure, events that, however, are not followed by growth arrest or death. For this reason, we suggest that the GO nanoparticle can be used for therapeutic delivery to the brain tissue with minimal effects on healthy cells.

  10. Generation and Characterization of JCV Permissive Hybrid Cell Lines

    PubMed Central

    Sariyer, Ilker K.; Safak, Mahmut; Gordon, Jennifer; Khalili, Kamel

    2009-01-01

    JC virus (JCV) is a human neurotropic polyomavirus whose replication in the central nervous system induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV particles have been detected primarily in oligodendrocytes and astrocytes of the brains of patients with PML and in the laboratory its propagation is limited to primary cultures of human fetal glial cells. In this short communication, the development of a new cell culture system is described through the fusion of primary human fetal astrocytes with the human glioblastoma cell line, U-87MG. The new hybrid cell line obtained from this fusion has the capacity to support efficiently expression of JCV and replication of viral DNA in vitro up to 16 passages. This cell line can serve as a reliable culture system to study the biology of JCV host cell interaction, determine the mechanisms involved in cell type specific replication of JCV, and provide a convenient cell culture system for high throughput screening of anti-viral agents. PMID:19442856

  11. Effect of ambrex (a herbal formulation) on oxidative stress in hyperlipidemic rats and differentiation of 3T3-L1 preadipocytes

    PubMed Central

    Devi, A. Jamuna; Ravindran, Rekha; Sankar, M.; Rajkumar, Johanna

    2014-01-01

    Background: Ambrex is a polyherbal formulation which consists of Withania somnifera, Orchis mascula, Cycas circirnalis, Shorea robusta with amber. Objective: The present study was designed to explore the potential effects of ambrex on the antioxidant status in high fat diet fed rats and to investigate the possible mechanisms focusing on the gene expression involved in adipogenesis and inflammation in 3T3-L1 cell line. Materials and Methods: Male Wistar rats were divided into four groups (n = 6); Group A received normal diet, Group B received high fat diet for 30 days, Group C and D received high fat diet for 30 days and treated with ambrex (40 mg/kg b.w) and atorvastatin (10 mg/kg b.w) for successive 15 days respectively. This study also assesses the effect of ambrex on adipogenesis in 3T3-L1 adipocytes. Results: The serum total cholesterol and triglycerides were significantly decreased in ambrex treated hyperlipidemic animals when compared to untreated animals. The activities of catalase, superoxide dismutase and reduced glutathione were significantly augmented in the serum, liver, and heart of hyperlipidemic rats treated with ambrex when compared to control. Ambrex treated rats had significant reductions in malondiadehyde levels in the serum, liver and heart compared to untreated rats. In addition, we observed that treatment with ambrex resulted in a major inhibition of pre-adipocyte differentiation of 3T3-L1 cells in vitro by suppression of peroxisome proliferator activated receptor gamma, sterol regulatory binding proteins, tumor necrosis factor-α, inducible nitricoxide synthase, leptin, and upregulation of thioredoxin 1 (TRX1) and TRX2 mRNA expression. Conclusion: Therefore, ambrex may be a potential drug for treatment of hyperlipidemia and related disorders. PMID:24914283

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

    PubMed

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

    2009-12-01

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

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

    PubMed

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

    2016-06-01

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

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

    SciTech Connect

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

    1995-12-01

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

  15. Generation of cell lines for monoclonal antibody production.

    PubMed

    Alvin, Krista; Ye, Jianxin

    2014-01-01

    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.

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

    PubMed

    Ishiuchi, Takashi; Torres-Padilla, Maria-Elena

    2014-07-01

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

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

    PubMed Central

    Krampe, Britta

    2010-01-01

    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. PMID:20502964

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

    PubMed

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

    1990-03-05

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

  19. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells

    SciTech Connect

    Lai, Jing; Liu, Gexiu; Yan, Guoyao; He, Dongmei; Zhou, Ying; Chen, Shengting

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells – a murine acute myelomonocytic leukemia cell line – we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPARγ, C/EBPα, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

  20. Increased EGF receptors on human squamous carcinoma cell lines.

    PubMed Central

    Cowley, G. P.; Smith, J. A.; Gusterson, B. A.

    1986-01-01

    Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours. PMID:2420349

  1. Sphingosine-1-phosphate inhibits the adipogenic differentiation of 3T3-L1 preadipocytes.

    PubMed

    Moon, Myung-Hee; Jeong, Jae-Kyo; Lee, You-Jin; Seol, Jae-Won; Park, Sang-Youel

    2014-10-01

    Sphingosine-1-phosphate (S1P) is a pluripotent lipid mediator that transmits signals through G-protein-coupled receptors to control diverse biological processes. The novel biological activity of S1P in the adipogenesis of 3T3-L1 preadipocytes was identified in the present study. S1P significantly decreased lipid accumulation in maturing preadipocytes in a dose‑dependent manner. In order to understand the anti‑adipogenic effects of S1P, preadipocytes were treated with S1P, and the change in the expression of several adipogenic transcription factors and enzymes was investigated using quantitative RT-PCR. S1P downregulated the transcriptional levels of the peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding proteins and adiponectin, which are markers of adipogenic differentiation. The effects of S1P on the levels of mitogen‑activated protein kinase (MAPK) signals in preadipocytes were also investigated. The activation of JNK and p38 were downregulated by S1P treatment in human preadipocytes. In conclusion, the results of this study suggest that S1P alters fat mass by directly affecting adipogenesis. This is mediated by the downregulation of adipogenic transcription factors and by inactivation of the JNK and p38 MAPK pathways. Thus, selective targeting of the S1P receptors and sphingosine kinases may have clinical applications for the treatment of obesity.

  2. Transthyretin expression in medulloblastomas and medulloblastoma cell lines.

    PubMed

    Albrecht, S; Bayer, T A; Kraus, J A; Pietsch, T

    1995-10-01

    Transthyretin is a protein crucial to the transport of lipophilic molecules such as thyroid hormones and retinoids. In the central nervous system, large amounts of transthyretin are synthesized by the choroid plexus and are secreted into the cerebrospinal fluid. The choroid plexus is the only site of transthyretin synthesis in the brain. Transthyretin is expressed by most benign and malignant choroid plexus tumours while gliomas and meningiomas do not express transthyretin. Other major sites of transthyretin synthesis are the retinal pigment epithelium and hepatocytes. Medulloblastoma is the prototypical primitive neuroectodermal tumour of the cerebellum and can show multiple lines of differentiation, including the expression of retinal markers. In this study, we examined transthyretin expression both at the RNA and protein level in four medulloblastomas and six medulloblastoma cell lines using Northern and Western blot analysis, reverse transcription polymerase chain reaction (PCR), RNA in situ hybridization, and immunohistochemistry. All four medulloblastomas and five of the six medulloblastoma cell lines expressed transthyretin-mRNA as demonstrated by reverse PCR and in situ hybridization while three medulloblastomas and one cell line were positive on Northern blot. The medulloblastoma with the most abundant RNA expression was transthyretin-immunoreactive on cryosections and the medulloblastoma cell line that was positive on Northern blot also expressed transthyretin at levels detectable by Western blot. No transthyretin-immunoreactivity was seen in 16 additional medulloblastomas studied on paraffin sections. These findings indicate that low-level expression of transthyretin-mRNA is common in medulloblastomas and medulloblastoma cell lines. Expression of transthyretin protein occurs rarely but can reach significant levels. Transthyretin expression in medulloblastoma is consistent with retinal pigment epithelium differentiation in medulloblastomas and reflects

  3. Maslinic Acid Inhibits Proliferation of Renal Cell Carcinoma Cell Lines and Suppresses Angiogenesis of Endothelial Cells

    PubMed Central

    Thakor, Parth; Song, Wenzhe; Subramanian, Ramalingam B.; Thakkar, Vasudev R.; Vesey, David A.

    2017-01-01

    Despite the introduction of many novel therapeutics in clinical practice, metastatic renal cell carcinoma (RCC) remains a treatment-resistant cancer. As red and processed meat are considered risk factors for RCC, and a vegetable-rich diet is thought to reduce this risk, research into plant-based therapeutics may provide valuable complementary or alternative therapeutics for the management of RCC. Herein, we present the antiproliferative and antiangiogenic effects of maslinic acid, which occurs naturally in edible plants, particularly in olive fruits, and also in a variety of medicinal plants. Human RCC cell lines (ACHN, Caki-1, and SN12K1), endothelial cells (human umbilical vein endothelial cell line [HUVEC]), and primary cultures of kidney proximal tubular epithelial cells (PTEC) were treated with maslinic acid. Maslinic acid was relatively less toxic to PTEC when compared with RCC under similar experimental conditions. In RCC cell lines, maslinic acid induced a significant reduction in proliferation, proliferating cell nuclear antigen, and colony formation. In HUVEC, maslinic acid induced a significant reduction in capillary tube formation in vitro and vascular endothelial growth factor. This study provides a rationale for incorporating a maslinic acid–rich diet either to reduce the risk of developing kidney cancer or as an adjunct to existing antiangiogenic therapy to improve efficacy.

  4. Engineering Retina from Human Retinal Progenitors (Cell Lines)

    PubMed Central

    Cao, Yang

    2009-01-01

    Retinal degeneration resulting in the loss of photoreceptors is the leading cause of blindness. Several therapeutic protocols are under consideration for treatment of this disease. Tissue replacement is one such strategy currently being explored. However, availability of tissues for transplant poses a major obstacle. Another strategy with great potential is the use of adult stem cells, which could be expanded in culture and then utilized to engineer retinal tissue. In this study, we have explored a spontaneously immortalized human retinal progenitor cell line for its potential in retinal engineering using rotary cultures to generate three-dimensional (3D) structures. Retinal progenitors cultured alone or cocultured with retinal pigment epithelial cells form aggregates. The aggregate size increases between days 1 and 10. The cells grown as a 3D culture rotary system, which promotes cell–cell interaction, retain a spectrum of differentiation capability. Photoreceptor differentiation in these cultures is confirmed by significant upregulation of rhodopsin and AaNat, an enzyme implicated in melatonin synthesis (immunohistochemistry and Western blot analysis). Photoreceptor induction and differentiation is further attested to by the upregulation of rod transcription factor Nrl, Nr2e3, expression of interstitial retinal binding protein, and rhodopsin kinase by reverse transcription–polymerase chain reaction. Differentiation toward other cell lineages is confirmed by the expression of tyrosine hydroxylase in amacrine cells, thy 1.1 expression in ganglion cells and calbindin, and GNB3 expression in cone cells. The capability of retinal progenitors to give rise to several retinal cell types when grown as aggregated cells in rotary culture offers hope that progenitor stem cells under appropriate culture conditions will be valuable to engineer retinal constructs, which could be further tested for their transplant potential. The fidelity with which this multipotential cell

  5. The silver lining of induced pluripotent stem cell variation

    PubMed Central

    Jain, Tanya; Sevilla, Ana

    2016-01-01

    Induced pluripotent stem cells (iPSCs) are being generated using various reprogramming methods and from different cell sources. Hence, a lot of effort has been devoted to evaluating the differences among iPSC lines, in particular with respect to their differentiation capacity. While line-to-line variability should mainly reflect the genetic diversity within the human population, here we review some studies that have brought attention to additional variation caused by genomic and epigenomic alterations. We discuss strategies to evaluate aberrant changes and to minimize technical and culture-induced noise, in order to generate safe cells for clinical applications. We focus on the findings from a recent study, which compared the differentiation capacity of several iPSC lines committed to the hematopoietic lineage and correlated the differential maturation capacity with aberrant DNA methylations. Although iPSC variation represents a challenge for the field, we embrace the authors’ perspective that iPSC variations should be used to our advantage for predicting and selecting the best performing iPSC lines, depending on the desired application. PMID:28066788

  6. Peroxisome proliferator-activated receptor alpha (PPARalpha) activators, bezafibrate and Wy-14,643, increase uncoupling protein-3 mRNA levels without modifying the mitochondrial membrane potential in primary culture of rat preadipocytes.

    PubMed

    Cabrero, A; Alegret, M; Sánchez, R; Adzet, T; Laguna, J C; Vázquez, M

    2000-08-15

    Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which act as pores for H(+) ions, dissipating the electrochemical gradient that develops during mitochondrial respiration at the expense of ATP synthesis. We have studied the effects of two fibrates, bezafibrate and Wy-14,643, on UCP-3 and UCP-2 mRNA levels in primary monolayer cultures of rat adipocytes and undifferentiated preadipocytes. Treatment with both PPARalpha activators for 24 h up-regulated UCP-3 mRNA levels. Thus, bezafibrate treatment resulted in an 8-fold induction in UCP-3 mRNA levels in preadipocytes compared with the 3.5-fold induction observed in adipocytes. Differences in the induction of UCP-3 between these cells correlated well with the higher expression of PPARalpha and RXRalpha mRNA values in preadipocytes compared to adipocytes. Wy-14,643 caused similar effects on UCP-3 mRNA expression. In contrast to UCP-3, UCP-2 mRNA levels were only slightly modified by bezafibrate in adipocytes. The induction in UCP-3 expression was not accompanied by changes in the mitochondrial membrane potential of rat primary preadipocytes after bezafibrate or Wy-14,643 treatment. Since it has been proposed that UCP-3 could be involved in the regulation of the use of fatty acids as fuel substrates, the UCP-3 induction achieved after bezafibrate and Wy-14, 643 treatment may indicate a higher oxidation of fatty acids, limiting their availability to be stored as triglycerides. This change may result in a reduced rate of conversion of preadipocytes to adipocytes, which directly affects fat depots.

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

    Cancer.gov

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

  8. Volatile metabolomic signature of human breast cancer cell lines

    PubMed Central

    Silva, Catarina L.; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S.

    2017-01-01

    Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC–MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways. PMID:28256598

  9. Volatile metabolomic signature of human breast cancer cell lines.

    PubMed

    Silva, Catarina L; Perestrelo, Rosa; Silva, Pedro; Tomás, Helena; Câmara, José S

    2017-03-03

    Breast cancer (BC) remains the most prevalent oncologic pathology in women, causing huge psychological, economic and social impacts on our society. Currently, the available diagnostic tools have limited sensitivity and specificity. Metabolome analysis has emerged as a powerful tool for obtaining information about the biological processes that occur in organisms, and is a useful platform for discovering new biomarkers or make disease diagnosis using different biofluids. Volatile organic compounds (VOCs) from the headspace of cultured BC cells and normal human mammary epithelial cells, were collected by headspace solid-phase microextraction (HS-SPME) and analyzed by gas chromatography combined with mass spectrometry (GC-MS), thus defining a volatile metabolomic signature. 2-Pentanone, 2-heptanone, 3-methyl-3-buten-1-ol, ethyl acetate, ethyl propanoate and 2-methyl butanoate were detected only in cultured BC cell lines. Multivariate statistical methods were used to verify the volatomic differences between BC cell lines and normal cells in order to find a set of specific VOCs that could be associated with BC, providing comprehensive insight into VOCs as potential cancer biomarkers. The establishment of the volatile fingerprint of BC cell lines presents a powerful approach to find endogenous VOCs that could be used to improve the BC diagnostic tools and explore the associated metabolomic pathways.

  10. MicroRNA-125b-5p inhibits proliferation and promotes adipogenic differentiation in 3T3-L1 preadipocytes.

    PubMed

    Ouyang, Dan; Ye, Yaqiong; Guo, Dongguang; Yu, Xiaofang; Chen, Jian; Qi, Junjie; Tan, Xiaotong; Zhang, Yuan; Ma, Yongjiang; Li, Yugu

    2015-05-01

    Previous evidence has indicated that the microRNA-125b (miR-125b) family plays important roles in the regulation of cancer cell growth, development, differentiation, and apoptosis. However, whether they contribute to the process of adipocyte differentiation remains unclear. In the present study, we revealed that the expression level of miR-125b-5p, a member of miR-125b family, was dramatically up-regulated during differentiation of 3T3-L1 preadipocyte into mature adipocyte. Supplement of miR-125b-5p into 3T3-L1 cells promoted adipogenic differentiation as evidenced by increased lipid droplets and mRNA levels of adipocyte-specific molecular markers, including peroxisome proliferators-activated receptor γ, CCAAT/enhancer-binding protein α, fatty acid-binding protein 4, and lipoprotein lipase, and by triglyceride accumulation. CCK-8 assay showed that miR-125b-5p supplementation significantly inhibited cell proliferation. Flow cytometry analysis showed that miR-125b-5p impaired G1/S phase transition as well as the mRNA and protein expression of G1/S-related genes, such as Cyclin D2, Cyclin D3, and CDK4. Nevertheless, it had no effect on apoptosis. Additionally, by target gene prediction, we demonstrated that smad4 may be a potential target of miR-125b-5p in mouse 3T3-L1 preadipocytes, accounting for some of miR-125b-5p's functions. Taken together, these data indicated that miR-125b-5p may serve as an important positive regulator in adipocyte differentiation, at least partially through down-regulating smad4.

  11. Antiproliferative Properties of Clausine-B against Cancer Cell Lines

    PubMed Central

    Wan Mohd Zain, Wan Nor I’zzah; Rahmat, Asmah; Othman, Fauziah; Yap, Taufiq Yun Hin

    2009-01-01

    Background: Clausine B, a carbazole alkaloid isolated from the stem bark of Clausena excavata, was investigated for its antiproliferative activities against five human cancer cell lines: HepG2 (hepatic cancer), MCF-7 (hormone-dependent breast cancer), MDA-MB-231 (non-hormone-dependent breast cancer), HeLa (cervical cancer), and CAOV3 (ovarian cancer). Methods: Chang liver (normal cells) was used as a control. The effect of clausine-B was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Results: Clausine-B was found to be active (IC50<30 μg/mL) against four of the cancer cell lines tested. The IC50 values for these four lines were: 21.50 μg/mL (MDA-MB-231), 22.90 g/ml (HeLa), 27.00 μg/mL (CAOV3) and 28.94 μg/mL (HepG2). Clausine-B inhibited the MCF-7 cancer cell line at 52.90 μg/mL, and no IC50 value was obtained against Chang liver. Conclusion: It is possible that the phenolic group in clausine-B responsible for the antiproliferative activities found in this study. PMID:22589662

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

    PubMed Central

    Zhang, Wei; Dolan, M. Eileen

    2009-01-01

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

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

    EPA Science Inventory

    Diversity of arsenic metabolism in cultured human cancer cell lines.

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

  14. METHYLATION OF ARSENITE BY SOME MAMMALIAN CELL LINES

    EPA Science Inventory

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

    Methylation of Arsenite by Some Mammalian Cell Lines.

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

  15. Curbing rampant cross-contamination and misidentification of cell lines.

    PubMed

    Nardone, Roland M

    2008-09-01

    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.

  16. 76 FR 16609 - Proposed Information Collection; Comment Request; Identification of Human Cell Lines Project

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-03-24

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

  17. Biological characteristics of side population cells in a self-established human ovarian cancer cell line

    PubMed Central

    WEI, ZHENTONG; LV, SHUANG; WANG, YISHU; SUN, MEIYU; CHI, GUANGFAN; GUO, JUN; SONG, PEIYE; FU, XIAOYU; ZHANG, SONGLING; LI, YULIN

    2016-01-01

    The aim of the present study was to establish an ovarian cancer (OC) cell line from ascites of an ovarian serous cystadenocarcinoma patient and investigate the biological characteristics of its side population (SP) cells. The OC cell line was established by isolating, purifying and subculturing primary cells from ascites of an ovarian serous cystadenocarcinoma patient (stage IIIc; grade 3). SP and non-SP (NSP) cells were isolated by fluorescence-activated cell sorting and cultured in serum-free medium and soft agar to compare the tumorsphere and colony formation capacities. Furthermore, SP and NSP cell tumorigenesis was examined by subcutaneous and intraperitoneal injection of the cells to non-obese diabetic/severe combined immune deficiency (NOD/SCID) mice. Drug resistance to cisplatin was examined by cell counting kit-8. The OC cell line was successfully established from ascites of an ovarian serous cystadenocarcinoma patient, which exhibited properties similar to primary tumors subsequent to >50 passages and >2 years of culture. The SP cell ratio was 0.38% in the OC cell line, and a similar SP cell ratio (0.39%) was observed when sorted SP cells were cultured for 3 weeks. Compared with NSP cells, SP cells exhibited increased abilities in differentiation and tumorsphere and colony formation, in addition to the formation of xenografted tumors and ascites and metastasis of the tumors in NOD/SCID mice, even at low cell numbers (3.0×103 cells). The xenografted tumors demonstrated histological features similar to primary tumors and expressed the ovarian serous cystadenocarcinoma marker CA125. In addition, SP cells demonstrated a significantly stronger drug resistance to cisplatin compared with NSP and unsorted cells, while treatment with verapamil, an inhibitor of ATP-binding cassette transporters, potently abrogated SP cell drug resistance. In conclusion, the present study verified SP cells from an established OC cell line and characterized the cells with self

  18. Establishment a CHO Cell Line Expressing Human CD52 Molecule

    PubMed Central

    Kadijeh, Tati; Mahsa, Yazdanpanah-Samani; Amin, Ramezani; Elham, Mahmoudi Maymand; Abbas, Ghaderi

    2016-01-01

    Background: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details Methods: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. Results: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. Conclusion: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids PMID:28070536

  19. The YUMM lines: a series of congenic mouse melanoma cell lines with defined genetic alterations

    PubMed Central

    Meeth, Katrina; Wang, Jake; Micevic, Goran; Damsky, William; Bosenberg, Marcus W.

    2017-01-01

    Summary The remarkable success of immune therapies emphasizes the need for immune competent cancer models. Elegant genetically engineered mouse models of a variety of cancers have been established, but their effective use is limited by cost and difficulties in rapidly generating experimental data. Some mouse cancer cell lines are transplantable to immunocompetent host mice and have been utilized extensively to study cancer immunology. Here we describe a comprehensive system of mouse melanoma cell lines that are syngeneic to C57Bl/6J, have well-defined human-relevant driver mutations, and are genomically stable. These will be a useful tool for the study of tumor immunology and genotype-specific cancer biology. PMID:27287723

  20. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing

    PubMed Central

    Quentmeier, Hilmar; Drexler, Hans G.; Hauer, Vivien; MacLeod, Roderick A. F.; Pommerenke, Claudia; Uphoff, Cord C.; Zaborski, Margarete; Berglund, Mattias

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members. PMID:27907212

  1. Diffuse Large B Cell Lymphoma Cell Line U-2946: Model for MCL1 Inhibitor Testing.

    PubMed

    Quentmeier, Hilmar; Drexler, Hans G; Hauer, Vivien; MacLeod, Roderick A F; Pommerenke, Claudia; Uphoff, Cord C; Zaborski, Margarete; Berglund, Mattias; Enblad, Gunilla; Amini, Rose-Marie

    2016-01-01

    Diffuse large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin lymphoma worldwide. We describe the establishment and molecular characteristics of the DLBCL cell line U-2946. This cell line was derived from a 52-year-old male with DLBCL. U-2946 cells carried the chromosomal translocation t(8;14) and strongly expressed MYC, but not the mature B-cell lymphoma associated oncogenes BCL2 and BCL6. Instead, U-2946 cells expressed the antiapoptotic BCL2 family member MCL1 which was highly amplified genomically (14n). MCL1 amplification is recurrent in DLBCL, especially in the activated B cell (ABC) variant. Results of microarray expression cluster analysis placed U-2946 together with ABC-, but apart from germinal center (GC)-type DLBCL cell lines. The 1q21.3 region including MCL1 was focally coamplified with a short region of 17p11.2 (also present at 14n). The MCL1 inhibitor A-1210477 triggered apoptosis in U-2946 (MCL1pos/BCL2neg) cells. In contrast to BCL2pos DLBCL cell lines, U-2946 did not respond to the BCL2 inhibitor ABT-263. In conclusion, the novel characteristics of cell line U-2946 renders it a unique model system to test the function of small molecule inhibitors, especially when constructing a panel of DLBCL cell lines expressing broad combinations of antiapoptotic BCL2-family members.

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

    PubMed

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

    2013-10-01

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

  3. 9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

    SciTech Connect

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

    1992-06-01

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

  4. Cranberries (Oxycoccus quadripetalus) inhibit adipogenesis and lipogenesis in 3T3-L1 cells.

    PubMed

    Kowalska, Katarzyna; Olejnik, Anna; Rychlik, Joanna; Grajek, Włodzimierz

    2014-04-01

    Cranberries (Oxycoccus quadripetalus) are a valuable source of bioactive substances with high antioxidant potential and well documented beneficial health properties. In the present study, the activity of cranberries, in terms of the inhibiting effects of adipogenesis, was investigated using the 3T3-L1 cell line. The obtained results showed that cranberries reduced proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with cranberries decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that cranberries directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPARγ, C/EBPα and SREBP1. These findings indicate that cranberries are capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation.

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

    PubMed Central

    2014-01-01

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

  6. [Sorting of side population cells from multiple myeloma cell lines and analysis of their biological characteristics].

    PubMed

    Zhang, Xiao-Li; Zhang, Li-Na; Huang, Hong-Ming; Ding, Run-Sheng; Shi, Wei; Xu, Rui-Rong; Yu, Xiao-Tang; Jiang, Sheng-Hua

    2014-06-01

    This study was aimed to sort the side population (SP) cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescence-activated cell sorting (FACS) technology was used to isolate SP cells and main population (MP) cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells (7.10 ± 2.69)%, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells (P < 0.05). The expression levels of CD138 in SP and MP cells were not significantly different (P > 0.05). RT-PCR results showed that SP cells expressed the drug-resistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells (P < 0.05), however, the difference was not significant (P > 0.05) at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.

  7. Study of lactoferrin gene expression in human and mouse adipose tissue, human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers.

    PubMed

    Moreno-Navarrete, José María; Serrano, Marta; Sabater, Mònica; Ortega, Francisco; Serino, Matteo; Pueyo, Neus; Luche, Elodie; Waget, Aurelie; Rodriguez-Hermosa, José Ignacio; Ricart, Wifredo; Burcelin, Remy; Fernández-Real, José Manuel

    2013-07-01

    Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.

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

    DOEpatents

    Bradbury, Andrew M.

    2012-07-24

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

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

    PubMed Central

    Dalton, Annamarie C; Barton, William A

    2014-01-01

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

  10. Androglobin knockdown inhibits growth of glioma cell lines

    PubMed Central

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

    2014-01-01

    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. PMID:24966926

  11. Fucose-targeted glycoengineering of pharmaceutical cell lines.

    PubMed

    Ogorek, Christiane; Jordan, Ingo; Sandig, Volker; von Horsten, Hans Henning

    2012-01-01

    Glycosylation is known to have an impact on pharmacokinetics and pharmacodynamics of therapeutic proteins. While the production of pharmaceutically desirable glycosylation forms of a therapeutic protein can in certain cases be influenced by the upstream process parameters, certain specialized glycan structures can only be produced in large quantities from cell lines that have been genetically engineered.One particular case where a specialized glycostructure has a major impact on pharmacodynamic mode of action is the enhanced ADCC-effector function of afucosylated IgG1-type monoclonal antibodies. Here we describe the methodological details of a powerful yet simple glycoengineering approach targeted at the fucosylation machinery within eukaryotic cells. As an example we demonstrate the modification of the permanent avian cell line AGE1.CR.pIX which is characterized by a unique glycosylation machinery.

  12. Isolation, Characterization, and Establishment of Spontaneously Immortalized Cell Line HRPE-2S With Stem Cell Properties.

    PubMed

    Shams Najafabadi, Hoda; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Ranaei Pirmardan, Ehsan; Masoumi, Maryam

    2016-12-12

    The retinal pigment epithelium is a monolayer of highly specialized pigmented cells located between the neural retina and the Bruch's membrane of the choroid. RPE cells play a crucial role in the maintenance and function of the underlying photoreceptors. This study introduces a spontaneously arising human retinal pigment epithelial cell line, HRPE-2S, which was isolated from primary RPE cell culture of 2 days old male donor. We characterized morphology and functional properties of the new cell line. The immortalized cell line was maintained in culture for more than 70 passages and 240 divisions. The average doubling time of the cells was approximately 22 h and got freezed at 26th passage. The cell line expressed RPE-specific markers RPE65 and cell junction protein ZO1 as an epithelial cell marker. It also expressed CHX10, PAX6, Nestin, SOX2 as stem and retinal progenitor cell markers. Ki67 as a marker of cell proliferation was expressed in all HRPE-2S cells. It represented typical epithelial cobblestone morphology and did not phenotypically change through several passages. Stem cell-like aggregations (neurospheres) were observed in SEM microscopy. The cells represented high mitotic index. They could be viable under hypoxic conditions and serum deprivation. According to functional studies, the cell line exhibited stem cell-like behaviors with particular emphasis on its self-renewal capacity. LDH isoenzymes expression pattern confirmed the same cellular source for both of the HRPE-2S cells and primary RPE cells. Characteristics of HRPE-2S cells promise it as an in vitro model for RPE stem cell-based researches. J. Cell. Physiol. 9999: 1-15, 2016. © 2016 Wiley Periodicals, Inc.

  13. Anti-adipogenic Effects and Mechanisms of Ginsenoside Rg3 in Pre-adipocytes and Obese Mice.

    PubMed

    Zhang, Longyun; Zhang, Lijuan; Wang, Xiaoyong; Si, Hongwei

    2017-01-01

    Red or black ginseng has been reported more powerful than white/fresh ginseng in dealing with various diseases/conditions including obesity. The major reason is that heating/steaming, the process of making red or black ginseng, produces large amount of bioactive compounds including ginsenoside Rg3 (Rg3), which are trace in fresh or white ginseng. In the present study, Rg3 was applied both in pre-adipocytes and obese mice to investigate the anti-adipogenic effects and relevant mechanisms. Our results show that Rg3 dose-dependently inhibited cell differentiation both in 3T3-L1 cells (30, 50, and 100 μM) and human primary pre-adipocytes (10, 20, and 30 μM). This inhibitory effect is accompanied by the attenuation of the expressions of adipogenic markers including peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer binding protein alpha (C/EBP-α), fatty acid synthase (FAS), fatty acid binding protein 4 (FABP4) and perilipin. Although dietary intake of Rg3 (0.1 mg Rg3/kg diet, 8 weeks) did not significantly affect body weight gain, fat pads and food intake as well as of PPAR-γ expression in fat tissues, we found that hepatic PPAR-γ and C/EBP-α protein expressions and hepatic glutathione reductase and glutathione S-transferase, two major antioxidants molecules were significantly reduced by Rg3. These results suggest that ginsenoside Rg3 may be a potential agent in reducing/preventing obesity.

  14. Promoting effect of triterpenoid compound from Agrimonia pilosa Ledeb on preadipocytes differentiation via up-regulation of PPARγ expression

    PubMed Central

    Guo, Tingwang; Zhu, Liancai; Tan, Jun; Zhou, Xuemei; Xiao, Ling; Liu, Xi; Wang, Bochu

    2015-01-01

    Background: Agrimonia Pilosa Ledeb (APL), a traditional Chinese medicine, has been reported a variety of biological activities, including treating T2DM. Objective: Triterpenoid compound (TC) was collected from APL. The aim of this study was to investigate the effects of TC on 3T3-L1 preadipocytes differentiation and genes related to differentiation and IR. Materials and Methods: Column chromatography was used to collect TC from ALP. 3T3-L1 cell differentiation was induced typically in the presence of various concentrations of TC or pioglitazone. Oil red O staining and measurement of intracellular TG content were performed on the seventh day of differentiation. Then quantitative polymerase chain reaction (Q-PCR) was used to test the expressions of three transcription factors (PPARγ, CCAAT enhancer binding protein-α (C/EBP-α), and sterol regulatory element-binding protein 1 (SREBP-1)) and the target genes of PPARγ including glucose transporter (GLUT4), lipoprotein lipase (LPL), fat acid binding protein (AP2), and adiponectin in 3T3-L1 cells. Results: At the concentration of 5, 25 and 125 μg/mL, TC significantly promoted triglyceride accumulation. Further study showed that TC could promote the expression of PPARγ, C/EBPα and ADD1/SREBP1 significantly at 125 μg/mL. As for downstream genes controlled by PPARγ, TC at 25 and 125 μg/mL could significantly promote the expression of GLUT4 and adiponectin. However, the expression of aP2 related to lipid metabolism and adiposity in the TC group was significantly lower than that in the pioglitazone group. Conclusion: TC could promote preadipocytes differentiation through activating PPARγ and downstream controlled genes. TC has the ideal insulin sensitization with lower adipogenic action than classical TZDs in vitro. So TC from Agrimonia Pilosa Ledeb has a good prospect as a natural drug for IR and T2DM. PMID:25709235

  15. Anti-adipogenic Effects and Mechanisms of Ginsenoside Rg3 in Pre-adipocytes and Obese Mice

    PubMed Central

    Zhang, Longyun; Zhang, Lijuan; Wang, Xiaoyong; Si, Hongwei

    2017-01-01

    Red or black ginseng has been reported more powerful than white/fresh ginseng in dealing with various diseases/conditions including obesity. The major reason is that heating/steaming, the process of making red or black ginseng, produces large amount of bioactive compounds including ginsenoside Rg3 (Rg3), which are trace in fresh or white ginseng. In the present study, Rg3 was applied both in pre-adipocytes and obese mice to investigate the anti-adipogenic effects and relevant mechanisms. Our results show that Rg3 dose-dependently inhibited cell differentiation both in 3T3-L1 cells (30, 50, and 100 μM) and human primary pre-adipocytes (10, 20, and 30 μM). This inhibitory effect is accompanied by the attenuation of the expressions of adipogenic markers including peroxisome proliferator-activated receptor gamma (PPAR-γ), CCAAT/enhancer binding protein alpha (C/EBP-α), fatty acid synthase (FAS), fatty acid binding protein 4 (FABP4) and perilipin. Although dietary intake of Rg3 (0.1 mg Rg3/kg diet, 8 weeks) did not significantly affect body weight gain, fat pads and food intake as well as of PPAR-γ expression in fat tissues, we found that hepatic PPAR-γ and C/EBP-α protein expressions and hepatic glutathione reductase and glutathione S-transferase, two major antioxidants molecules were significantly reduced by Rg3. These results suggest that ginsenoside Rg3 may be a potential agent in reducing/preventing obesity. PMID:28337143

  16. Inhibitory effect and transcriptional impact of berberine and evodiamine on human white preadipocyte differentiation.

    PubMed

    Hu, Yueshan; Fahmy, Hesham; Zjawiony, Jordan K; Davies, Gareth E

    2010-06-01

    It has been reported that the botanical alkaloids, berberine and evodiamine inhibit mouse preadipocyte 3T3-L1 differentiation. The aim of this study was to investigate the effect and transcriptional impact of berberine and evodiamine individually and in combination on human white preadipocyte (HWP) differentiation. We have shown that treatment with 8 microM berberine or 4 microM evodiamine resulted in a major inhibition of HWP differentiation accompanied by up-regulation of both GATA binding protein 2 and 3 (GATA-2 and GATA-3) mRNA and protein expression, suggesting that both compounds may have excellent potential as agents to prevent obesity.

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

    PubMed

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

    2014-01-01

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

  18. Rosiglitazone inhibits cell proliferation by inducing G1 cell cycle arrest and apoptosis in ADPKD cyst-lining epithelia cells.

    PubMed

    Liu, Yawei; Dai, Bing; Fu, Lili; Jia, Jieshuang; Mei, Changlin

    2010-06-01

    Abnormal proliferation is an important pathological feature of autosomal dominant polycystic kidney disease (ADPKD). Many drugs inhibiting cell proliferation have been proved to be effective in slowing the disease progression in ADPKD. Recent evidence has suggested that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have anti-neoplasm effects through inhibiting cell growth and inducing cell apoptosis in various cancer cells. In the present study, we examined the expression of PPARgamma in human ADPKD kidney tissues and cyst-lining epithelial cell line, and found that the expression of PPARgamma was greater in ADPKD kidney tissues and cyst-lining epithelial cell line than in normal kidney tissues and human kidney cortex (HKC) cell line. Rosiglitazone inhibited significantly proliferation of cyst-lining epithelial cells in a concentration- and time-dependent manner. These effects were diminished by GW9662, a specific PPARgamma antagonist. Cell cycle analysis showed a G0/G1 arrest in human ADPKD cyst-lining epithelial cells with rosiglitazone treatment. Analysis of cell cycle regulatory proteins revealed that rosiglitazone decreased the protein levels of proliferating cell nuclear antigen, pRb, cyclin D1, cyclin D2 and Cdk4 but increased the levels of p21 and p27 in a dose-dependent manner. Rosiglitazone also induced apoptosis in cyst-lining epithelial cells, which was correlated with increased bax expression and decreased bcl-2 expression. These results suggest PPARgamma agonist might serve as a promising drug for the treatment of ADPKD.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. A murine stromal cell line promotes the proliferation of the human factor-dependent leukemic cell line UT-7.

    PubMed

    Auffray, I; Dubart, A; Izac, B; Vainchenker, W; Coulombel, L

    1994-05-01

    In long-term human bone marrow cultures, stromal cells of human origin are usually used on the assumption that human primitive progenitor cells do not respond to cytokines produced by stromal cells from other species. There is accumulating evidence, however, that murine stromal cells also promote maintenance and differentiation of very primitive human stem cells, which suggests the existence of novel stromal activities that cross species barriers. In this study, we show that a murine bone marrow-derived stromal cell line, MS-5, allows the proliferation of the human leukemic cell line UT-7. The long-term growth of UT-7 is usually supported only by human interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo). None of these three cytokines was involved in the observed effect, since murine GM-CSF and IL-3 do not act on human cells and MS-5 cells do not produce Epo. Soluble stem cell factor (SCF) induced UT-7 cell proliferation. However, S1/S1 mutant fibroblasts also supported UT-7 cell growth and anti-c-kit antibodies only partially abolished UT-7 cell proliferative response to MS-5 cells. These observations excluded a major role of SCF in this system. MS-5-derived growth-promoting activity was diffusible, but attempts to grow UT-7 cells in high levels of known soluble murine stromal-derived cytokines active on human cells showed no or minimal response, suggesting that MS-5's proliferative effect was not mediated by known cytokines. Finally, involvement of an autocrine loop of activation induced by MS-5 was excluded: RT-PCR analysis did not detect increased transcripts for GM-CSF, IL-3, IL-6, SCF, or Epo in UT-7 cells cocultured for 2 to 6 days with MS-5. In addition, UT-7 cell proliferation on MS-5 was not inhibited by neutralizing antibodies against the human GM-CSF receptor or the human IL-6 receptor alpha chain. Whether UT-7 cell proliferation triggered by MS-5 reflects the existence of novel stromal cytokines or

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

    PubMed Central

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

    2016-01-01

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

  2. Regulation of cholesterol synthesis in four colonic adenocarcinoma cell lines.

    PubMed

    Cerda, S R; Wilkinson, J; Broitman, S A

    1995-12-01

    Colon tumor cells, unlike normal human fibroblasts, exhibited an uncoupling of low density lipoprotein (LDL)-derived cholesterol from cellular growth, when endogenous cholesterol synthesis was inhibited by mevinolin, a hydroxymethylglutaryl-CoA reductase (HMG-CoAR) competitive inhibitor [Fabricant, M., and Broitman, S.A. (1990) Cancer Res. 50, 632-636]. Further evaluation of cholesterol metabolism was conducted in two undifferentiated (SW480, SW1417) and two differentiated (HT29, CACO2) colonic adenocarcinoma (adeno-CA) cell lines and an untransformed human fibroblast, AG1519A. Cells grown in monolayer culture to near subconfluency were used to assess endogenous cholesterol synthesis by 14C-acetate incorporation, in response to the following treatments in lipoprotein-deficient serum (LPDS)-supplemented minimum essential medium (MEM): LPDS alone, LDL, mevinolin, mevinolin with LDL, and 25-hydroxy-cholesterol (25-OH-CH). Complete fetal bovine serum (FBS)-supplemented MEM was used as control. All colon tumor lines exhibited similarly high endogenous cholesterol synthesis in both FBS and LPDS relative to the fibroblasts which demonstrated low basal levels in FBS and maximal synthesis in LPDS. LDL treatment did not inhibit cholesterol synthesis in colon tumor cells, but suppressed that in the fibroblast by 70%. Sterol repression of cholesterol synthesis mediated by 25-OH-CH occurred in all cells. Mevinolin caused a reduction in cholesterol synthesis in the colonic cancer cell lines, which was not further decreased by concurrent addition of LDL. In contrast, in mevinolin-treated fibroblasts, LDL further inhibited cholesterol synthesis. When the effect of cell density on cholesterol synthesis regulation was evaluated under conditions of sparse density in SW480 and SW147, results indicated that (i) basal rates of cholesterol synthesis were higher, (ii) LDL inhibited cholesterol synthesis more effectively, and (iii) mevinolin or 25-OH-CH had a more pronounced effect than in

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

    PubMed

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

    2015-06-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. The dual role of TLR3 in metastatic cell line.

    PubMed

    Matijevic, Tanja; Pavelic, Jasminka

    2011-10-01

    Toll-like receptors (TLRs) are members of transmembrane proteins that recognize conserved molecular motifs of viral and bacterial origin and initiate innate immune response. As the role of TLRs in tumors cells is still not clear, our aim was to investigate the role of TLR3 in primary tumor and metastatic cells (SW480, SW620, FaDu and Detroit 562). We have reported here on the dual role of TLR3 in pharynx metastatic cell line (Detroit 562); on one hand TLR3 activation drove cells to apoptosis while on the other its stimulation contributed to tumor progression by altering the expression of tumor promoting genes (PLAUR, RORB) and enhancing the cell migration potential. In addition, we have shown TLR3 signaling pathway is functional in another metastatic cancer cell line (SW620) suggesting TLR3 might be important in the process of tumor metastasis. Since TLR3 agonists have been used in tumor therapy with the aim to activate immune system, scientific contribution of this work is drawing attention to the importance of further work on this topic, especially pro-tumor effect of TLR3, in order to avoid possible side-effects.

  6. Growth dynamics and cyclin expression in cutaneous T-cell lymphoma cell lines

    PubMed Central

    Biskup, Edyta; Manfé, Valentina; Kamstrup, Maria R.; Gniadecki, Robert

    2010-01-01

    We have investigated cell growth dynamics and cyclins B1 and E expression in cell lines derived from mycosis fungoides (MyLa), Sézary syndrome (SeAx), and CD30+ lympho-proliferative diseases (Mac1, Mac2a, JK). Mac1 and Mac2a had the highest growth rate (doubling time 18–28 h, >90% cycling cells) whereas SeAx was proliferating slowly (doubling time 55 h, approximately 35% cycling cells). Expression of cyclin B1 correlated positively with doubling time whereas expression of cyclin E was unscheduled and constant across the investigated cell lines. All cell lines exhibited high expression of PCNA. Thus, we concluded that cyclin B1 could be used for rapid screening of cell proliferation in malignant lymphocytes derived from cutaneous T-cell lymphoma. PMID:25386244

  7. Bisphosphonates induce apoptosis in human breast cancer cell lines

    PubMed Central

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

    2000-01-01

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

  8. Designing of promiscuous inhibitors against pancreatic cancer cell lines

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

    Pancreatic cancer remains the most devastating disease with worst prognosis. There is a pressing need to accelerate the drug discovery process to identify new effective drug candidates against pancreatic cancer. We have developed QSAR models for predicting promiscuous inhibitors using the pharmacological data. Our models achieved maximum Pearson correlation coefficient of 0.86, when evaluated on 10-fold cross-validation. Our models have also successfully validated the drug-to-oncogene relationship and further we used these models to screen FDA approved drugs and tested them in vitro. We have integrated these models in a webserver named as DiPCell, which will be useful for screening and designing novel promiscuous drug molecules. We have also identified the most and least effective drugs for pancreatic cancer cell lines. On the other side, we have identified resistant pancreatic cancer cell lines, which need investigative scanner on them to put light on resistant mechanism in pancreatic cancer.

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

    PubMed Central

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

    1979-01-01

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

  10. Efficient Genetic Method for Establishing Drosophila Cell Lines Unlocks the Potential to Create Lines of Specific Genotypes

    PubMed Central

    Truesdell, Sharon; Paul, Litty; Chen, Ting; Butchar, Jonathan P.; Justiniano, Steven

    2008-01-01

    Analysis of cells in culture has made substantial contributions to biological research. The versatility and scale of in vitro manipulation and new applications such as high-throughput gene silencing screens ensure the continued importance of cell-culture studies. In comparison to mammalian systems, Drosophila cell culture is underdeveloped, primarily because there is no general genetic method for deriving new cell lines. Here we found expression of the conserved oncogene RasV12 (a constitutively activated form of Ras) profoundly influences the development of primary cultures derived from embryos. The cultures become confluent in about three weeks and can be passaged with great success. The lines have undergone more than 90 population doublings and therefore constitute continuous cell lines. Most lines are composed of spindle-shaped cells of mesodermal type. We tested the use of the method for deriving Drosophila cell lines of a specific genotype by establishing cultures from embryos in which the warts (wts) tumor suppressor gene was targeted. We successfully created several cell lines and found that these differ from controls because they are primarily polyploid. This phenotype likely reflects the known role for the mammalian wts counterparts in the tetraploidy checkpoint. We conclude that expression of RasV12 is a powerful genetic mechanism to promote proliferation in Drosophila primary culture cells and serves as an efficient means to generate continuous cell lines of a given genotype. PMID:18670627

  11. Evidence for preadipocyte proliferation during culture of subcutaneous and intramuscular adipose tissues from Angus and Wagyu crossbred steers.

    PubMed

    May, S G; Savell, J W; Lunt, D K; Wilson, J J; Laurenz, J C; Smith, S B

    1994-12-01

    The primary objective of this study was to provide evidence for preadipocyte proliferation during culture of adipose tissue explants; a secondary objective was to compare the lipogenic activity and cellularity of adipose tissues from American Wagyu crossbred steers. Subcutaneous (s.c.) and intramuscular (i.m.) adipose tissues were obtained at slaughter from the 2nd to 6th lumbar region of the loin from Angus (n = 10) and Wagyu crossbred steers (n = 10) that had been fed for 552 d by typical Japanese production standards. Adipose tissue explants were incubated 36 h with [3H]thymidine in the absence and presence of aphidicolin (a specific inhibitor of genomic DNA replication). Adipocytes were liberated by collagenase treatment and [3H]thymidine incorporation into DNA was measured. Whereas there were no significant differences between adipose tissue depots, Wagyu s.c. and i.m. preadipocytes and stromal-vascular cells exhibited greater (P < .05) [3H]thymidine incorporation into DNA than adipocytes from Angus steers. Intramuscular adipose tissue from both breeds exhibited lower (P < .05) rates of lipogenesis from acetate both before and after long-term (36-h) incubation than s.c. adipose tissue. Furthermore, i.m. adipocytes were smaller (P < .05) than s.c. adipocytes. The activities of fatty acid synthetase and glucose-6-phosphate dehydrogenase were greater (P < .05) in Wagyu s.c. adipose tissue and less in Wagyu i.m. adipose tissue than in corresponding Angus tissues. There were no differences between breed types (P = .17) in rates of lipogenesis from acetate, either before or after explant culture.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Apoptosis induced by propolis in human hepatocellular carcinoma cell line.

    PubMed

    Choi, Y H; Lee, W Y; Nam, S Y; Choi, K C; Park, Y E

    1999-07-01

    Propolis has been reported to exhibit a wide spectrum of activities including antibiotic, antiviral, anti-inflammatory, immunostimulatory and tumor carcinostatic properties. We showed propolis induced apoptosis in a human hepatoma cell line (SNU449) by FITC-Annexin V/PI staining. We also compared the apoptosis inducing effect between Korean and Commercial (Sigma # p-1010) propolis. There was no difference on apoptosis between them.

  13. Comparing N-glycan processing in mammalian cell lines to native and engineered lepidopteran insect cell lines.

    PubMed

    Tomiya, Noboru; Narang, Someet; Lee, Yuan C; Betenbaugh, Michael J

    2004-01-01

    In the past decades, a large number of studies in mammalian cells have revealed that processing of glycoproteins is compartmentalized into several subcellular organelles that process N-glycans to generate complex-type oligosaccharides with terminal N -acetlyneuraminic acid. Recent studies also suggested that processing of N-glycans in insect cells appear to follow a similar initial pathway but diverge at subsequent processing steps. N-glycans from insect cell lines are not usually processed to terminally sialylated complex-type structures but are instead modified to paucimannosidic or oligomannose structures. These differences in processing between insect cells and mammalian cells are due to insufficient expression of multiple processing enzymes including glycosyltransferases responsible for generating complex-type structures and metabolic enzymes involved in generating appropriate sugar nucleotides. Recent genomics studies suggest that insects themselves may include many of these complex transferases and metabolic enzymes at certain developmental stages but expression is lost or limited in most lines derived for cell culture. In addition, insect cells include an N -acetylglucosaminidase that removes a terminal N -acetylglucosamine from the N-glycan. The innermost N -acetylglucosamine residue attached to asparagine residue is also modified with alpha(1,3)-linked fucose, a potential allergenic epitope, in some insect cells. In spite of these limitations in N-glycosylation, insect cells have been widely used to express various recombinant proteins with the baculovirus expression vector system, taking advantage of their safety, ease of use, and high productivity. Recently, genetic engineering techniques have been applied successfully to insect cells in order to enable them to produce glycoproteins which include complex-type N-glycans. Modifications to insect N-glycan processing include the expression of missing glycosyltransferases and inclusion of the metabolic

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

    PubMed Central

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

    2015-01-01

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

  15. Hepatitis C virus infection of cholangiocarcinoma cell lines.

    PubMed

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

    2015-06-01

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

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

    PubMed Central

    2013-01-01

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

  17. HIV-1 latency in actively dividing human T cell lines

    PubMed Central

    Jeeninga, Rienk E; Westerhout, Ellen M; van Gerven, Marja L; Berkhout, Ben

    2008-01-01

    Background Eradication of HIV-1 from an infected individual cannot be achieved by current drug regimens. Viral reservoirs established early during the infection remain unaffected by anti-retroviral therapy and are able to replenish systemic infection upon interruption of the treatment. Therapeutic targeting of viral latency will require a better understanding of the basic mechanisms underlying the establishment and long-term maintenance of HIV-1 in resting memory CD4 T cells, the most prominent reservoir of transcriptional silent provirus. However, the molecular mechanisms that permit long-term transcriptional control of proviral gene expression in these cells are still not well understood. Exploring the molecular details of viral latency will provide new insights for eventual future therapeutics that aim at viral eradication. Results We set out to develop a new in vitro HIV-1 latency model system using the doxycycline (dox)-inducible HIV-rtTA variant. Stable cell clones were generated with a silent HIV-1 provirus, which can subsequently be activated by dox-addition. Surprisingly, only a minority of the cells was able to induce viral gene expression and a spreading infection, eventhough these experiments were performed with the actively dividing SupT1 T cell line. These latent proviruses are responsive to TNFα treatment and alteration of the DNA methylation status with 5-Azacytidine or genistein, but not responsive to the regular T cell activators PMA and IL2. Follow-up experiments in several T cell lines and with wild-type HIV-1 support these findings. Conclusion We describe the development of a new in vitro model for HIV-1 latency and discuss the advantages of this system. The data suggest that HIV-1 proviral latency is not restricted to resting T cells, but rather an intrinsic property of the virus. PMID:18439275

  18. Bombesin stimulates insulin secretion by a pancreatic islet cell line.

    PubMed Central

    Swope, S L; Schonbrunn, A

    1984-01-01

    The amphibian tetradecapeptide, bombesin (BBS) has been shown to stimulate insulin secretion both in vivo and by pancreatic islet cells in vitro. To determine whether BBS can act directly on pancreatic beta cells, we examined its effects on insulin secretion by HIT-T15 cells (HIT cells), a clonal islet cell line. Addition of 100 nM BBS to HIT cells stimulated insulin release 25-fold within 30 sec. The rapid stimulatory effect of BBS on insulin release was short-lived: the secretory rate returned to basal levels after 90 min of BBS treatment. The decrease in the rate of insulin release in the continued presence of BBS was due not to depletion of intracellular insulin stores but to specific desensitization to this peptide. Stimulation of insulin secretion by BBS was dose dependent with an ED50 value (0.51 +/- 0.15 nM) similar to the concentration of BBS-like immunoreactive material in rat plasma. Five BBS analogs, including porcine gastrin-releasing peptide, were as powerful as BBS in stimulating insulin release. The relative potencies of the analogs tested indicated that the COOH-terminal octapeptide sequence in BBS was sufficient for stimulation of release. In contrast, 14 peptides structurally unrelated to BBS did not alter insulin secretion. BBS action was synergistic with that of glucagon; insulin secretion in the presence of maximal concentrations of both peptides was greater than the additive effects of the two peptides added individually. Somatostatin inhibited BBS-stimulated release by 69 +/- 1% with an ID50 value of 3.2 +/- 0.3 nM. These results show that BBS stimulation of insulin secretion by a clonal pancreatic cell line closely parallels its effects in vivo and support the hypothesis that BBS stimulates insulin secretion by a direct effect on the pancreatic beta cell. The clonal HIT cell line provides a homogeneous cell preparation amenable for studies on the biochemical mechanisms of BBS action in the endocrine pancreas. PMID:6143320

  19. Cytogenetic instability of dental pulp stem cell lines.

    PubMed

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

    2012-02-01

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

  20. [Mechanisms of gamma-inducible death of Jurkat cells line].

    PubMed

    Gamkrelidze, M M; Bezhitashvili, N D; Pavliashvili, A T; Mchedlishvili, T V; Sanikidze, T V

    2008-06-01

    Mechanisms of radio-inducible death of Jurkat cells were investigated. Human lymphoblastoid T-cell line Jurkat is widely established model for studying apoptosis mechanisms. The cell was radiated by "Teragam" (Czech Republic) by dose 2 g during 1 minute. After radiation cells were incubated at standard conditions during 24 hours. After gamma radiation in cell population amount of cells in gaplois (apoptotic G 0) stage was increased 8,2 folds, in diplois (G 0/G1) stage - by 17%, in synthetic (S) stage decreased by 35% and tetraploid (G2/M) stage by 73% in comparison to control group. It was revealed intensive production of free radicals of oxygen and nitric oxide and decreasing activity of antioxidant enzymes (superoxidismutasa, catalasa and glutathione peroxidase). Revealed dependence between intensification of apoptosis and radiation-induced arrest of cell cycle G2/M phase may be determined by excess amount of free oxygen and nitrogen radicals generated in Jurkat cells as a result of nondirect effects of low doses of gamma radiation.

  1. Establishment of lal-/- myeloid lineage cell line that resembles myeloid-derived suppressive cells.

    PubMed

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

    2015-01-01

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

  2. Sirt1 attenuates camptothecin-induced apoptosis through caspase-3 pathway in porcine preadipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adipose tissue is an important energy reservoir, and its over-development results in obesity in humans or body fat over-deposition in livestock. Loss of preadipocytes through apoptosis has been proposed as an alternative way to reduce adipose tissue mass. At present, the effect and regulatory mechan...

  3. Board Sponsored Invited Review: The biology and regulation of preadipocytes and adipocytes in meat animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The quality and value of the carcass in domestic meat animals is reflected in its protein and fat content. Preadipocytes and adipocytes are important in establishing the overall fatness of a carcass, as well as being the main contributors to the marbling component needed for consumer preference of ...

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

    PubMed

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

    2012-10-01

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

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling.

    PubMed

    Kassotis, Christopher D; Masse, Lauren; Kim, Stephanie; Schlezinger, Jennifer J; Webster, Thomas F; Stapleton, Heather M

    2017-02-08

    The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies.

  7. Characterization of Adipogenic Chemicals in Three Different Cell Culture Systems: Implications for Reproducibility Based on Cell Source and Handling

    PubMed Central

    Kassotis, Christopher D.; Masse, Lauren; Kim, Stephanie; Schlezinger, Jennifer J.; Webster, Thomas F.; Stapleton, Heather M.

    2017-01-01

    The potential for chemical exposures to exacerbate the development and/or prevalence of metabolic disorders, such as obesity, is currently of great societal concern. Various in vitro assays are available to assess adipocyte differentiation, though little work has been done to standardize protocols and compare models effectively. This study compares several adipogenic cell culture systems under a variety of conditions to assess variability in responses. Two sources of 3T3-L1 preadipocytes as well as OP9 preadipocytes were assessed for cell proliferation and triglyceride accumulation following different induction periods and using various tissue culture plates. Both cell line and cell source had a significant impact on potencies and efficacies of adipogenic chemicals. Gene expression analyses suggested that differential expression of nuclear receptors involved in adipogenesis underlie the differences between OP9 and 3T3-L1 cells; however, there were also differences based on 3T3-L1 cell source. Induction period modulated potency and efficacy of response depending on cell line and test chemical, and large variations were observed in triglyceride accumulation and cell proliferation between brands of tissue culture plates. Our results suggest that the selection of a cell system and differentiation protocol significantly impacts the detection of adipogenic chemicals, and therefore, influences reproducibility of these studies. PMID:28176856

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

    PubMed

    Kniss, Douglas A; Summerfield, Taryn L

    2014-08-01

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

  9. Cross-contamination of cell lines as revealed by DNA fingerprinting in the IFO animal cell bank.

    PubMed

    Satoh, M; Takeuchi, M

    1993-01-01

    For quality control of cell lines, the Institute for Fermentation, Osaka (IFO) animal cell bank recently introduced DNA fingerprinting analysis, which enables verification of cell lines at the individual level, to detect cross-culture contamination. By using this analysis, we found two cases of cross-contamination of cell lines.

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

    PubMed

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

    2016-08-05

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

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

    PubMed

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

    2016-08-01

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

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

    PubMed Central

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

    2014-01-01

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

  13. Transcriptional signature of accessory cells in the lateral line, using the Tnk1bp1:EGFP transgenic zebrafish line

    PubMed Central

    2012-01-01

    Background Because of the structural and molecular similarities between the two systems, the lateral line, a fish and amphibian specific sensory organ, has been widely used in zebrafish as a model to study the development/biology of neuroepithelia of the inner ear. Both organs have hair cells, which are the mechanoreceptor cells, and supporting cells providing other functions to the epithelium. In most vertebrates (excluding mammals), supporting cells comprise a pool of progenitors that replace damaged or dead hair cells. However, the lack of regenerative capacity in mammals is the single leading cause for acquired hearing disorders in humans. Results In an effort to understand the regenerative process of hair cells in fish, we characterized and cloned an egfp transgenic stable fish line that trapped tnks1bp1, a highly conserved gene that has been implicated in the maintenance of telomeres' length. We then used this Tg(tnks1bp1:EGFP) line in a FACsorting strategy combined with microarrays to identify new molecular markers for supporting cells. Conclusions We present a Tg(tnks1bp1:EGFP) stable transgenic line, which we used to establish a transcriptional profile of supporting cells in the zebrafish lateral line. Therefore we are providing a new set of markers specific for supporting cells as well as candidates for functional analysis of this important cell type. This will prove to be a valuable tool for the study of regeneration in the lateral line of zebrafish in particular and for regeneration of neuroepithelia in general. PMID:22273551

  14. Construction and characterization of deltaretrovirus indicator cell lines.

    PubMed

    Jewell, Nancy A; Mansky, Louis M

    2005-01-01

    The deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), replicate poorly in culture and the molecular details of their life cycles are limited. To facilitate the analysis of virus replication, mammalian cell lines were created with the long terminal repeats (LTRs) of each virus driving expression of the enhanced green fluorescent protein gene (egfp). The BLGFP, H1GFP and H2GFP cell lines detect virus infection by the expression of GFP via the transactivation of the LTR via the Tax protein of BLV, HTLV-1 or HTLV-2, respectively. GFP expression was measured by flow cytometry, yielding sensitive and rapid detection of virus infectivity. Interestingly, we observed that the Tax proteins of HTLV-1 and HTLV-2 could transactivate the BLV LTR at levels that were comparable to that of BLV Tax. In contrast, the BLV Tax showed low levels of transactivation in H1GFP and H2GFP cells. HTLV-1 and HTLV-2 Tax proteins efficiently transactivated both the HTLV-1 and HTLV-2 LTRs. Finally, spinoculation of BLV resulted in only a two-fold increase in viral titer.

  15. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    SciTech Connect

    Itoigawa, Yoshiaki; Kishimoto, Koshi N.; Okuno, Hiroshi; Sano, Hirotaka; Kaneko, Kazuo; Itoi, Eiji

    2010-09-03

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

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

    SciTech Connect

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

    2012-07-20

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

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

    PubMed

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

    2016-01-07

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

  18. Perfluorooctane sulfonate induces apoptosis in N9 microglial cell line.

    PubMed

    Zhang, Ling; Li, Yuan-yuan; Zeng, Huai-cai; Li, Miao; Wan, Yan-Jian; Schluesener, Hermann J; Zhang, Zhi-yuan; Xu, Shun-qing

    2011-03-01

    Perfluorooctane sulfonate (PFOS) is an environmental persistent acid found at low levels in human, wildlife, and environmental media samples. To study the apoptosis effects of PFOS on microglia, murine N9 cell line was used as a model in current research. The results showed that PFOS could reduce the cell viability significantly, and the cellular apoptosis induced by PFOS was closely accompanied with dissipation of mitochondria membrane potential, upregulation messenger RNAs (mRNAs) of p53, Bax, caspase 9, and caspase 3, and decreased expression of Bcl-2 mRNA. These results suggested that PFOS could disturb homeostasis of N9 cells, impact mitochondria, and affect gene expression of apoptotic regulators, all of which resulted in a start-up of apoptosis.

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

    SciTech Connect

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

    2012-10-19

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2012-04-01

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

  3. Adhesion of Actinobacillus actinomycetemcomitans to a human oral cell line.

    PubMed Central

    Mintz, K P; Fives-Taylor, P M

    1994-01-01

    Two quantitative, rapid assays were developed to study the adhesion of Actinobacillus actinomycetemcomitans, an oral bacterium associated with periodontal disease, to human epithelial cells. The human oral carcinoma cell line KB was grown in microtiter plates, and adherent bacteria were detected by an enzyme-linked immunosorbent assay with purified anti-A. actinomycetemcomitans serum and horseradish peroxidase-conjugated secondary antibody or [3H]thymidine-labeled bacteria. Adhesion was found to be time dependent and increased linearly with increasing numbers of bacteria added. Variation in the level of adhesion was noted among strains of A. actinomycetemcomitans. Adhesion was not significantly altered by changes in pH (from pH 5 to 9) but was sensitive to sodium chloride concentrations greater than 0.15 M. Pooled human saliva was inhibitory for adhesion when bacteria were pretreated with saliva before being added to the cells. Pretreatment of the KB cells with saliva did not inhibit adhesion. Protease treatment of A. actinomycetemcomitans reduced adhesion of the bacteria to KB cells. The data are consistent with the hypothesis that a protein(s) is required for bacterial adhesion and that host components may play a role in modulating adhesion to epithelial cells. Images PMID:8063383

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

    SciTech Connect

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

    1988-07-01

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

  5. Retinal Pigment Epithelial Cell Line Suppression of Phagolysosome Activation.

    PubMed

    Taylor, A W; Dixit, S; Yu, J

    2015-01-29

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

  6. Ferulic acid promoting apoptosis in human osteosarcoma cell lines

    PubMed Central

    Zhang, Xu-dong; Wu, Qiang; Yang, Shu-hua

    2017-01-01

    Objective: To explore the promoting apoptosis and antitumor activities of ferulic acid (FA) in human osteosarcoma and its potential mechanism. Methods: The SaOS-2 and MG63 osteosarcoma cell lines were opted to experiment and these cells were, respectively, cultured with various concentrations of FA (0 μM, 10 μM, 20 μM, 40 μM) for 72 hours at 37°C. The viabilities of the FA treated cells were monitored by MTT. Apoptosis cells were evaluated using annexin V/PI by flow cytometry. Apoptosis proteins caspase-3, procaspase-3, Bcl-2 and Bax were detected by western blot. Expressions of apoptotic genes Bcl-2 and Bax were quantified by qPCR. Results: The cell viabilities were critically declined in the concentration-dependent manner in FA groups (P < 0.01). The apoptosis cells were increased proportionately with the concentration of FA (P < 0.05). The procaspase-3 protein contents, and Bcl-2 mRNA and protein contents were significantly decreased while caspase-3 protein contents, and Bax mRNA and protein contents were concomitantly increased in the concentration-dependent manner in FA groups (P < 0.05). The response to FA by the SaOS-2 osteosarcoma cell was similar with the MG63 osteosarcoma cell (P > 0.05). Conclusion: Ferulic acid could significantly descend osteosarcoma cell viability through the promoting apoptosis pathway in which FA activates both caspase-3 and Bax and inactivates Bcl-2. PMID:28367185

  7. Hodgkin's disease cell lines: a model for interleukin-1-independent accessory cell function.

    PubMed

    McKenzie, J L; Egner, W; Calder, V L; Hart, D N

    1992-11-01

    The haemopoietic origins of the Hodgkin's disease (HD)-derived cell lines L428, KM-H2 and HDLM-2 remain controversial. Analysis of T-cell receptor (TcR) and Ig rearrangements cannot resolve this, and lineage promiscuity limits the interpretation of isolated surface antigen expression. Nonetheless the cell marker profile of L428 has similarities with human dendritic cells (DC), and L428 strongly stimulates in the mixed leucocyte reaction (MLR). We therefore undertook an extended immunophenotypic comparison of the HD lines with that recently defined for DC, prior to examining their ability to stimulate allogenic T lymphocytes, and comparing the molecular interactions involved with those of primary MLR stimulatory cells. The immunophenotype of the HD lines failed to establish either a lymphoid or monocytoid derivation. The profile of L428 appeared similar to the human DC. All three lines were potent stimulators in the primary MLR, and each expressed relevant adhesion and signal-transducing molecules important for co-stimulating T lymphocytes. Inhibition studies using monoclonal antibodies indicated similar contributions within HD line-T cell MLR to that documented in human tonsil DC-T cell MLR. The HD lines produced no detectable interleukin-1 (IL-1) by biological or immunological analysis. Moreover they stimulated allogeneic T lymphocytes in the presence of anti-IL-1 antibodies. Thus although IL-1 mRNA can be detected in both HDLM-2 and KM-H2 by polymerase chain reaction, these lines, and L428, share with DC the ability to stimulate allogeneic T lymphocytes in an IL-1-independent manner [corrected]. HD lines, particularly L428, may provide a standardized, reproducible, IL-1-independent model for dissection of the co-stimulatory requirements of the human primary MLR.

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

    PubMed

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

    2009-08-01

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

  9. Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures.

    PubMed

    Chung, Soobin; Kim, Seol-Hee; Seo, Yuri; Kim, Sook-Kyung; Lee, Ji Youn

    2017-04-04

    Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

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

    PubMed

    Mehta, Rajvi H

    2014-11-01

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

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

    PubMed Central

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

    2013-01-01

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

  12. Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

    PubMed

    Arndt, Nadia X; Tiralongo, Joe; Madge, Paul D; von Itzstein, Mark; Day, Christopher J

    2011-09-01

    Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan-binding lectins. The screening of breast cancer MDA-MB435 cells, cervical cancer HeLa cells and colon cancer Caco-2, HCT116 and HCT116-FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4-deoxy-4-guanidino-Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell-lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan-lectin interactions that are exhibited by immortalised cell lines.

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

    PubMed Central

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

    2002-01-01

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

  14. Chicoric acid induces apoptosis in 3T3-L1 preadipocytes through ROS-mediated PI3K/Akt and MAPK signaling pathways.

    PubMed

    Xiao, Haifang; Wang, Jing; Yuan, Li; Xiao, Chunxia; Wang, Yutang; Liu, Xuebo

    2013-02-20

    Chicoric acid has been reported to possess various bioactivities. However, the antiobesity effects of chicoric acid remain poorly understood. In this study, we investigated the effects of chicoric acid on 3T3-L1 preadipocytes and its molecular mechanisms of apoptosis. Chicoric acid inhibited cell viability and induced apoptosis in 3T3-L1 preadipocytes which was characterized by chromatin condensation and poly ADP-ribose-polymerase (PARP) cleavage. Mitochondrial membrane potential (MMP) loss, Bax/Bcl-2 dysregulation, cytochrome c release, and caspase-3 activation were observed, indicating mitochondria-dependent apoptosis induced by chicoric acid. Furthermore, PI3K/Akt and MAPK (p38 MAPK, JNK, and ERK1/2) signaling pathways were involved in chicoric acid-induced apoptosis. The employment of protein kinase inhibitors LY294002, SB203580, SP600125, and U0126 revealed that PI3K/Akt signaling pathway interplayed with MAPK signaling pathways. Moreover, chicoric acid induced reactive oxygen species (ROS) generation. Pretreatment with the antioxidant N-acetylcysteine (NAC) significantly blocked cell death and changes of Akt and MAPK signalings induced by chicoric acid. In addition, chicoric acid down regulated HO-1 and COX-2 via the PI3K/Akt pathway.

  15. Tumorigenic potential of mononucleated small cells of Hodgkin lymphoma cell lines.

    PubMed

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

    2010-12-01

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

  16. Aldehyde dehydrogenase activity in cancer stem cells from canine mammary carcinoma cell lines.

    PubMed

    Michishita, M; Akiyoshi, R; Suemizu, H; Nakagawa, T; Sasaki, N; Takemitsu, H; Arai, T; Takahashi, K

    2012-08-01

    Increasing evidence suggests that diverse solid tumours arise from a small population of cells known as cancer stem cells or tumour-initiating cells. Cancer stem cells in several solid tumours are enriched for aldehyde dehydrogenase (ALDH) activity. High levels of ALDH activity (ALDH(high)) were detected in four cell lines derived from canine mammary carcinomas. ALDH(high) cells were enriched in a CD44(+)CD24(-) population having self-renewal capacity. Xenotransplantation into immunodeficient mice demonstrated that 1×10(4) ALDH(high) cells were sufficient for tumour formation in all injected mice, whereas 1×10(4) ALDH(low) cells failed to initiate any tumours. ALDH(high)-derived tumours contained both ALDH(+) and ALDH(-) cells, indicating that these cells had cancer stem cell-like properties.

  17. Edwardsiella tarda invasion of fish cell lines and the activation of divergent cell death pathways.

    PubMed

    Wang, Bin; Yu, Tong; Dong, Xue; Zhang, Zenghu; Song, Lin; Xu, Ying; Zhang, Xiao-Hua

    2013-05-03

    Edwardsiella tarda is an important gram-negative intracellular pathogen of fish. However, the invasive features of E. tarda to fish cells and the pathogenesis of host cell death have not been thoroughly investigated. In this study, two fish cell models were used to investigate the interactions between E. tarda and its cellular hosts. E. tarda invaded and replicated in both cell lines. Epithelioma papulosum cyprini (EPC) cells were more sensitive to E. tarda infection than the flounder gill cell line FG-9307, with higher levels of intracellular bacteria in the former. The invasion and intracellular replication of E. tarda in FG-9307 cells were studied at the ultrastructural level, and infected cells with large amounts of replicated bacteria and destroyed organelles were observed. Apoptosis was observed in EPC cells upon infection, characterized by the occurrence of apoptotic bodies, DNA ladder, increased Annexin V binding and the activation of caspase-3, whereas E. tarda infected FG-9307 cells were negative for all of those features. E. tarda infection in FG-9307 cells failed to protect the staurosporine-induced apoptosis. Moreover, both intrinsic and extrinsic pathways were activated in EPC cells upon E. tarda infection. The present study revealed that E. tarda interacts with fish cells in different manners, and divergent pathways were activated in these cellular hosts to mediate cell death. These results provided new information on the interactions between E. tarda and fish cells.

  18. Effects of oxalate on IMCD cells: a line of mouse inner medullary collecting duct cells.

    PubMed

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Chandhoke, Paramjit S; Koul, Hari K

    2004-12-01

    Oxalate, a metabolic end product and a major constituent of the majority of renal stones, has been shown to be toxic to renal epithelial cells of cortical origin. However, it is unknown whether inner medullary collecting duct (IMCD) cells that are physiologically exposed to higher concentrations of oxalate also behave in a similar manner. In the present study, we examined the effects of oxalate on IMCD cells. IMCD cells from the mouse were maintained in DMEM/F12 media supplemented with fetal bovine serum and antibiotics. Exposure of IMCD cells to oxalate produced time- and concentration-dependent changes in the light microscopic appearance of the cells. Long-term exposure to oxalate resulted in alterations in cell viability, with net cell loss after exposure to concentrations of 2 mM or greater. The production of free radicals was directly related to the exposure time and the concentration of oxalate. Crystal formation occurred in less than 1 h and cells in proximity to crystals would lose membrane integrity. Compared with IMCD cells, LLC-PK1 cells as well as HK-2 cells showed significant toxicity starting at lower oxalate concentrations (0.4 mM or greater). These results provide the first direct demonstration of toxic effects of oxalate in IMCD cells, a line of renal epithelial cells of the inner medullary collecting duct, and suggest that the cells lining the collecting duct are relatively resistant to oxalate toxicity.

  19. Highly tumorigenic hepatocellular carcinoma cell line with cancer stem cell-like properties

    PubMed Central

    Cassim, Shamir; Lapierre, Pascal; Bilodeau, Marc

    2017-01-01

    There are limited numbers of models to study hepatocellular carcinoma (HCC) in vivo in immunocompetent hosts. In an effort to develop a cell line with improved tumorigenicity, we derived a new cell line from Hepa1-6 cells through an in vivo passage in C57BL/6 mice. The resulting Dt81Hepa1-6 cell line showed enhanced tumorigenicity compared to Hepa1-6 with more frequent (28±12 vs. 0±0 lesions at 21 days) and more rapid tumor development (21 (100%) vs. 70 days (10%)) in C57BL/6 mice. The minimal Dt81Hepa1-6 cell number required to obtain visible tumors was 100,000 cells. The Dt81Hepa1-6 cell line showed high hepatotropism with subcutaneous injection leading to liver tumors without development of tumors in lungs or spleen. In vitro, Dt81Hepa1-6 cells showed increased anchorage-independent growth (34.7±6.8 vs. 12.3±3.3 colonies; P<0.05) and increased EpCAM (8.7±1.1 folds; P<0.01) and β-catenin (5.4±1.0 folds; P<0.01) expression. A significant proportion of Dt81Hepa1-6 cells expressed EpCAM compared to Hepa1-6 (34.8±1.1% vs 0.9±0.13%; P<0.001). Enriched EpCAM+ Dt81Hepa1-6 cells led to higher tumor load than EpCAM- Dt81Hepa1-6 cells (1093±74 vs 473±100 tumors; P<0.01). The in vivo selected Dt81Hepa1-6 cell line shows high liver specificity and increased tumorigenicity compared to Hepa1-6 cells. These properties are associated with increased expression of EpCAM and β-catenin confirming that EpCAM+ HCC cells comprise a subset with characteristics of tumor-initiating cells with stem/progenitor cell features. The Dt81Hepa1-6 cell line with its cancer stem cell-like properties will be a useful tool for the study of hepatocellular carcinoma in vivo. PMID:28152020

  20. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  1. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  2. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

  3. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

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

  4. 9 CFR 113.52 - Requirements for cell lines used for production of biologics.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Requirements for cell lines used for... STANDARD REQUIREMENTS Ingredient Requirements § 113.52 Requirements for cell lines used for production of... cell line used to prepare a biological product shall be tested as prescribed in this section. A...

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

    NASA Technical Reports Server (NTRS)

    Passini, Cheryl A.; Goochee, Charles F.

    1989-01-01

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

  6. Increased transversions in a novel mutator colon cancer cell line.

    PubMed

    Eshleman, J R; Donover, P S; Littman, S J; Swinler, S E; Li, G M; Lutterbaugh, J D; Willson, J K; Modrich, P; Sedwick, W D; Markowitz, S D; Veigl, M L

    1998-03-05

    We describe a novel mutator phenotype in the Vaco411 colon cancer cell line which increases the spontaneous mutation rate 10-100-fold over background. This mutator results primarily in transversion base substitutions which are found infrequently in repair competent cells. Of the four possible types of transversions, only three were principally recovered. Spontaneous mutations recovered also included transitions and large deletions, but very few frameshifts were recovered. When compared to known mismatch repair defective colon cancer mutators, the distribution of mutations in Vaco411 is significantly different. Consistent with this difference, Vaco411 extracts are proficient in assays of mismatch repair. The Vaco411 mutator appears to be novel, and is not an obvious human homologue of any of the previously characterized bacterial or yeast transversion phenotypes. Several hypotheses by which this mutator may produce transversions are presented.

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

    NASA Astrophysics Data System (ADS)

    Dulbecco, Renato; Bologna, Mauro; Unger, Michael

    1980-03-01

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

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

    PubMed

    Klingbeil, Ma Fátima G; Xavier, Flávia C A; Sardinha, Luiz R; Severino, Patricia; Mathor, Monica B; Rodrigues, Rodrigo V; Pinto, Décio S

    2013-11-01

    Head and neck squamous cell carcinoma is a complex disease with several etiologic factors and different molecular changes that may trigger certain events; it is also globally one of the most common malignancies in this topography. Extracts from Viscum album L. (VA) (mistletoe) have been used as adjuvant therapies with promising results in several types of cancer, mainly in European countries. In vitro studies have demonstrated that various types of VA may have cytotoxicity in carcinoma cells, activating the apoptotic cascade or leading cells to necrosis. This study aimed to verify the effects of three types of VA extracts (Iscador Qu Spezial, Iscador P and Iscador M) in squamous cell carcinoma of the tongue cell lines SCC9 and SCC25, not previously studied. A concentration of 0.3 mg/ml (IC50) of the drugs induced apoptosis, affecting gene expression and protein levels of AKT, PTEN and CYCLIN D1. It was concluded that VA extracts have a cytotoxic effect on SCC9 and SCC25 cell lines, but while SCC9 cell line was more resistant to the action of the drugs, Iscador Qu Spezial and Iscador M have higher cytotoxic potential in both cell lines compared to Iscador P.

  9. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

    PubMed

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

    2010-05-28

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

  10. 3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

    SciTech Connect

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

    2010-05-28

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

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

    PubMed

    Chan, S W; Bando, Y; Warr, G W; Middleton, D L; Higgins, D A

    1999-04-01

    The T strain of reticuloendotheliosis virus (REV-T) obtained, along with the helper chicken syncytia virus (CSV), from the CSO4 cell line was highly oncogenic and rapidly fatal in ducks. Tumours were mainly seen in spleen, but neoplastic cells were observed microscopically in many organs. In vitro REV transformation of duck lymphocytes failed to yield stable cell lines, so cells from organs (blood, bone marrow, spleen, lymph node, bursa of Fabricius) of infected birds were used to establish cell lines. Some of these cell lines have been cloned. The success rates of establishment and cloning were increased if cells were cultured in a range of media containing different supplements; however, medium containing 5% foetal calf serum (FCS) and 5% duck serum was generally most efficacious for initial establishment, while spent medium from the parental line supplemented with a further 20% FCS gave best results for cloning. Cloned cell lines had the morphology of lymphoblastoid cells, with irregular nuclei and diffuse chromatin. Analysis of mRNA extracted from these cell lines showed that the uncloned lines were strongly expressing the β chain of the T cell antigen receptor (TCR) and weakly expressing immunoglobulin (Ig) polypeptides [λ light chain and μ, υ, υ (ΔFc) and α heavy chains in various proportions], suggesting the presence of T and B cells. The cloned cell lines that could be classified were TCR β+ ve T cells. This is the first report of the establishment, cloning and partial characterization of duck lymphoblastoid cell lines.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2008-11-01

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

  14. Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines.

    PubMed

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

    2011-02-01

    Colorectal cancer (CRC) is the second most fatal and the third most diagnosed type of cancer worldwide. Despite having multifactorial causes, most CRC cases are mainly determined by dietary factors. In recent years, a large number of studies have attributed a protective effect to polyphenols and foods containing these compounds (fruits and vegetables) against CRC. Indeed, polyphenols have been reported to interfere with cancer initiation, promotion, and progression, acting as chemopreventive agents. The aim of this review is to summarize the main chemopreventive properties of some polyphenols (quercetin, rutin, myricetin, chrysin, epigallocatechin-3-gallate, epicatechin, catechin, resveratrol, and xanthohumol) against CRC, observed in cell culture models. From the data reviewed in this article, it can be concluded that these compounds inhibit cell growth, by inducing cell cycle arrest and/or apoptosis; inhibit proliferation, angiogenesis, and/or metastasis; and exhibit anti-inflammatory and/or antioxidant effects. In turn, these effects involve multiple molecular and biochemical mechanisms of action, which are still not completely characterized. Thus, caution is mandatory when attempting to extrapolate the observations obtained in CRC cell line studies to humans.

  15. Growth inhibitory activity of Ankaferd hemostat on primary melanoma cells and cell lines

    PubMed Central

    Turk, Seyhan; Malkan, Umit Yavuz; Ghasemi, Mehdi; Hocaoglu, Helin; Mutlu, Duygu; Gunes, Gursel; Aksu, Salih; Haznedaroglu, Ibrahim Celalettin

    2017-01-01

    Objective: Ankaferd hemostat is the first topical hemostatic agent about the red blood cell–fibrinogen relations tested in the clinical trials. Ankaferd hemostat consists of standardized plant extracts including Alpinia officinarum, Glycyrrhiza glabra, Thymus vulgaris, Urtica dioica, and Vitis vinifera. The aim of this study was to determine the effect of Ankaferd hemostat on viability of melanoma cell lines. Methods: Dissimilar melanoma cell lines and primary cells were used in this study. These cells were treated with different concentrations of Ankaferd hemostat to assess the impact of different dosages of the drug. All cells treated with different concentrations were incubated for different time intervals. After the data had been obtained, one-tailed T-test was used to determine whether the Ankaferd hemostat would have any significant inhibitory impact on cell growth. Results: We demonstrated in this study that cells treated with Ankaferd hemostat showed a significant decrease in cell viability compared to control groups. The cells showed different resistances against Ankaferd hemostat which depended on the dosage applied and the time treated cells had been incubated. We also demonstrated an inverse relationship between the concentration of the drug and the incubation time on one hand and the viability of the cells on the other hand, that is, increasing the concentration of the drug and the incubation time had a negative impact on cell viability. Conclusion: The findings in our study contribute to our knowledge about the anticancer impact of Ankaferd hemostat on different melanoma cells. PMID:28293423

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

    PubMed

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

    2015-11-10

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

  17. In vitro cell-toxicity of Peganum harmala alkaloids on cancerous cell-lines.

    PubMed

    Lamchouri, F; Settaf, A; Cherrah, Y; Hassar, M; Zemzami, M; Atif, N; Nadori, E B; Zaid, A; Lyoussi, B

    2000-02-01

    The alkaloidic fraction of the methanol extract of Peganum harmala seeds was tested in vitro on three tumoral cell-lines: UCP-Med and Med-mek carcinoma, and UCP-Med sarcoma. Proliferation was significantly reduced at all tested concentrations (20-120 micrograms/ml) during the first 24 h of contact. A cell lysis effect occurred after 24 h and increased thereafter to complete cell death within 48-72 h, depending on tested concentration.

  18. Investigation of Cross-Contamination and Misidentification of 278 Widely Used Tumor Cell Lines

    PubMed Central

    Huang, Yaqing; Liu, Yuehong; Zheng, Congyi; Shen, Chao

    2017-01-01

    In recent years, biological research involving human cell lines has been rapidly developing in China. However, some of the cell lines are not authenticated before use. Therefore, misidentified and/or cross-contaminated cell lines are unfortunately commonplace. In this study, we present a comprehensive investigation of cross-contamination and misidentification for a panel of 278 cell lines from 28 institutes in China by using short tandem repeat profiling method. By comparing the DNA profiles with the cell bank databases of ATCC and DSMZ, a total of 46.0% (128/278) cases with cross-contamination/misidentification were uncovered coming from 22 institutes. Notably, 73.2% (52 out of 71) of the cell lines established by the Chinese researchers were misidentified and accounted for 40.6% of total misidentification (52/128). Further, 67.3% (35/52) of the misidentified cell lines established in laboratories of China were HeLa cells or a possible hybrid of HeLa with another kind of cell line. Furthermore, the bile duct cancer cell line HCCC-9810 and degenerative lung cancer Calu-6 exhibited 88.9% match in the ATCC database (9-loci), indicating that they were from the same origin. However, when we used 21-loci to compare these two cell lines with the same algorithm, the percent match was only 48.2%, indicating that these two cell lines were different. The SNP profiles of HCCC-9810 and Calu-6 also revealed that they were different cell lines. 150 cell lines with unique profiles demonstrated a wide range of in vitro phenotypes. This panel of 150 genomically validated cancer cell lines represents a valuable resource for the cancer research community and will advance our understanding of the disease by providing a standard reference for cell lines that can be used for biological as well as preclinical studies. PMID:28107433

  19. Killing of human myelomonocytic leukemia and lymphocytic cell lines by Actinobacillus actinomycetemcomitans leukotoxin.

    PubMed Central

    Simpson, D L; Berthold, P; Taichman, N S

    1988-01-01

    The purified leukotoxin of Actinobacillus actinomycetemcomitans kills human leukemic cell lines (e.g., HL-60, U937, and KG-1) and human T- and B-cell lines (e.g., JURKAT, MOLT-4, Daudi, and Raji) in a dose- and time-dependent manner. The 50% effective doses for these cell lines are similar to those established for human polymorphonuclear leukocytes and monocytes. In contrast, other human and nonhuman tumor cell lines are not susceptible to the leukotoxin. These human leukemia and lymphoid cell lines will serve as useful model systems with which to study the molecular specificity and mechanism(s) of action of the actinobacillus leukotoxin. Images PMID:3258584

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

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

    SciTech Connect

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

    2005-09-01

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

  3. Etomoxir, sodium 2-[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylate, up-regulates uncoupling protein-3 mRNA levels in primary culture of rat preadipocytes.

    PubMed

    Cabrero, A; Alegret, M; Sánchez, R; Adzet, T; Laguna, J C; Vázquez, M

    1999-09-16

    Uncoupling proteins (UCPs) are mitochondrial membrane proton transporters that uncouple respiration from oxidative phosphorylation by dissipating the proton gradient across the membrane. Treatment of primary culture of rat preadipocytes for 24 h with 40 microM etomoxir, an irreversible inhibitor of carnitine palmitoyltransferase I (CPT-I), up-regulated UCP-3 mRNA levels (3. 6-fold induction), whereas changes in UCP-2 mRNA levels were not significant. As a consequence of increased UCP-3 expression, a fall in the mitochondrial membrane potential was detected by flow cytometry. Etomoxir treatment modified neither L-CPT-I (liver-type) nor PPARalpha mRNA levels in preadipocytes. In contrast, mRNA expression of acyl-CoA oxidase (ACO), the rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, whose transcription is controlled by PPARalpha, was significantly induced (1.3-fold induction, P = 0.015). These findings suggest that the effects of etomoxir were mediated by PPARalpha. Since it has been reported that the intracellular accumulation of lipids following the inhibition of CPT-I by etomoxir leads to a PPARalpha-mediated metabolic response that increases the expression of genes involved in alternate fatty acid oxidation pathways, these results seem to implicate UCP-3 in this protective metabolic response. It remains to be studied whether reductions in the expression of UCP-3 could compromise this response, giving rise to lipotoxic effects on cells.

  4. Production of angiotensin II receptors type one (AT1) and type two (AT2) during the differentiation of 3T3-L1 preadipocytes.

    PubMed

    Mallow, H; Trindl, A; Löffler, G

    2000-01-01

    During their development from progenitor cells, adipocytes not only express enzymatic activities necessary for the storage of triglycerides, but also achieve the capability to produce a number of endocrine factors such as leptin, tumor necrosis factor alpha (TNFalpha), complement factors, adiponectin/adipoQ, plasminogen activator inhibitor-1 (PAI-1), angiotensin II and others. Angiotensin II is produced from angiotensinogen by the proteolytic action of renin and angiotensin-converting enzyme; and several data point to the existence of a complete local renin-angiotensin system in adipose tissue, including angiotensin II receptors. In this study, we directly monitored the production of angiotensin II type one receptor (AT1) and angiotensin II type two receptor (AT2) proteins during the adipose conversion of murine 3T3-L1 preadipocytes by immunodetection with specific antibodies. AT1 receptors could be detected throughout the whole differentiation period. The strong AT2 signal in preadipocytes however was completely lost during the course of differentiation, which suggests that expression of AT2 receptors is inversely correlated to the adipose conversion program.

  5. Trans, trans-farnesol as a mevalonate-derived inducer of murine 3T3-F442A pre-adipocyte differentiation

    PubMed Central

    Torabi, Sheida

    2015-01-01

    Based on our finding that depletion of mevalonate-derived metabolites inhibits adipocyte differentiation, we hypothesize that trans, trans-farnesol (farnesol), a mevalonate-derived sesquiterpene, induces adipocyte differentiation. Farnesol dose-dependently (25–75 μmol/L) increased intracellular triglyceride content of murine 3T3-F442A pre-adipocytes measured by AdipoRed™ Assay and Oil Red-O staining. Concomitantly, farnesol dose-dependently increased glucose uptake and glucose transport protein 4 (GLUT4) expression without affecting cell viability. Furthermore, quantitative real-time polymerase chain reaction and Western blot showed that farnesol increased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, and the mRNA levels of PPARγ-regulated fatty acid-binding protein 4 and adiponectin; in contrast, farnesol downregulated Pref-1 gene, a marker of pre-adipocytes. GW9662 (10 µmol/L), an antagonist of PPARγ, reversed the effects of farnesol on cellular lipid content, suggesting that PPARγ signaling pathway may mediate the farnesol effect. Farnesol (25–75 μmol/L) did not affect the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway. Farnesol may be the mevalonate-derived inducer of adipocyte differentiation and potentially an insulin sensitizer via activation of PPARγ and upregulation of glucose uptake. PMID:26660152

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

    PubMed Central

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

    2013-01-01

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

  7. MicroRNA-15a fine-tunes the level of Delta-like 1 homolog (DLK1) in proliferating 3T3-L1 preadipocytes

    SciTech Connect

    Andersen, Ditte C.; Schneider, Mikael; Eskildsen, Tilde; Teisner, Borge; and others

    2010-06-10

    Delta like 1 homolog (Dlk1) exists in both transmembrane and soluble molecular forms, and is implicated in cellular growth and plays multiple roles in development, tissue regeneration, and cancer. Thus, DLK1 levels are critical for cell function, and abnormal DLK1 expression can be lethal; however, little is known about the underlying mechanisms. We here report that miR-15a modulates DLK1 levels in preadipocytes thus providing a mechanism for DLK1 regulation that further links it to cell cycle arrest and cancer since miR-15a is deregulated in these processes. In preadipocytes, miR-15a increases with cell density, and peaks at the same stage where membrane DLK1{sup M} and soluble DLK1{sup S} are found at maximum levels. Remarkably, miR-15a represses the amount of all Dlk1 variants at the mRNA level but also the level of DLK1{sup M} protein while it increases the amount of DLK1{sup S} supporting a direct repression of DLK1 and a parallel effect on the protease that cleaves off the DLK1 from the membrane. In agreement with previous studies, we found that miR-15a represses cell numbers, but additionally, we report that miR-15a also increases cell size. Conversely, anti-miR-15a treatment decreases cell size while increasing cell numbers, scenarios that were completely rescued by addition of purified DLK1{sup S}. Our data thus imply that miR-15a regulates cell size and proliferation by fine-tuning Dlk1 among others, and further emphasize miR-15a and DLK1 levels to play important roles in growth signaling networks.

  8. Assessment of tumor characteristic gene expression in cell lines using a tissue similarity index (TSI).

    PubMed

    Sandberg, Rickard; Ernberg, Ingemar

    2005-02-08

    The gene expression profiles of 60 cell lines, derived from nine different tissues, were compared with their corresponding in vivo tumors and tissues. Cell lines expressed few tissue-specific (2%) or tumor-specific (5%) genes when analyzed group-wise. A tissue similarity index (TSI) was designed based upon singular value decomposition that measured in vivo tumor characteristic gene expression in each cell line independently. Only 34 of the 60 cell lines received the highest TSI toward its tumor of origin. In addition, we identified the most appropriate cell lines to be used as model systems for different in vivo tumors. Seven cell lines were identified as being of another origin than the originally presumed one. The proposed TSI will likely become an important tool for the selection of the most appropriate cell lines in pharmaceutical screening programs and experimental and biomedical research.

  9. Serum Preadipocyte Factor 1 Levels Are Not Associated with Bone Mineral Density among Healthy Postmenopausal Korean Women

    PubMed Central

    Choi, Hoon Sung; Kim, Sang-Wook

    2017-01-01

    Background Multipotent mesenchymal stem cells can differentiate into adipocytes or osteoblasts through closely regulated lineage-control processes. However, adipocyte precursor cells release preadipocyte factor 1 (Pref-1), which inhibits the differentiation of mesenchymal stem cells into mature adipocytes and osteoblasts. Previous studies have also reported an inverse association between Pref-1 levels and bone mineral density (BMD) among patients with anorexia nervosa. Methods In this retrospective study, we examined the correlations between Pref-1 levels and BMD among 124 healthy postmenopausal women (>50 years old). The patients had provided information regarding their clinical characteristics, and underwent blood testing and serum Pref-1 testing. Results The subjects' mean age was 59.9±7.1 years and the median time since menopause onset was 9.1 years. A history of osteoporotic fracture was identified in 23 subjects (19%). Serum Pref-1 levels were not significantly correlated with BMD values at the lumbar spine (R2=0.038, P=0.109), femur neck (R2=0.017, P=0.869), and total hip (R2=0.041, P=0.09), and multivariate analyses with adjustment for age and body mass index also did not detect any significant correlations. Subgroup analyses according to a history of fracture also did not detect significant associations between Pref-1 levels and BMD values. Conclusion In our study population, it does not appear that serum Pref-1 levels are significantly associated with BMD values and osteoporosis. PMID:28256115

  10. Sulphamoylated 2-Methoxyestradiol Analogues Induce Apoptosis in Adenocarcinoma Cell Lines

    PubMed Central

    Visagie, Michelle; Theron, Anne; Mqoco, Thandi; Vieira, Warren; Prudent, Renaud; Martinez, Anne; Lafanechère, Laurence; Joubert, Annie

    2013-01-01

    2-Methoxyestradiol (2ME2) is a naturally occurring estradiol metabolite which possesses antiproliferative, antiangiogenic and antitumor properties. However, due to its limited biological accessibility, synthetic analogues have been synthesized and tested in attempt to develop drugs with improved oral bioavailability and efficacy. The aim of this study was to evaluate the antiproliferative effects of three novel in silico-designed sulphamoylated 2ME2 analogues on the HeLa cervical adenocarcinoma cell line and estrogen receptor-negative breast adenocarcinoma MDA-MB-231 cells. A dose-dependent study (0.1–25 μM) was conducted with an exposure time of 24 hours. Results obtained from crystal violet staining indicated that 0.5 μM of all 3 compounds reduced the number of cells to 50%. Lactate dehydrogenase assay was used to assess cytotoxicity, while the mitotracker mitochondrial assay and caspase-6 and -8 activity assays were used to investigate the possible occurrence of apoptosis. Tubulin polymerization assays were conducted to evaluate the influence of these sulphamoylated 2ME2 analogues on tubulin dynamics. Double immunofluorescence microscopy using labeled antibodies specific to tyrosinate and detyrosinated tubulin was conducted to assess the effect of the 2ME2 analogues on tubulin dynamics. An insignificant increase in the level of lactate dehydrogenase release was observed in the compounds-treated cells. These sulphamoylated compounds caused a reduction in mitochondrial membrane potential, cytochrome c release and caspase 3 activation indicating apoptosis induction by means of the intrinsic pathway in HeLa and MDA-MB-231 cells. Microtubule depolymerization was observed after exposure to these three sulphamoylated analogues. PMID:24039728

  11. Evaluation of the change in sphingolipids in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 treated with arsenic trioxide.

    PubMed

    Zou, Jianhua; Ma, Xiaoqiong; Zhang, Guangji; Shen, Li; Zhou, Liting; Yu, Yu; Zhu, Fanfan; Chen, Zhe

    2015-11-01

    Arsenic trioxide (As2O3) has been found to display anticancer activity against many types of tumors and has been developed into an anticancer drug in clinical treatments. Sphingolipids are membrane lipids that participate in many signal transduction pathways. In this paper, the changes in sphingolipids of the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803 treated with arsenic trioxide were investigated using an HPLC-ESI-MS/MS method. Analytes were separated by an XBridge BEH C8 column used for Cer, HexCer, LacCer and SM chromatographic separation, and a Capcell PAK MG II C18 column was used for Sph, dhSph, S1P and dhS1P chromatographic separation and gradient elution with acetonitrile-water containing 0.1% formic acid as a mobile phase. A tandem mass spectrometer QTrap in SRM mode was employed in combination with RPLC as a detector for quantitative analysis. The ceramide/sphingolipid internal standard (IS) mixture was used to quantify the levels of sphingolipids. The distributions of sphingolipids were found to be different in the human multiple myeloma cell line U266 and the gastric cancer cell line MGC-803. Ceramide (Cer), hexosylceramide (HexCer) and dihexosylceramide (Hex2Cer) levels in U266 cell line are higher than those in MGC-803 cell line. Additionally, sphingomyelin (SM), sphingosine-1-phosphate (S1P) and sphinganine-1-phosphate (dhS1P) levels in the MGC-803 cell line are higher than those in the U266 cell line. When treated with arsenic trioxide (1-5μM iAs(III)(As(III) ions)), the levels of Hex2Cer in the human multiple myeloma cell line U266 decreased, and the levels of S1P and dhS1P in the human gastric cancer cell line MGC-803 decreased. The decrease of Hex2Cer, S1P and dhS1P in the human multiple myeloma cell line U266 and gastric cancer cell line MGC-803 were observed when the concentration of iAs(III) is 1.0μM. Therefore, arsenic trioxide exhibits anti-cancer activity by altering the sphingolipid pathway in the

  12. Development of improved vaccine cell lines against rotavirus

    PubMed Central

    Wu, Weilin; Orr-Burks, Nichole; Karpilow, Jon; Tripp, Ralph A.

    2017-01-01

    Rotavirus is a major cause of severe gastroenteritis among very young children. In developing countries, rotavirus is the major cause of mortality in children under five years old, causing up to 20% of all childhood deaths in countries with high diarrheal disease burden, with more than 90% of these deaths occurring in Africa and Asia. Rotavirus vaccination mimics the first infection without causing illness, thus inducing strong and broad heterotypic immunity against prospective rotavirus infections. Two live vaccines are available, Rotarix and RotaTeq, but vaccination efforts are hampered by high production costs. Here, we present a dataset containing a genome-wide RNA interference (RNAi) screen that identified silencing events that enhanced rotavirus replication. Evaluated against several rotavirus vaccine strains, hits were validated in a Vero vaccine cell line as well as CRISPR/Cas9 generated cells permanently and stably lacking the genes that affect RV replication. Knockout cells were dramatically more permissive to RV replication and permitted an increase in rotavirus replication. These data show a means to improve manufacturing of rotavirus vaccine. PMID:28248921

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

    DOE PAGES

    Stoiber, Marcus; Celniker, Susan; Cherbas, Lucy; ...

    2016-01-15

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

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

    SciTech Connect

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

    2016-01-15

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

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

    PubMed Central

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

    2016-01-01

    Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate . Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone . In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. We found that the early transcriptional response mirrors the distinctiveness of physiological origins: genes respond in restricted patterns, conditional on the expression levels of dozens of transcription factors. Only a small cohort of genes is constitutively modulated independent of initial cell state. Ecdysone-responsive genes tend to organize into directional same-stranded units, with consecutive genes induced from the same strand. Here, we identify half of the ecdysone receptor heterodimer as the primary rate-limiting step in the response, and find that initial receptor isoform levels modulate the activated cohort of target transcription factors. This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly. PMID:26772746

  16. Analysis of differential protein expression in normal and neoplastic human breast epithelial cell lines

    SciTech Connect

    Williams, K.; Chubb, C.; Huberman, E.; Giometti, C.S.

    1997-07-01

    High resolution two dimensional get electrophoresis (2DE) and database analysis was used to establish protein expression patterns for cultured normal human mammary epithelial cells and thirteen breast cancer cell lines. The Human Breast Epithelial Cell database contains the 2DE protein patterns, including relative protein abundances, for each cell line, plus a composite pattern that contains all the common and specifically expressed proteins from all the cell lines. Significant differences in protein expression, both qualitative and quantitative, were observed not only between normal cells and tumor cells, but also among the tumor cell lines. Eight percent of the consistently detected proteins were found in significantly (P < 0.001) variable levels among the cell lines. Using a combination of immunostaining, comigration with purified protein, subcellular fractionation, and amino-terminal protein sequencing, we identified a subset of the differentially expressed proteins. These identified proteins include the cytoskeletal proteins actin, tubulin, vimentin, and cytokeratins. The cell lines can be classified into four distinct groups based on their intermediate filament protein profile. We also identified heat shock proteins; hsp27, hsp60, and hsp70 varied in abundance and in some cases in the relative phosphorylation levels among the cell lines. Finally, we identified IMP dehydrogenase in each of the cell lines, and found the levels of this enzyme in the tumor cell lines elevated 2- to 20-fold relative to the levels in normal cells.

  17. Tualang Honey Promotes Apoptotic Cell Death Induced by Tamoxifen in Breast Cancer Cell Lines

    PubMed Central

    Yaacob, Nik Soriani; Nengsih, Agustine; Norazmi, Mohd. Nor

    2013-01-01

    Tualang honey (TH) is rich in flavonoids and phenolic acids and has significant anticancer activity against breast cancer cells comparable to the effect of tamoxifen (TAM), in vitro. The current study evaluated the effects of TH when used in combination with TAM on MCF-7 and MDA-MB-231 cells. We observed that TH promoted the anticancer activity of TAM in both the estrogen receptor-(ER-)responsive and ER-nonresponsive human breast cancer cell lines. Flow cytometric analyses indicated accelerated apoptosis especially in MDA-MB-231 cells and with the involvement of caspase-3/7, -8 and -9 activation as shown by fluorescence microscopy. Depolarization of the mitochondrial membrane was also increased in both cell lines when TH was used in combination with TAM compared to TAM treatment alone. TH may therefore be a potential adjuvant to be used with TAM for reducing the dose of TAM, hence, reducing TAM-induced adverse effects. PMID:23476711

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

    PubMed Central

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

    2015-01-01

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

  19. Development of cell lines from the sheep used to construct the CHORI-243 ovine BAC library

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two cell lines, designated MARC.OVSM and MARC.OKF, were initiated from the aorta and kidney, respectively, obtained from the Texel ram used to make the CHORI-243 Ovine BAC library. These cell lines have been submitted to the NIA Aging Cell Repository at the Coriell Cell Respositories, Camden, NJ, U...

  20. Bracken-fern extracts induce cell cycle arrest and apoptosis in certain cancer cell lines.

    PubMed

    Roudsari, Motahhareh Tourchi; Bahrami, Ahmad Reza; Dehghani, Hesam

    2012-01-01

    Bracken fern [Pteridium aquilinem (L.) kuhn (Dennstaedtiaceae)] is one of the most common species on the planet. It has been consumed by humans and animals for centuries. Use by some human groups is because they believe bracken fern is good for health as plant medicine. However, it is also one of the few known plants that can cause tumors in farm animals. Many interested groups have focused their attention on bracken fern because of these interesting features. In order to evaluate the biological effects of exposure to this plant in cellular level, human cancer cell lines were treated with the fern dichloromethane extracts and the genotoxic and cytotoxic effects were studied. Anti-proliferative/cytotoxic effects were evaluated by cell count, MTT assay and flow cytometry methods with three different cancer cell lines, TCC, NTERA2, and MCF-7, and two normal cells, HDF1 and HFF3. Pro-apoptotic effects of the extracts were determined by DAPI staining and comet assay, on TCC cancer cells compared to the normal control cell lines. Cellular morphology was examined by light microscopy. Our present study showed that the extract caused DNA damage and apoptosis at high concentrations (200 μg/mL) and also it may induce cell cycle arrest (G2/M phase) at mild concentrations (50 and 30 μg/mL) depending on the cell type and tumor origin. These results indicate that bracken fern extract is a potent source of anticancer compounds that could be utilized pharmaceutically.

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

    PubMed

    Othon, Christina M; Wu, Xingjia; Anders, Juanita J; Ringeisen, Bradley R

    2008-09-01

    Biological laser printing (BioLP) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes ( approximately microLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 microm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth.

  2. Differences in lipid characteristics of autologous human melanoma cell lines with distinct biological properties.

    PubMed

    Le Bivic, A; Sari, H; Reynier, M; Lebec, S; Bardin, F

    1987-12-01

    Significant differences in lipid composition were found when six established human melanoma cell lines were compared. A pair of cell lines was initiated from a superficial spreading melanoma and the lymph node of the same patient; four others were also autologous, three of which originated from the same nodular melanoma and the other from its metastasis. Cell lines varied in pigmentation level and ability to grow in nude mice. Cell lines contained similar amounts of total cholesterol, glycerides, and phospholipids but different amounts of free cholesterol and cholesterol esters. In particular, the molar ratio of free cholesterol to phospholipid was increased in highly tumorigenic cell lines. No changes in phospholipid profiles were noted among cell lines, except an increase in sphingomyelin with a concomitant decrease in phosphatidylcholine in one cell line compared to the profiles of its counterpart cell line. The saturated-to-unsaturated fatty acid ratios in phosphatidylcholine and phosphatidylethanolamine were similar in all cell lines, but the monounsaturated-to-polyunsaturated fatty acid ratio in phosphatidylcholine was increased in highly tumorigenic cell lines. A significant variation in the latter ratio in phosphatidylethanolamine was also observed in the pair of autologous cell lines. These changes were unrelated to a depletion in linoleic acid in culture medium. Results obtained by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene were consistent with the differences in lipid composition between two autologous cell lines. The present results indicate that two lipid characteristics were significantly changed in highly tumorigenic cell lines as compared to cell lines with low tumorigenicity, but no correlation was found between either pigmentation level or origin (primary or metastatic) and lipid composition.

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

    PubMed Central

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

    1998-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  5. [Neuronal differentiation of human small cell lung cancer cell line PC-6 by Solcoseryl].

    PubMed

    Shimizu, T

    1997-11-01

    Solcoseryl is composed of extracts from calf blood, and is a drug known to activate tissue respiration. In the present study, I demonstrated the cell biological effects of Solcoseryl on a human small cell lung cancer cell line, PC-6, by analyzing cell morphology, cell growth, expression of neuronal differentiation markers, and the ras proto-oncogene product(ras p21). Exposure of PC-6 cells to Solcoseryl at the concentration of 200 microliters/ml induced (1) cell morphological changes, including neurodendrite-like projections from the cell surface, and (2) complete inhibition of cell growth, that was shown by the loss of Ki-67 expression. Solcoseryl also induced the expression of neurofilament protein and acetylcholinesterase, both of which are markers of neuronal differentiation. Moreover, it upregulated the expression of the ras proto-oncogene product, ras p21. Taken together, these data suggest that Solcoseryl is composed of component(s) which can induce neuronal differentiation of the human small cell lung cancer cell line, PC-6.

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

    PubMed Central

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

    2010-01-01

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

  7. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  8. Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5.

    PubMed

    Wiper-Bergeron, Nadine; Salem, Houssein Abdou; Tomlinson, Julianna J; Wu, Dongmei; Haché, Robert J G

    2007-02-20

    Preadipocyte differentiation in culture is driven by an insulin and cAMP dependant transcriptional cascade which induces the bzip transcription factors C/EBPbeta and C/EBPdelta. We have previously shown that glucocorticoid treatment, which strongly potentiates this differentiation pathway, stimulates the titration of the corepressor histone deacetylase 1 (HDAC1) from C/EBPbeta. This results in a dramatic enhancement of C/EBPbeta-dependent transcription from the C/EBPalpha promoter, concomitant with potentiation of preadipocyte differentiation. Here, we show that C/EBPbeta is acetylated by GCN5 and PCAF within a cluster of lysine residues between amino acids 98-102 and that this acetylation is strongly induced by glucocorticoid treatment. Arginine substitution of the lysine residues within the acetylation motif of C/EBPbeta prevented acetylation and blocked the ability of glucocorticoids to enhance C/EBPbeta-directed transcription and to potentiate C/EBPbeta-dependent preadipocyte differentiation. Moreover, acetylation of C/EBPbeta appeared to directly interfere with the interaction of HDAC1 with C/EBPbeta, suggesting that PCAF/GCN5-dependent acetylation of C/EBPbeta serves as an important molecular switch in determining the transcriptional regulatory potential of this transcription factor.

  9. Stimulation of preadipocyte differentiation by steroid through targeting of an HDAC1 complex.

    PubMed

    Wiper-Bergeron, Nadine; Wu, Dongmei; Pope, Louise; Schild-Poulter, Caroline; Haché, Robert J G

    2003-05-01

    Glucocorticoids potentiate the early steps of preadipocyte differentiation and promote obesity in Cushing's syndrome and during prolonged steroid therapy. We show that glucocorticoids stimulate 3T3 L1 preadipocyte differentiation through a non-transcriptional mechanism mediated through the ligand-binding domain of the glucocorticoid receptor. This enhanced the onset of CCAAT/enhancer binding protein (C/EBPalpha) expression by potentiating its initial transcriptional activation by C/EBPbeta. In the absence of steroid, C/EBPbeta associated with a transcriptional corepressor complex containing mSin3A and histone deacetylase 1 (HDAC1), but lacking HDAC2 and RbAp46/48. HDAC1/mSin3A were recruited to the C/EBPalpha promoter with C/EBPbeta and promoted the deacetylation of histone H4. Steroid induced the specific depletion of this corepressor by targeting the HDAC1 within the complex for degradation through the 26S proteasome. Treatment with histone deacetylase inhibitors replaced the effects of steroid treatment on preadipocyte differentiation and C/EBPalpha expression, while overexpression of HDAC1 abrogated the stimulatory effects of steroid. Recapitulation of the glucocorticoid effect by progestin treatment in the presence of the progesterone receptor ligand-binding domain suggests a conserved mechanism relevant to many aspects of steroid-mediated differentiation.

  10. DNA Methylation Heterogeneity Patterns in Breast Cancer Cell Lines

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2000-12-01

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

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

    SciTech Connect

    Blattmann, Claudia; Oertel, Susanne; Ehemann, Volker

    2010-09-01

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

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

    PubMed Central

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

    2015-01-01

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

  14. Germline transmission of a novel rat embryonic stem cell line derived from transgenic rats.

    PubMed

    Men, Hongsheng; Bauer, Beth A; Bryda, Elizabeth C

    2012-09-20

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

  15. Lines

    ERIC Educational Resources Information Center

    Mires, Peter B.

    2006-01-01

    National Geography Standards for the middle school years generally stress the teaching of latitude and longitude. There are many creative ways to explain the great grid that encircles our planet, but the author has found that students in his college-level geography courses especially enjoy human-interest stories associated with lines of latitude…

  16. Drug treatment of cancer cell lines: a way to select for cancer stem cells?

    PubMed

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A Ivana; Mondello, Chiara

    2011-03-04

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

  17. Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

    PubMed Central

    Chiodi, Ilaria; Belgiovine, Cristina; Donà, Francesca; Scovassi, A. Ivana; Mondello, Chiara

    2011-01-01

    Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs. PMID:24212655

  18. Photodynamic therapy-induced programmed cell death in carcinoma cell lines

    NASA Astrophysics Data System (ADS)

    He, Xiao-Yan; Sikes, Robert A.; Thomsen, Sharon L.; Chung, L.; Jacques, Steven L.

    1993-06-01

    The mode of cell death following photodynamic therapy (PDT) was investigated from the perspective of programmed cell death (apoptosis). Human prostate carcinoma cells (PC3), human non-small cell lung carcinoma (H322a), and rat mammary carcinoma (MTF7) were treated by PDT following sensitization with dihematoporphyrin ether (DHE). The response of these carcinoma cell lines to PDT was variable. An examination of extracted cellular DNA by gel electrophoresis showed the characteristic DNA ladder pattern indicative of internucleosomal cleavage of DNA during apoptosis. MTF7 and PC3 responded to PDT by inducing apoptosis while H322a had no apoptotic response. The magnitude of the response and the PDT dosage required to induce the effect were different in PC3 and MTF7. MTF7 cells responded with rapid apoptosis at the dose of light and drug that yielded 50% cell death (LD50). In contrast, PC3 showed only marginal apoptosis at the LD50 but had a marked response at the LD85. Furthermore, the onset of apoptosis followed slower kinetics in PC3 (2 hr - 4 hr) than in MTF7 (< 1 hr). H322a cells were killed by PDT but failed to exhibit any apoptotic response. This study indicates that apoptosis may occur during PDT induced cell death, but this pathway is not universal for all cancer cell lines.

  19. Rabies virus interaction with various cell lines is independent of the acetylcholine receptor.

    PubMed

    Reagan, K J; Wunner, W H

    1985-01-01

    Rabies virus infects most cells in vitro. The presence of the nicotinic acetylcholine receptor on the plasma membrane of various cell lines is not an obligate factor for rabies virus susceptibility of those cells.

  20. Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes

    SciTech Connect

    Yarmo, Michelle N.; Landry, Anne; Molgat, Andre S.D.; Gagnon, AnneMarie; Sorisky, Alexander

    2009-02-01

    This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27{sup kip1}, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBP{beta} were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

  1. Transplantation and differentiation of donor cells in the cloned pigs

    SciTech Connect

    Shimada, Arata; Tomii, Ryo; Kano, Koichiro; Nagashima, Hiroshi . E-mail: hnagas@isc.meiji.ac.jp

    2006-06-02

    The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal.

  2. Macrophage cell lines derived from major histocompatibility complex II-negative mice

    NASA Technical Reports Server (NTRS)

    Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

    1998-01-01

    Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

  3. N-linked glycan profiling in neuroblastoma cell lines.

    PubMed

    Hu, Yunli; Mayampurath, Anoop; Khan, Saira; Cohen, Joanna K; Mechref, Yehia; Volchenboum, Samuel L

    2015-05-01

    Although MYCN amplification has been associated with aggressive neuroblastoma, the molecular mechanisms that differentiate low-risk, MYCN-nonamplified neuroblastoma from high-risk, MYCN-amplified disease are largely unknown. Genomic and proteomic studies have been limited in discerning differences in signaling pathways that account for this heterogeneity. N-Linked glycosylation is a common protein modification resulting from the attachment of sugars to protein residues and is important in cell signaling and immune response. Aberrant N-linked glycosylation has been routinely linked to various cancers. In particular, glycomic markers have often proven to be useful in distinguishing cancers from precancerous conditions. Here, we perform a systematic comparison of N-linked glycomic variation between MYCN-nonamplified SY5Y and MYCN-amplified NLF cell lines with the aim of identifying changes in sugar abundance linked to high-risk neuroblastoma. Through a combination of liquid chromatography-mass spectrometry and bioinformatics analysis, we identified 16 glycans that show a statistically significant change in abundance between NLF and SY5Y samples. Closer examination revealed the preference for larger (in terms of total monosaccharide count) and more sialylated glycan structures in the MYCN-amplified samples in comparison to smaller, nonsialylated glycans that are more dominant in the MYCN-nonamplified samples. These results offer clues for deriving marker candidates for accurate neuroblastoma risk diagnosis.

  4. Opioid binding site in EL-4 thymoma cell line

    SciTech Connect

    Fiorica, E.; Spector, S.

    1988-01-01

    Using EL-4 thymoma cell-line we found a binding site similar to the k opioid receptor of the nervous system. The Scatchard analysis of the binding of (/sup 3/H) bremazocine indicated a single site with a K/sub D/ = 60 +/- 17 nM and Bmax = 2.7 +/- 0.8 pmols/10/sup 6/ cells. To characterize this binding site, competition studies were performed using selective compounds for the various opioid receptors. The k agonist U-50,488H was the most potent displacer of (/sup 3/H) bremazocine with an IC/sub 50/ value = 0.57..mu..M. The two steroisomers levorphanol and dextrorphan showed the same affinity for this site. While morphine, (D-Pen/sup 2/, D-Pen/sup 5/) enkephalin and ..beta..-endorphin failed to displace, except at very high concentrations, codeine demonstrated a IC/sub 50/ = 60..mu..M, that was similar to naloxone. 32 references, 3 figures, 2 tables.

  5. Uveal Melanoma Cell Lines: Where do they come from? (An American Ophthalmological Society Thesis)

    PubMed Central

    Jager, Martine J.; Magner, J. Antonio Bermudez; Ksander, Bruce R.; Dubovy, Sander R.

    2016-01-01

    Purpose To determine whether some of the most often used uveal melanoma cell lines resemble their original tumor. Methods Analysis of the literature, patient charts, histopathology, mutations, chromosome status, HLA type, and expression of melanocyte markers on cell lines and their primary tumors. We examined five cell lines and the primary tumors from which they were derived. Results Four of the five examined primary tumors were unusual: one occupied the orbit, two were recurrences after prior irradiation, and one developed in an eye with a nevus of Ota. One cell line did not contain the GNA11 mutation, but it was present in the primary tumor. Three of the primary tumors had monosomy 3 (two of these lacked BAP1 expression); however, all five cell lines showed disomy 3 and BAP1 expression. All of the cell lines had gain of 8q. Two cell lines lacked expression of melanocyte markers, although these were present in the corresponding primary tumor. Conclusions All cell lines could be traced back to their original uveal melanoma. Four of the five primary tumors were unusual. Cell lines often differed from their primary tumor in chromosome status and melanocyte markers. However, their specific chromosome aberrations and capacity to continue proliferation characterize them as uveal melanoma cell lines. PMID:28018010

  6. Efficient derivation of extraembryonic endoderm stem cell lines from mouse postimplantation embryos

    PubMed Central

    Lin, Jiangwei; Khan, Mona; Zapiec, Bolek; Mombaerts, Peter

    2016-01-01

    Various types of stem cell lines have been derived from preimplantation or postimplantation mouse embryos: embryonic stem cell lines, epiblast stem cell lines, and trophoblast stem cell lines. It is not known if extraembryonic endoderm stem (XEN) cell lines can be derived from postimplantation mouse embryos. Here, we report the derivation of 77 XEN cell lines from 85 postimplantation embryos at embryonic day E5.5 or E6.5, in parallel to the derivation of 41 XEN lines from 69 preimplantation embryos at the blastocyst stage. We attain a success rate of 100% of XEN cell line derivation with our E5.5 whole-embryo and E6.5 disaggregated-embryo methods. Immunofluorescence and NanoString gene expression analyses indicate that the XEN cell lines that we derived from postimplantation embryos (post-XEN) are very similar to the XEN cell lines that we derived from preimplantation embryos (pre-XEN) using a conventional method. After injection into blastocysts, post-XEN cells contribute to extraembryonic endoderm in chimeras at E6.5 and E7.5. PMID:27991575

  7. Comparative In Vitro Immune Stimulation Analysis of Primary Human B Cells and B Cell Lines

    PubMed Central

    Van Belle, Kristien; Herman, Jean; Boon, Louis; Waer, Mark

    2016-01-01

    B cell specific immunomodulatory drugs still remain an unmet medical need. Utilisation of validated simplified in vitro models would allow readily obtaining new insights in the complexity of B cell regulation. For this purpose we investigated which human B lymphocyte stimulation assays may be ideally suited to investigate new B lymphocyte immunosuppressants. Primary polyclonal human B cells underwent in vitro stimulation and their proliferation, production of immunoglobulins (Igs) and of cytokines, and expression of cell surface molecules were analysed using various stimuli. ODN2006, a toll-like receptor 9 (TLR9) agonist, was the most potent general B cell stimulus. Subsequently, we investigated on which human B cell lines ODN2006 evoked the broadest immunostimulatory effects. The Namalwa cell line proved to be the most responsive upon TLR9 stimulation and hence may serve as a relevant, homogeneous, and stable B cell model in an in vitro phenotypic assay for the discovery of new targets and inhibitors of the B cell activation processes. As for the read-out for such screening assay, it is proposed that the expression of activation and costimulatory surface markers reliably reflects B lymphocyte activation. PMID:28116319

  8. Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

    PubMed Central

    Xu, Lu; Shen, Qiwen; Mao, Zhongqi; Lee, L. James; Ziouzenkova, Ouliana

    2015-01-01

    Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants. PMID:26066392

  9. Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y.

    PubMed

    Attoff, K; Kertika, D; Lundqvist, J; Oredsson, S; Forsby, A

    2016-09-01

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10pM. Acrylamide significantly reduced the number of neurons starting at 1μM and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

  10. Sorafenib inhibits cell growth but fails to enhance radio- and chemosensitivity of glioblastoma cell lines

    PubMed Central

    Riedel, Matthias; Struve, Nina; Müller-Goebel, Justus; Köcher, Sabrina; Petersen, Cordula; Dikomey, Ekkehard; Rothkamm, Kai; Kriegs, Malte

    2016-01-01

    Background Glioblastomas (GBM) are the most common malignant type of primary brain tumor. GBM are intensively treated with surgery and combined radiochemotherapy using X-irradiation and temozolomide (TMZ) but they are still associated with an extremely poor prognosis, urging for the development of new treatment strategies. To improve the outcome of GBM patients, the small molecule multi-kinase inhibitor sorafenib has moved into focus of recent research. Sorafenib has already been shown to enhance the radio- and radiochemosensitivity of other tumor entities. Whether sorafenib is also able to sensitize GBM cells to radio- and chemotherapy is still an unsolved question which we have addressed in this study. Methods The effect of sorafenib on signaling, proliferation, radiosensitivity, chemosensitivity and radiochemosensitivity was analyzed in six glioblastoma cell lines using Western blot, proliferation- and colony formation assays. Results In half of the cell lines sorafenib clearly inhibited MAPK signaling. We also observed a strong blockage of proliferation, which was, however, not associated with MAPK pathway inhibition. Sorafenib had only minor effects on cell survival when administered alone. Most importantly, sorafenib treatment failed to enhance GBM cell killing by irradiation, TMZ or combined treatment, and instead rather caused resistance in some cell lines. Conclusion Our data suggest that sorafenib treatment may not improve the efficacy of radiochemotherapy in GBM. PMID:27542273

  11. Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

    PubMed Central

    Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

    2014-01-01

    Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

  12. 188Rhenium-induced cell death and apoptosis in a panel of tumor cell lines

    NASA Astrophysics Data System (ADS)

    Antoccia, Antonio; Banzato, Alessandra; Bello, Michele; Bollini, Dante; De Notaristefani, Francesco; Giron, Cecilia; Mazzi, Ulderico; Alafort, Laura Melendez; Moschini, Giuliano; Nadali, Anna; Navarria, Francesco; Perrotta, Andrea; Rosato, Antonio; Tanzarella, Caterina; Uzunov, Nikolay

    2007-02-01

    Assessment of "in vitro" tumor growth inhibition and radiobiological effects, such as apoptosis, have been evaluated in human neoplastic cells of different histotypes (H460 lung cancer cells, U87 glioblastoma, LnCaP prostate tumor cells) treated using solutions of 188Rhenium-perrhenate. The MTT assay, which measures mitochondrial metabolism in the entire cell culture is a recognized test for cytotoxicity and was used in cells exposed 48-72 h to specific activities ranged from 37 to 148 GBq/l. Whereas H460 and LnCaP were particularly sensitive to treatment, U87 glioblastoma cells behaved as radioresistant ones. However, evaluation of 188Re-induced apoptosis indicated that this kind of cell death contributed only marginally to the reduction in cell viability of H460 and LNCaP lines, suggesting the existence of protective mechanisms against apoptosis. In this respect, the membrane receptor, CD44, whose expression is dysregulated in most malignant cell types has proven to alter the response of cancer cells to apoptotic stimuli, including ionizing radiation. Cell samples decorated with a FITC-labelled CD44 antibody indicated, that in H460 and U87 cells the CD44(+) correlated well with an apoptosis-resistant response. Conversely, LnCap cells proven as CD44(-) did not display however sensitivity to radio-induced apoptosis.

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

    SciTech Connect

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

    2013-11-15

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

  14. Sphere Culture of Murine Lung Cancer Cell Lines Are Enriched with Cancer Initiating Cells

    PubMed Central

    Morrison, Brian J.

    2012-01-01

    Cancer initiating cells (CICs) represent a unique cell population essential for the maintenance and growth of tumors. Most in vivo studies of CICs utilize human tumor xenografts in immunodeficient mice. These models provide limited information on the interaction of CICs with the host immune system and are of limited value in assessing therapies targeting CICs, especially immune-based therapies. To assess this, a syngeneic cancer model is needed. We examined the sphere-forming capacity of thirteen murine lung cancer cell lines and identified TC-1 and a metastatic subclone of Lewis lung carcinoma (HM-LLC) as cell lines that readily formed and maintained spheres over multiple passages. TC-1 tumorspheres were not enriched for expression of CD133 or CD44, putative CIC markers, nor did they demonstrate Hoechst 33342 side population staining or Aldefluor activity compared to adherent TC-1 cells. However, in tumorsphere culture, these cells exhibited self-renewal and long-term symmetric division capacity and expressed more Oct-4 compared to adherent cells. HM-LLC sphere-derived cells exhibited increased Oct-4, CD133, and CD44 expression, demonstrated a Hoechst 33342 side population and Aldefluor activity compared to adherent cells or a low metastatic subclone of LLC (LM-LLC). In syngeneic mice, HM-LLC sphere-derived cells required fewer cells to initiate tumorigenesis compared to adherent or LM-LLC cells. Similarly TC-1 sphere-derived cells were more tumorigenic than adherent cells in syngeneic mice. In contrast, in immunocompromised mice, less than 500 sphere or adherent TC-1 cells and less than 1,000 sphere or adherent LLC cells were required to initiate a tumor. We suggest that no single phenotypic marker can identify CICs in murine lung cancer cell lines. Tumorsphere culture may provide an alternative approach to identify and enrich for murine lung CICs. Furthermore, we propose that assessing tumorigenicity of murine lung CICs in syngeneic mice better models the

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

    SciTech Connect

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

    1995-01-01

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

  16. Tubby-like protein superfamily member PLSCR3 functions as a negative regulator of adipogenesis in mouse 3T3-L1 preadipocytes by suppressing induction of late differentiation stage transcription factors

    PubMed Central

    Inokawa, Akira; Inuzuka, Tatsutoshi; Takahara, Terunao; Shibata, Hideki; Maki, Masatoshi

    2015-01-01

    PLSCR3 (phospholipid scramblase 3, Scr3) belongs to the superfamily of membrane-associated transcription regulators named Tubby-like proteins (TULPs). Physiological phospholipid scrambling activities of PLSCRs in vivo have been skeptically argued, and knowledge of the biological functions of Scr3 is limited. We investigated the expression of Scr3 during differentiation of mouse 3T3-L1 preadipocytes by Western blotting (WB) and by reverse-transcription and real-time quantitative PCR (RT-qPCR). The Scr3 protein decreased during 3T3-L1 differentiation accompanied by a reduction in the mRNA level, and there was a significant increase in the amount of Scr3 protein secreted into the culture medium in the form of extracellular microvesicles (exosomes). On the other hand, Scr3 expression did not significantly decrease, and the secretion of Scr3 in 3T3 Swiss-albino fibroblasts (a parental cell-line of 3T3-L1) was not increased by differentiation treatment. Overexpression of human Scr3 during 3T3-L1 differentiation suppressed triacylglycerol accumulation and inhibited induction of the mRNAs of late stage pro-adipogenic transcription factors [CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ)] and X-box-binding protein 1 (XBP1). Expression of early stage pro-adipogenic transcription factors (C/EBPβ and C/EBPδ) was not significantly affected. These results suggest that Scr3 functions as a negative regulator of adipogenesis in 3T3-L1 cells at a specific differentiation stage and that decrease in the intracellular amount of Scr3 protein caused by reduction in Scr3 mRNA expression and enhanced secretion of Scr3 protein appears to be important for appropriate adipocyte differentiation. PMID:26677203

  17. Chromium VI-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and a lymphoblastic leukemia cell line (MOLT-4).

    PubMed

    Gambelunghe, Angela; Piccinini, Renza; Abbritti, Giuseppe; Ambrogi, Maura; Ugolini, Barbara; Marchetti, Cristina; Migliorati, Graziella; Balducci, Chiara; Muzi, Giacomo

    2006-03-01

    Hexavalent chromium compounds are well-documented human carcinogens. In vitro experiments show Cr (VI) induces cell death by apoptosis by activating p53 protein. The aim of this study was to evaluate Cr (VI)-induced apoptosis in a human bronchial epithelial cell line (BEAS-2B) and in a lymphoblastic leukemia cell line (MOLT-4). Cr (VI) caused a dose- and time-dependent increase in the apoptosis rate in both cell lines. Western blotting showed increased p53 protein expression in MOLT-4 cells, but not in BEAS-2B cells, after exposure to 0.5 and 3 muM hexavalent chromium for 12 hours and 4 hours, respectively. Apoptotic cell death induced by Cr (VI) was not decreased by pretreatment with caspase-3, -8, and -9 inhibitors. These preliminary results provide evidence of Cr (VI)-induced apoptosis, which deserves further investigation in occupationally exposed workers.

  18. Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast

    PubMed Central

    Ortega, Francisco G; Fernández-Baldo, Martín A; Fernández, Jorge G; Serrano, María J; Sanz, María I; Diaz-Mochón, Juan J; Lorente, José A; Raba, Julio

    2015-01-01

    In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors. PMID:25844035

  19. Study of antitumor activity in breast cell lines using silver nanoparticles produced by yeast.

    PubMed

    Ortega, Francisco G; Fernández-Baldo, Martín A; Fernández, Jorge G; Serrano, María J; Sanz, María I; Diaz-Mochón, Juan J; Lorente, José A; Raba, Julio

    2015-01-01

    In the present article, we describe a study of antitumor activity in breast cell lines using silver nanoparticles (Ag NPs) synthesized by a microbiological method. These Ag NPs were tested for their antitumor activity against MCF7 and T47D cancer cells and MCF10-A normal breast cell line. We analyzed cell viability, apoptosis induction, and endocytosis activity of those cell lines and we observed that the effects of the biosynthesized Ag NPs were directly related with the endocytosis activity. Moreover, Ag NPs had higher inhibition efficacy in tumor lines than in normal lines of breast cells, which is due to the higher endocytic activity of tumor cells compared to normal cells. In this way, we demonstrate that biosynthesized Ag NPs can be an alternative for the treatment of tumors.

  20. Hesa-A Effects on Cell Cycle Signaling in Esophageal Carcinoma Cell Line

    PubMed Central

    Ahmadian, Nasser; Pashaei-Asl, Roghiyeh; Samadi, Nasser; Rahmati-yamchi, Mohammad; Rashidi, Mohammad-Reza; Ahmadian, Masomeh; Esmaeili, Moosa; Salamat, Faezeh; Besharat, Sima; Joshaghani, Hamid Reza

    2016-01-01

    BACKGROUND Hesa-A is a natural compound with anticancer properties. The exact mechanism of its action in esophageal cancer is not clear, yet. The aim of this study was to evaluate the cell toxicity effect of Hesa-A on the esophageal carcinoma cell lines, KYSE-30, and cell cycle genes expression. METHODS In this study, we tested cell toxicity with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay and flow cytometry to evaluatet he cell cycle arrest. Real time polymerase chain reaction was used to assess the expression of P53, P16, P21, cyclin D1, and cyclin B1 genes. RESULTS Our results showed that Hesa-A is effective in the expression of cell cycling check point proteins. Hesa-A induced an arrest in G2 phase of esophageal cell cycle. The levels of P53 (>13 times), P21 (>21 times), P16, cyclin B1, and cyclin D1 genes were increased 48 hours after Hesa-A treatment. CONCLUSION P21 and P16 expression were the potential mechanisms for G2 arrest of KYSE-30 esophageal cancer cell line by Hesa-A. PMID:27957293

  1. Development and characterization of two porcine monocyte-derived macrophage cell lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell lines Cdelta2+ and Cdelta2- were developed from monocytes obtained from a 10-month-old, crossbred, female pig. These cells morphologically resembled macrophages, stained positively for a-naphthyl esterase and negatively for peroxidase. The cell lines were bactericidal and highly phagocytic. ...

  2. Molecular Integrative Clustering of Asian Gastric Cell Lines Revealed Two Distinct Chemosensitivity Clusters

    PubMed Central

    Choong, Meng Ling; Tan, Shan Ho; Tan, Tuan Zea; Manesh, Sravanthy; Ngo, Anna; Yong, Jacklyn W. Y.; Yang, Henry He; Lee, May Ann

    2014-01-01

    Cell lines recapitulate cancer heterogeneity without the presence of interfering tissue found in primary tumor. Their heterogeneous characteristics are reflected in their multiple genetic abnormalities and variable responsiveness to drug treatments. In order to understand the heterogeneity observed in Asian gastric cancers, we have performed array comparative genomic hybridization (aCGH) on 18 Asian gastric cell lines. Hierarchical clustering and single-sample Gene Set Enrichment Analysis were performed on the aCGH data together with public gene expression data of the same cell lines obtained from the Cancer Cell Line Encyclopedia. We found a large amount of genetic aberrations, with some cell lines having 13 fold more aberrations than others. Frequently mutated genes and cellular pathways are identified in these Asian gastric cell lines. The combined analyses of aCGH and expression data demonstrate correlation of gene copy number variations and expression profiles in human gastric cancer cells. The gastric cell lines can be grouped into 2 integrative clusters (ICs). Gastric cells in IC1 are enriched with gene associated with mitochondrial activities and oxidative phosphorylation while cells in IC2 are enriched with genes associated with cell signaling and transcription regulations. The two clusters of cell lines were shown to have distinct responsiveness towards several chemotherapeutics agents such as PI3 K and proteosome inhibitors. Our molecular integrative clustering provides insight into critical genes and pathways that may be responsible for the differences in survival in response to chemotherapy. PMID:25343454

  3. Chapter 9. routine maintenance and storage of lepidopteran insect cell lines and baculoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The various methods for maintaining (a.k.a., subculturing, splitting, or passaging) established lepidopteran cell lines are described. Three procedures are presented that are appropriate for different cell lines dependent upon the growth characteristics (in particular, cell attachment properties) o...

  4. Genomic Landscape of Primary Mediastinal B-Cell Lymphoma Cell Lines

    PubMed Central

    Nagel, Stefan; Eberth, Sonja; Pommerenke, Claudia; Dirks, Wilhelm G.; Geffers, Robert; Kalavalapalli, Srilaxmi; Kaufmann, Maren; Meyer, Corrina; Faehnrich, Silke; Chen, Suning; Drexler, Hans G.; MacLeod, Roderick A. F.

    2015-01-01

    Primary mediastinal B-Cell lymphoma (PMBL) is a recently defined entity comprising ~2–10% non-Hodgkin lymphomas (NHL). Unlike most NHL subtypes, PMBL lacks recurrent gene rearrangements to serve as biomarkers or betray target genes. While druggable, late chemotherapeutic complications warrant the search for new targets and models. Well characterized tumor cell lines provide unlimited material to serve as preclinical resources for verifiable analyses directed at the discovery of new biomarkers and pathological targets using high throughput microarray technologies. The same cells may then be used to seek intelligent therapies directed at clinically validated targets. Four cell lines have emerged as potential PMBL models: FARAGE, KARPAS-1106P, MEDB-1 and U-2940. Transcriptionally, PMBL cell lines cluster near c(lassical)-HL and B-NHL examples showing they are related but separate entities. Here we document genomic alterations therein, by cytogenetics and high density oligonucleotide/SNP microarrays and parse their impact by integrated global expression profiling. PMBL cell lines were distinguished by moderate chromosome rearrangement levels undercutting cHL, while lacking oncogene translocations seen in B-NHL. In total 61 deletions were shared by two or more cell lines, together with 12 amplifications (≥4x) and 72 homozygous regions. Integrated genomic and transcriptional profiling showed deletions to be the most important class of chromosome rearrangement. Lesions were mapped to several loci associated with PMBL, e.g. 2p15 (REL/COMMD1), 9p24 (JAK2, CD274), 16p13 (SOCS1, LITAF, CIITA); plus new or tenuously associated loci: 2p16 (MSH6), 6q23 (TNFAIP3), 9p22 (CDKN2A/B), 20p12 (PTPN1). Discrete homozygous regions sometimes substituted focal deletions accompanied by gene silencing implying a role for epigenetic or mutational inactivation. Genomic amplifications increasing gene expression or gene-activating rearrangements were respectively rare or absent. Our findings

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

    PubMed Central

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

    2016-01-01

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

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

    SciTech Connect

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

    2010-05-04

    Methylation of CpG islands within the DNA promoter regions is one mechanism that leads to aberrant gene expression in cancer. In particular, the abnormal methylation of CpG islands may silence associated genes. Therefore, using high-throughput microarrays to measure CpG island methylation will lead to better understanding of tumor pathobiology and progression, while revealing potentially new biomarkers. We have examined a recently developed high-throughput technology for measuring genome-wide methylation patterns called mTACL. Here, we propose a computational pipeline for integrating gene expression and CpG island methylation profles to identify epigenetically regulated genes for a panel of 45 breast cancer cell lines, which is widely used in the Integrative Cancer Biology Program (ICBP). The pipeline (i) reduces the dimensionality of the methylation data, (ii) associates the reduced methylation data with gene expression data, and (iii) ranks methylation-expression associations according to their epigenetic regulation. Dimensionality reduction is performed in two steps: (i) methylation sites are grouped across the genome to identify regions of interest, and (ii) methylation profles are clustered within each region. Associations between the clustered methylation and the gene expression data sets generate candidate matches within a fxed neighborhood around each gene. Finally, the methylation-expression associations are ranked through a logistic regression, and their significance is quantified through permutation analysis. Our two-step dimensionality reduction compressed 90% of the original data, reducing 137,688 methylation sites to 14,505 clusters. Methylation-expression associations produced 18,312 correspondences, which were used to further analyze epigenetic regulation. Logistic regression was used to identify 58 genes from these correspondences that showed a statistically signifcant negative correlation between methylation profles and gene expression in the

  7. High-throughput SNP-based authentication of human cell lines

    PubMed Central

    Castro, Felipe; Dirks, Wilhelm G.; Fähnrich, Silke; Hotz-Wagenblatt, Agnes; Pawlita, Michael; Schmitt, Markus

    2012-01-01

    Use of false cell lines remains a major problem in biological research. Short tandem repeat (STR) profiling represents the gold standard technique for cell line authentication. However, mismatch repair (MMR) deficient cell lines are characterized by microsatellite instability, which could force allelic drifts in combination with a selective outgrowth of otherwise persisting side lines, and thus, are likely to be misclassified by STR-profiling. Based on the high-throughput Luminex platform, we developed a 24-plex SNP-profiling assay, called Multiplex Cell Authentication (MCA), for determining authentication of human cell lines. MCA was evaluated by analysing a collection of 436 human cell lines from the DSMZ, previously characterised by eight loci STR profiling. Both assays showed a very high degree of concordance and similar average matching probabilities (~1 × 10−8 for STR-profiling and ~1 × 10−9 for MCA). MCA enabled the detection of less than 3% contaminating human cells. Analysing MMR deficient cell lines, evidence was obtained for a higher robustness of the MCA compared to STR profiling. In conclusion, MCA could complement routine cell line authentication and replace the standard authentication STR technique in case of MSI cell lines. PMID:22700458

  8. The Importance of Physiologically Relevant Cell Lines for Studying Virus–Host Interactions

    PubMed Central

    Hare, David; Collins, Susan; Cuddington, Breanne; Mossman, Karen

    2016-01-01

    Viruses interact intimately with the host cell at nearly every stage of replication, and the cell model that is chosen to study virus infection is critically important. Although primary cells reflect the phenotype of healthy cells in vivo better than cell lines, their limited lifespan makes experimental manipulation challenging. However, many tumor-derived and artificially immortalized cell lines have defects in induction of interferon-stimulated genes and other antiviral defenses. These defects can affect virus replication, especially when cells are infected at lower, more physiologically relevant, multiplicities of infection. Understanding the selective pressures and mechanisms underlying the loss of innate signaling pathways is helpful to choose immortalized cell lines without impaired antiviral defense. We describe the trials and tribulations we encountered while searching for an immortalized cell line with intact innate signaling, and how directed immortalization of primary cells avoids many of the pitfalls of spontaneous immortalization. PMID:27809273

  9. Restriction of human adenovirus replication in Chinese hamster cell lines and their hybrids with human cells.

    PubMed

    Radna, R L; Foellmer, B; Feldman, L A; Francke, U; Ozer, H L

    1987-11-01

    We have found that the replication of human adenovirus (Ad2) is restricted in multiple Chinese hamster cell lines including CHO and V79. The major site of restriction involves differential accumulation of late viral proteins as demonstrated by immunofluorescence assay and polyacrylamide gel electrophoresis with and without prior immunoprecipitation. Synthesis of fiber and penton base are markedly reduced, whereas others, such as the 100K polypeptide, are synthesized efficiently. This pattern of restriction is similar to that previously reported for Ad2 infection of several monkey cell lines; however, the restriction is more marked in the Chinese hamster cell lines. The restriction is most likely due to a deficient cellular function since stable cell hybrids between V79 or CHO and human cells are permissive for virus replication. By analysis of a series of hybrids with reduced numbers of human chromosomes, fiber synthesis was correlated with the presence of the short arm of human chromosome 3. More hybrids showed restoration of fiber synthesis than production of progeny virus, suggesting that more than one unlinked function is required for the latter.

  10. A Human Corneal Epithelial Cell Line Model for Limbal Stem Cell Biology and Limbal Immunobiology.

    PubMed

    Shaharuddin, Bakiah; Ahmad, Sajjad; Md Latar, Nani; Ali, Simi; Meeson, Annette

    2016-10-14

    : Limbal stem cell (LSC) deficiency is a visually debilitating condition caused by abnormal maintenance of LSCs. It is treated by transplantation of donor-derived limbal epithelial cells (LECs), the success of which depends on the presence and quality of LSCs within the transplant. Understanding the immunobiological responses of these cells within the transplants could improve cell engraftment and survival. However, human corneal rings used as a source of LSCs are not always readily available for research purposes. As an alternative, we hypothesized that a human telomerase-immortalized corneal epithelial cell (HTCEC) line could be used as a model for studying LSC immunobiology. HTCEC constitutively expressed human leukocyte antigen (HLA) class I but not class II molecules. However, when stimulated by interferon-γ, HTCECs then expressed HLA class II antigens. Some HTCECs were also migratory in response to CXCL12 and expressed stem cell markers, Nanog, Oct4, and Sox2. In addition because both HTCECs and LECs contain side population (SP) cells, which are an enriched LSC population, we used these SP cells to show that some HTCEC SP cells coexpressed ABCG2 and ABCB5. HTCEC SP and non-side population (NSP) cells also expressed CXCR4, but the SP cells expressed higher levels. Both were capable of colony formation, but the NSP colonies were smaller and contained fewer cells. In addition, HTCECs expressed ΔNp63α. These results suggest the HTCEC line is a useful model for further understanding LSC biology by using an in vitro approach without reliance on a supply of human tissue.

  11. Trans-10,cis-12 conjugated linoleic acid (CLA) interferes with lipid droplet accumulation during 3T3-L1 preadipocyte differentiation.

    PubMed

    Yeganeh, Azadeh; Taylor, Carla G; Tworek, Leslee; Poole, Jenna; Zahradka, Peter

    2016-07-01

    In this study, we hypothesize that the biologically active isomers of conjugated linoleic acid (CLA), cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12) CLA, have different effects on early and late stages 3T3-L1 preadipocyte differentiation. Both c9-t11 and t10-c12CLA stimulated early stage pre-adipocyte differentiation (day 2), while t10-c12CLA inhibited late differentiation (day 8) as determined by lipid droplet numbers and both perilipin-1 levels and phosphorylation state. At day 8, the adipokines adiponectin, chemerin and adipsin were all reduced in t10-c12CLA treated cells versus control cells. Immunofluorescence microscopy showed perilipin-1 was present solely on lipid droplets on day 8 in t10-c12 treated 3T3-L1 cells, whereas preilipin-1 was also located in the perinuclear region in control and c9-t11 treated cells. The t10-c12CLA isomer also decreased levels of hormone-sensitive lipase and inhibited lipolysis. These findings indicate that the decrease in lipid droplets caused by t10-c12CLA is the result of an inhibition of lipid droplet production during adipogenesis rather than a stimulation of lipolysis. Additionally, treatment with Gö6976 blocked the effect of t10-c12CLA on perilipin-1 phosphorylation, implicating PKCα in perilipin-1 phosphorylation, and thus a regulator of triglyceride catabolism. These data are supported by evidence that t10-c12CLA activated PKCα. These are the first data to show that CLA isomers can affect lipid droplet dynamics in adipocytes through PKCα.

  12. Growth Inhibition of Breast Tumor Cells by Hypodense and Normodense Eosinophilic Cell Lines

    DTIC Science & Technology

    2001-07-01

    with a Fluorescent Activated Cell Sorter using CCR3 (Eotaxin Receptor) and CD49d antibodies. 6 Supernatants (24hr, 48hr and 72hr) from FACS sorted...MDA-MB-23 1 cells (fig. 26). The last cell line tested was established from the GRC.014.24S subline (which is CCR3 ), using the FACS Sorter and...Abdelnaby A, Hunter KA, Howland C, Brown R, Awich J, and Oredipe 0. (Manuscript In Preparation) CCR3 +, CD49÷’ and CD 15’ EBV Transformed Sublines Inhibit

  13. Erythropoietic differentiation of a human embryonic stem cell line harbouring the sickle cell anaemia mutation.

    PubMed

    Pryzhkova, Marina V; Peters, Ann; Zambidis, Elias T

    2010-08-01

    Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and in-situ immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research.

  14. Hybrids between tobacco crown gall cells and normal somatic cells of Atropa belladonna : Isolation and characterization of cell lines.

    PubMed

    Gleba, Y Y; Kanevsky, I F; Skarzhynskaya, M V; Komarnitsky, I K; Cherep, N N

    1986-10-01

    Protoplast fusion of Nicotiana tabacum (B6S3) crown gall cells and Atropa belladonna leaf mesophyll cells was carried out. Hybrids were selected for their capacity to grow on hormone-free media and to green in light. Shoots incapable of rhizogenesis were regenerated on the same media and grafted onto normal plants of different species. 57 hybrid cell lines differing in their genetic constitution were produced. Analysis of hybrid lines involved the determination of the lysopine dehydrogenase (LpDH) activity and the molecular forms of esterase and amylase, a restriction analysis of chloroplast DNA and a cytogenetic study.

  15. Heterogeneity of cell lines derived after transformation of early passage rodent cells by the Ha-ras1 human oncogene.

    PubMed

    Spandidos, D A; Freshney, M; Wilkie, N M

    1985-01-01

    The chromosome patterns of Chinese hamster cell lines derived after immortalization or tumorigenic conversion of early passage cells with recombinants carrying the mutated T24 or the normal human Ha-ras1 gene have been characterized by trypsin-Giemsa banding. Whereas immortalized Chinese hamster cell lines exhibited a near normal karyotype, tumorigenic cell lines were found to have abnormal karyotypes carrying marker chromosomes. Moreover, chromosomal patterns correlated with growth in semisolid media and tumourigenicity in nude mice. Similarly, malignant conversion of early passage Syrian hamster cells, with a recombinant carrying the mutated T24 human Ha-ras1 gene, resulted in cells with a near diploid karyotype. On the other hand, tumorigenic conversion of early passage Wistar rat cells with the same oncogene produced cell lines with heteroploid karyotypes. More chromosomal alterations have been observed during further growth of these cells. It is suggested that the transformed phenotype in these cells may be dependent on the chromosomal instability.

  16. HO-1 inhibits preadipocyte proliferation and differentiation at the onset of obesity via ROS dependent activation of Akt2

    PubMed Central

    Wagner, Gabriel; Lindroos-Christensen, Josefine; Einwallner, Elisa; Husa, Julia; Zapf, Thea-Christin; Lipp, Katharina; Rauscher, Sabine; Gröger, Marion; Spittler, Andreas; Loewe, Robert; Gruber, Florian; Duvigneau, J. Catharina; Mohr, Thomas; Sutterlüty-Fall, Hedwig; Klinglmüller, Florian; Prager, Gerhard; Huppertz, Berthold; Yun, Jeanho; Wagner, Oswald; Esterbauer, Harald; Bilban, Martin

    2017-01-01

    Excessive accumulation of white adipose tissue (WAT) is a hallmark of obesity. The expansion of WAT in obesity involves proliferation and differentiation of adipose precursors, however, the underlying molecular mechanisms remain unclear. Here, we used an unbiased transcriptomics approach to identify the earliest molecular underpinnings occuring in adipose precursors following a brief HFD in mice. Our analysis identifies Heme Oxygenase-1 (HO-1) as strongly and selectively being upregulated in the adipose precursor fraction of WAT, upon high-fat diet (HFD) feeding. Specific deletion of HO-1 in adipose precursors of Hmox1fl/flPdgfraCre mice enhanced HFD-dependent visceral adipose precursor proliferation and differentiation. Mechanistically, HO-1 reduces HFD-induced AKT2 phosphorylation via ROS thresholding in mitochondria to reduce visceral adipose precursor proliferation. HO-1 influences adipogenesis in a cell-autonomous way by regulating events early in adipogenesis, during the process of mitotic clonal expansion, upstream of Cebpα and PPARγ. Similar effects on human preadipocyte proliferation and differentiation in vitro were observed upon modulation of HO-1 expression. This collectively renders HO-1 as an essential factor linking extrinsic factors (HFD) with inhibition of specific downstream molecular mediators (ROS & AKT2), resulting in diminished adipogenesis that may contribute to hyperplastic adipose tissue expansion. PMID:28102348

  17. Novel effect of helenalin on Akt signaling and Skp2 expression in 3T3-L1 preadipocytes

    SciTech Connect

    Auld, Corinth A.; Hopkins, Robin G.; Fernandes, Karishma M.; Morrison, Ron F. . E-mail: ron_morrison@uncg.edu

    2006-07-21

    We have previously shown that the F-box protein, Skp2, is highly regulated during preadipocyte proliferation and plays a mechanistic role in p27 degradation during cell cycle progression. Data presented here demonstrate that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 protein accumulation during early phases of 3T3-L1 adipocyte differentiation. Furthermore, helenalin was shown to completely block p27 degradation, cyclin A accumulation, and G{sub 1}/S transition resulting in G{sub 1} arrest. Helenalin was also shown to block Skp2 mRNA accumulation in a concentration-dependent manner and to completely suppress hormonally induced Skp2 promoter activity suggesting transcriptional mechanisms were involved. Examination of signaling events previously determined to be important for Skp2 upregulation during adipogenesis revealed impaired Akt phosphorylation immediately preceding the inhibitory effect of helenalin on Skp2 mRNA accumulation. These studies demonstrate a novel effect of helenalin on Skp2 regulation and growth factor receptor signaling during early stages of adipocyte differentiation.

  18. Specific estradiol biosynthetic pathway in choriocarcinoma (JEG-3) cell line.

    PubMed

    Samson, Mélanie; Labrie, Fernand; Luu-The, Van

    2009-09-01

    Estradiol (E2) plays a crucial role in all reproduction processes. In the placenta, it is well recognized that E2 is synthesized from fetal dehydroepiandrosterone sulfate (DHEAS). However, there is some controversy about the biosynthetic pathway involved, some authors suggest that E2 is produced by aromatization of testosterone (T), while others suggest that E2 is produced by the conversion of estrone (E1) into E2 by type 1 17beta-HSD, subsequent to the aromatization of 4-androstenedione (4-dione) into E1. In the present report, using the precursor [(14)C]DHEA, inhibitors of steroidogenic enzymes (chemical inhibitors and siRNA) and a choriocarcinoma (JEG-3) cell line that expresses all the enzymes necessary to transform DHEA into E2, we could determine the sequential steps and the specific steroidogenic enzymes involved in the transformation of DHEA into E2. Quantification of mRNA expression levels using real-time PCR, strongly suggests that type 1 3beta-hydroxysteroid dehydrogenase (3beta-HSD1), aromatase and type 1 17beta-HSD (17beta-HSD1) that are highly expressed in JEG-3 cells are the enzymes responsible for the transformation of DHEA into E2. Analysis of the intermediates produced in the absence and presence of 3beta-HSD, aromatase and 17beta-HSD1 inhibitors permits to determine the following sequential steps: DHEA is transformed into 4-dione by 3beta-HSD1, then 4-dione is aromatized into E1 by aromatase and E1 is finally transformed into E2 by 17beta-HSD1. Our data are clearly in favor of the pathway in which the step of aromatization precedes the step of reduction by 17beta-HSD.

  19. A Bovine Cell Line That Can Be Infected by Natural Sheep Scrapie Prions

    PubMed Central

    Oelschlegel, Anja M.; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schaetzl, Hermann; Groschup, Martin H.

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases. PMID:25565633

  20. Subclones in B-lymphoma cell lines: isogenic models for the study of gene regulation

    PubMed Central

    Quentmeier, Hilmar; Pommerenke, Claudia; Ammerpohl, Ole; Geffers, Robert; Hauer, Vivien; MacLeod, Roderick AF; Nagel, Stefan; Romani, Julia; Rosati, Emanuela; Rosén, Anders; Uphoff, Cord C; Zaborski, Margarete; Drexler, Hans G

    2016-01-01

    Genetic heterogeneity though common in tumors has been rarely documented in cell lines. To examine how often B-lymphoma cell lines are comprised of subclones, we performed immunoglobulin (IG) heavy chain hypermutation analysis. Revealing that subclones are not rare in B-cell lymphoma cell lines, 6/49 IG hypermutated cell lines (12%) consisted of subclones with individual IG mutations. Subclones were also identified in 2/284 leukemia/lymphoma cell lines exhibiting bimodal CD marker expression. We successfully isolated 10 subclones from four cell lines (HG3, SU-DHL-5, TMD-8, U-2932). Whole exome sequencing was performed to molecularly characterize these subclones. We describe in detail the clonal structure of cell line HG3, derived from chronic lymphocytic leukemia. HG3 consists of three subclones each bearing clone-specific aberrations, gene expression and DNA methylation patterns. While donor patient leukemic cells were CD5+, two of three HG3 subclones had independently lost this marker. CD5 on HG3 cells was regulated by epigenetic/transcriptional mechanisms rather than by alternative splicing as reported hitherto. In conclusion, we show that the presence of subclones in cell lines carrying individual mutations and characterized by sets of differentially expressed genes is not uncommon. We show also that these subclones can be useful isogenic models for regulatory and functional studies. PMID:27566572

  1. A bovine cell line that can be infected by natural sheep scrapie prions.

    PubMed

    Oelschlegel, Anja M; Geissen, Markus; Lenk, Matthias; Riebe, Roland; Angermann, Marlies; Schatzl, Herman; Schaetzl, Hermann; Groschup, Martin H

    2015-01-01

    Cell culture systems represent a crucial part in basic prion research; yet, cell lines that are susceptible to prions, especially to field isolated prions that were not adapted to rodents, are very rare. The purpose of this study was to identify and characterize a cell line that was susceptible to ruminant-derived prions and to establish a stable prion infection within it. Based on species and tissue of origin as well as PrP expression rate, we pre-selected a total of 33 cell lines that were then challenged with natural and with mouse propagated BSE or scrapie inocula. Here, we report the successful infection of a non-transgenic bovine cell line, a sub-line of the bovine kidney cell line MDBK, with natural sheep scrapie prions. This cell line retained the scrapie infection for more than 200 passages. Selective cloning resulted in cell populations with increased accumulation of PrPres, although this treatment was not mandatory for retaining the infection. The infection remained stable, even under suboptimal culture conditions. The resulting infectivity of the cells was confirmed by mouse bioassay (Tgbov mice, Tgshp mice). We believe that PES cells used together with other prion permissive cell lines will prove a valuable tool for ongoing efforts to understand and defeat prions and prion diseases.

  2. Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos

    SciTech Connect

    Fang, Zhen F.; Gai, Hui; Huang, You Z.; Li, Shan G.; Chen, Xue J.; Shi, Jian J.; Wu, Li; Liu, Ailian; Xu, Ping; Sheng, Hui Z. . E-mail: hzsheng2003@yahoo.com

    2006-11-01

    Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.

  3. Cell culture-induced aberrant methylation of the imprinted IG DMR in human lymphoblastoid cell lines.

    PubMed

    Saferali, Aabida; Grundberg, Elin; Berlivet, Soizik; Beauchemin, Hugues; Morcos, Lisanne; Polychronakos, Constantin; Pastinen, Tomi; Graham, Jinko; McNeney, Brad; Naumova, Anna K

    2010-01-01

    DNA methylation patterns are often poorly conserved through cell culturing. To determine the effect of cell immortalization and culture on DNA methylation profiles, we analyzed methylation in the differentially methylated regions (DMR) of five imprinted domains: the intergenic (IG) DMR on chromosome 14q32; potassium voltage-gated channel, KQT-like subfamily, member 1, (KCNQ1); small nuclear ribonucleoprotein polypeptide N (SNRPN), mesoderm specific transcript homolog (MEST); and H19 in lymphoblastoid cell lines (LCLs). In the IG DMR we found an aberrant methylation pattern that was consistent through all the cell lines tested and significantly different from that of noncultured peripheral blood cells. Using a generalized linear mixed model to compare methylation profiles, we show that recently derived LCLs significantly differ from the CEPH LCLs. This implies a gradual cell-culture related deterioration of DNA methylation in the IG DMR with at least two steps that may be identified: loss of methylation at CG sites 1 and 8; and loss of allelic differences in DNA methylation. The IG DMR methylation profile also confirms the high level of clonality of the CEPH LCLs. We conclude that non-transformed primary cells may be less susceptible to epigenetic anomalies and therefore may provide a more accurate reflection of gene expression in vivo.

  4. Tumor Suppressors Status in Cancer Cell Line Encyclopedia

    PubMed Central

    Sonkin, Dmitriy; Hassan, Mehedi; Murphy, Denis J.; Tatarinova, Tatiana V.

    2013-01-01

    Tumor suppressors play a major role in the etiology of human cancer, and typically achieve a tumor promoting effect upon complete functional inactivation. Bi-allelic inactivation of tumor suppressors may occur through genetic mechanisms (such as loss-of-function mutation, copy number (CN) loss, or loss-of-heterozygosity (LOH)), epigenetic mechanisms (such as promoter methylation or histone modification), or a combination of the two. We report systematically derived status of 69 known or putative tumor suppressors, across 799 samples of the Cancer Cell Line Encyclopedia. In order to generate such resource we constructed a novel comprehensive computational framework for the assessment of tumor suppressor functional “status”. This approach utilizes several orthogonal genomic data types, including mutation data, copy number, LOH and expression. Through correlation with additional data types (compound sensitivity and gene set activity) we show that this integrative method provides a more accurate assessment of tumor suppressor status than can be inferred by expression, copy number, or mutation alone. This approach has the potential for a more realistic assessment of tumor suppressor genes for both basic and translational oncology research. PMID:23639312

  5. Tumor suppressors status in cancer cell line Encyclopedia.

    PubMed

    Sonkin, Dmitriy; Hassan, Mehedi; Murphy, Denis J; Tatarinova, Tatiana V

    2013-08-01

    Tumor suppressors play a major role in the etiology of human cancer, and typically achieve a tumor-promoting effect upon complete functional inactivation. Bi-allelic inactivation of tumor suppressors may occur through genetic mechanisms (such as loss of function mutation, copy number (CN) loss, or loss of heterozygosity (LOH)), epigenetic mechanisms (such as promoter methylation or histone modification), or a combination of the two. We report systematically derived status of 69 known or putative tumor suppressors, across 799 samples of the Cancer Cell Line Encyclopedia. In order to generate such resource we constructed a novel comprehensive computational framework for the assessment of tumor suppressor functional "status". This approach utilizes several orthogonal genomic data types, including mutation data, copy number, LOH and expression. Through correlation with additional data types (compound sensitivity and gene set activity) we show that this integrative method provides a more accurate assessment of tumor suppressor status than can be inferred by expression, copy number, or mutation alone. This approach has the potential for a more realistic assessment of tumor suppressor genes for both basic and translational oncology research.

  6. Efficient DNA fingerprinting method for the identification of cross-culture contamination of cell lines.

    PubMed

    Matsuo, Y; Nishizaki, C; Drexler, H G

    1999-09-01

    In order to identify cross-culture contamination of cell lines, we applied DNA fingerprinting using variable number of tandem repeat (VNTR) loci and short tandem repeat (STR) loci amplified by polymerase chain reaction (PCR) instead of a radioisotope labeled multilocus probe. Eleven cell lines were used for the Apo B and D1S80 loci detection, and twelve cell lines were examined in the Y-chromosome analysis. The data obtained from the sister cell lines NALM-6 and B85, two MOLM-1 cultures from two cryopreserved tubes, and four subclones of BALM-9 and its sister cell line BALM-10, displayed clear and distinct bands of each PCR product for both Apo B and D1S80. Detection of a Y-chromosome DNA sequence is another very informative marker for the identification of cell lines, if the Y-chromosome is present. We examined eight cell lines for the expression of four STR loci; the data thus generated were compared with the results previously reported from other laboratories. The resulting electrophoretic banding patterns showed that our "home-made" STR detection system is a useful and efficient tool for the authentication of cell lines. PCR detection of VNTR and STR loci represents a simple, rapid and powerful DNA fingerprinting technique to authenticate human cell lines and to detect cross-culture contamination. This PCR technique may be used in lieu of the more time-consuming, labor-intensive and radioactive Southern blot multilocus method.

  7. Annona squamosa Linn: cytotoxic activity found in leaf extract against human tumor cell lines.

    PubMed

    Wang, De-Shen; Rizwani, Ghazala H; Guo, Huiqin; Ahmed, Mansoor; Ahmed, Maryam; Hassan, Syed Zeeshan; Hassan, Amir; Chen, Zhe-Sheng; Xu, Rui-Hua

    2014-09-01

    Cancer is a common cause of death in human populations. Surgery, chemotherapy and radiotherapy still remain the corner stone of treatment. However, herbal medicines are gaining popularity on account of their lesser harmful side effects on non-targeted human cells and biological environment. Annona squamosa Linn is a common delicious edible fruit and its leaf have been used for the treatment in various types of diseases. The objective of present study is to determine the anticancer potential of the organic and aqueous extracts of leaf of Annona squamosa L. MTT (3-(4, 5-dimethylthiazole-2yl)-2, 5-biphenyl tetrazolium bromide) assay against hepatocellular carcinoma cell line BEL-7404, lung cancer line H460, human epidermoid carcinoma cell line KB-3-1, prostatic cancer cell line DU145, breast carcinoma cell line MDA-MB-435, and colon cancer cell line HCT-116 Human primary embryonic kidney cell line HEK293 as control were used for the study. The crude extract (Zcd) and Ethyl acetate extract (ZE) were found significant anticancer activity only on human epidermoid carcinoma cell line KB-3-1 and colon cancer cell line HCT-116.

  8. Oncogenic mutation profiling in new lung cancer and mesothelioma cell lines

    PubMed Central

    Lam, David CL; Luo, Susan Y; Deng, Wen; Kwan, Johnny SH; Rodriguez-Canales, Jaime; Cheung, Annie LM; Cheng, Grace HW; Lin, Chi-Ho; Wistuba, Ignacio I; Sham, Pak C; Wan, Thomas SK; Tsao, Sai-Wah

    2015-01-01

    Background Thoracic tumor, especially lung cancer, ranks as the top cancer mortality in most parts of the world. Lung adenocarcinoma is the predominant subtype and there is increasing knowledge on therapeutic molecular targets, namely EGFR, ALK, KRAS, and ROS1, among lung cancers. Lung cancer cell lines established with known clinical characteristics and molecular profiling of oncogenic targets like ALK or KRAS could be useful tools for understanding the biology of known molecular targets as well as for drug testing and screening. Materials and methods Five new cancer cell lines were established from pleural fluid or biopsy tissues obtained from Chinese patients with primary lung adenocarcinomas or malignant pleural mesothelioma. They were characterized by immunohistochemistry, growth kinetics, tests for tumorigenicity, EGFR and KRAS gene mutations, ALK gene rearrangement and OncoSeq mutation profiling. Results These newly established lung adenocarcinoma and mesothelioma cell lines were maintained for over 100 passages and demonstrated morphological and immunohistochemical features as well as growth kinetics of tumor cell lines. One of these new cell lines bears EML4-ALK rearrangement variant 2, two lung cancer cell lines bear different KRAS mutations at codon 12, and known single nucleotide polymorphism variants were identified in these cell lines. Discussion Four new lung adenocarcinoma and one mesothelioma cell lines were established from patients with different clinical characteristics and oncogenic mutation profiles. These characterized cell lines and their mutation profiles will provide resources for exploration of lung cancer and mesothelioma biology with regard to the presence of known oncogenic mutations. PMID:25653542

  9. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery.

    PubMed

    Santana-Filho, Arquimedes Paixão de; Jacomasso, Thiago; Riter, Daniel Suss; Barison, Andersson; Iacomini, Marcello; Winnischofer, Sheila Maria Brochado; Sassaki, Guilherme Lanzi

    2017-02-15

    Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of (1)H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines.

  10. NMR metabolic fingerprints of murine melanocyte and melanoma cell lines: application to biomarker discovery

    PubMed Central

    Santana-Filho, Arquimedes Paixão de; Jacomasso, Thiago; Riter, Daniel Suss; Barison, Andersson; Iacomini, Marcello; Winnischofer, Sheila Maria Brochado; Sassaki, Guilherme Lanzi

    2017-01-01

    Melanoma is the most aggressive type of skin cancer and efforts to improve the diagnosis of this neoplasia are largely based on the use of cell lines. Metabolomics is currently undergoing great advancements towards its use to screening for disease biomarkers. Although NMR metabolomics includes both 1D and 2D methodologies, there is a lack of data in the literature regarding heteronuclear 2D NMR assignments of the metabolome from eukaryotic cell lines. The present study applied NMR-based metabolomics strategies to characterize aqueous and lipid extracts from murine melanocytes and melanoma cell lines with distinct tumorigenic potential, successfully obtaining fingerprints of the metabolites from the extracts of the cell lines by means of 2D NMR HSQC correlation maps. Relative amounts of the identified metabolites were compared between the 4 cell lines. Multivariate analysis of 1H NMR data was able not only to differentiate the melanocyte cell line from the tumorigenic ones but also distinguish among the 3 tumorigenic cell lines. We also investigated the effects of mitogenic agents, and found that they can markedly influence the metabolome of the melanocyte cell line, resembling the pattern of most proliferative cell lines. PMID:28198377

  11. Development, characterization, conservation and storage of fish cell lines: a review.

    PubMed

    Lakra, W S; Swaminathan, T Raja; Joy, K P

    2011-03-01

    Cell lines provide an important biological tool for carrying out investigations into physiology, virology, toxicology, carcinogenesis and transgenics. Teleost fish cell lines have been developed from a broad range of tissues such as ovary, fin, swim bladder, heart, spleen, liver, eye muscle, vertebrae, brain, skin. One hundred and twenty-four new fish cell lines from different fish species ranging from grouper to eel have been reported since the last review by Fryer and Lannan (J Tissue Culture Methods 16: 87-94, 1994). Among the cell lines listed, more than 60% were established from species from Asia, which contributes more than 80% of total fish production. This includes 59 cell lines from 19 freshwater, 54 from 22 marine and 11 from 3 brackish water fishes. Presently, about 283 cell lines have been established from finfish around the world. In addition to the listing and a scientific update on new cell lines, the importance of authentication, applications, cross-contamination and implications of overpassaged cell lines has also been discussed in this comprehensive review. The authors feel that the review will serve an updated database for beginners and established researchers in the field of fish cell line research and development.

  12. Cell surface glycopeptides from human intestinal epithelial cell lines derived from normal colon and colon adenocarcinomas

    SciTech Connect

    Youakim, A.; Herscovics, A.

    1985-11-01

    The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-(2-TH)mannose or L-(5,6-TH)fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with (2-TH)mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with (2-TH)mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-(1,6-TH)glucosamine and L-(1- UC)fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced TH-labeled N-acetylglucosamine and N-acetylgalactosamine.

  13. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

    PubMed

    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P < 0.05). After transferring cloned embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  14. Antisense bcl-2 treatment increases programmed cell death in non-small cell lung cancer cell lines.

    PubMed

    Koty, P P; Zhang, H; Levitt, M L

    1999-02-01

    Programmed cell death (PCD) is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the ratio of PCD inducers (Bax) or inhibitors (Bcl-2). An abnormally high ratio of Bcl-2 to Bax prevents PCD, thus contributing to resistance to chemotherapeutic agents, many of which are capable of inducing PCD. Non-small cell lung cancer (NSCLC) cells demonstrate resistance to these PCD-inducing agents. If Bcl-2 prevents NSCLC cells from entering the PCD pathway, then reducing the amount of endogenous Bcl-2 product may allow these cells to spontaneously enter the PCD pathway. Our purpose was to determine the effects of bcl-2 antisense treatment on the levels of programmed cell death in NSCLC cells. First, we determined whether bcl-2 and bax mRNA were expressed in three morphologically distinct NSCLC cell lines: NCI-H226 (squamous), NCI-H358 (adenocarcinoma), and NCI-H596 (adenosquamous). Cells were then exposed to synthetic antisense bcl-2 oligonucleotide treatment, after which programmed cell death was determined, as evidenced by DNA fragmentation. Bcl-2 protein expression was detected immunohistochemically. All three NSCLC cell lines expressed both bcl-2 and bax mRNA and had functional PCD pathways. Synthetic antisense bcl-2 oligonucleotide treatment resulted in decreased Bcl-2 levels, reduced cell proliferation, decreased cell viability, and increased levels of spontaneous PCD. This represents the first evidence that decreasing Bcl-2 in three morphologically distinct NSCLC cell lines allows the cells to spontaneously enter a PCD pathway. It also indicates the potential therapeutic use of antisense bcl-2 in the treatment of NSCLC.

  15. Generation of breast cancer stem cells by steroid hormones in irradiated human mammary cell lines.

    PubMed

    Vares, Guillaume; Cui, Xing; Wang, Bing; Nakajima, Tetsuo; Nenoi, Mitsuru

    2013-01-01

    Exposure to ionizing radiation was shown to result in an increased risk of breast cancer. There is strong evidence that steroid hormones influence radiosensitivity and breast cancer risk. Tumors may be initiated by a small subpopulation of cancer stem cells (CSCs). In order to assess whether the modulation of radiation-induced breast cancer risk by steroid hormones could involve CSCs, we measured by flow cytometry the proportion of CSCs in irradiated breast cancer cell lines after progesterone and estrogen treatment. Progesterone stimulated the expansion of the CSC compartment both in progesterone receptor (PR)-positive breast cancer cells and in PR-negative normal cells. In MCF10A normal epithelial PR-negative cells, progesterone-treatment and irradiation triggered cancer and stemness-associated microRNA regulations (such as the downregulation of miR-22 and miR-29c expression), which resulted in increased proportions of radiation-resistant tumor-initiating CSCs.

  16. Identifying anti-growth factors for human cancer cell lines through genome-scale metabolic modeling

    PubMed Central

    Ghaffari, Pouyan; Mardinoglu, Adil; Asplund, Anna; Shoaie, Saeed; Kampf, Caroline; Uhlen, Mathias; Nielsen, Jens

    2015-01-01

    Human cancer cell lines are used as important model systems to study molecular mechanisms associated with tumor growth, hereunder how genomic and biological heterogeneity found in primary tumors affect cellular phenotypes. We reconstructed Genome scale metabolic models (GEMs) for eleven cell lines based on RNA-Seq data and validated the functionality of these models with data from metabolite profiling. We used cell line-specific GEMs to analyze the differences in the metabolism of cancer cell lines, and to explore the heterogeneous expression of the metabolic subsystems. Furthermore, we predicted 85 antimetabolites that can inhibit growth of, or even kill, any of the cell lines, while at the same time not being toxic for 83 different healthy human cell types. 60 of these antimetabolites were found to inhibit growth in all cell lines. Finally, we experimentally validated one of the predicted antimetabolites using two cell lines with different phenotypic origins, and found that it is effective in inhibiting the growth of these cell lines. Using immunohistochemistry, we also showed high or moderate expression levels of proteins targeted by the validated antimetabolite. Identified anti-growth factors for inhibition of cell growth may provide leads for the development of efficient cancer treatment strategies. PMID:25640694

  17. Genomic and phenotypic profiles of two Brazilian breast cancer cell lines derived from primary human tumors

    PubMed Central

    CORRÊA, NATÁSSIA C.R.; KUASNE, HELLEN; FARIA, JERUSA A.Q.A.; SEIXAS, CIÇA C.S.; SANTOS, IRIA G.D.; ABREU, FRANCINE B.; NONOGAKI, SUELY; ROCHA, RAFAEL M.; SILVA, GERLUZA APARECIDA BORGES; GOBBI, HELENICE; ROGATTO, SILVIA R.; GOES, ALFREDO M.; GOMES, DAWIDSON A.

    2013-01-01

    Breast cancer is the most common type of cancer among women worldwide. Research using breast cancer cell lines derived from primary tumors may provide valuable additional knowledge regarding this type of cancer. Therefore, the aim of this study was to investigate the phenotypic profiles of MACL-1 and MGSO-3, the only Brazilian breast cancer cell lines available for comparative studies. We evaluated the presence of hormone receptors, proliferation, differentiation and stem cell markers, using immunohistochemical staining of the primary tumor, cultured cells and xenografts implanted in immunodeficient mice. We also investigated the ability of the cell lines to form colonies and copy number alterations by array comparative genomic hybridization. Histopathological analysis showed that the invasive primary tumor from which the MACL-1 cell line was derived, was a luminal A subtype carcinoma, while the ductal carcinoma in situ (DCIS) that gave rise to the MGSO-3 cell line was a HER2 subtype tumor, both showing different proliferation levels. The cell lines and the tumor xenografts in mice preserved their high proliferative potential, but did not maintain the expression of the other markers assessed. This shift in expression may be due to the selection of an ‘establishment’ phenotype in vitro. Whole-genome DNA evaluation showed a large amount of copy number alterations (CNAs) in the two cell lines. These findings render MACL-1 and MGSO-3 the first characterized Brazilian breast cancer cell lines to be potentially used for comparative research. PMID:23404580

  18. Whole-exome characterization of pancreatic neuroendocrine tumor cell lines BON-1 and QGP-1.

    PubMed

    Vandamme, Timon; Peeters, Marc; Dogan, Fadime; Pauwels, Patrick; Van Assche, Elvire; Beyens, Matthias; Mortier, Geert; Vandeweyer, Geert; de Herder, Wouter; Van Camp, Guy; Hofland, Leo J; Op de Beeck, Ken

    2015-04-01

    The human BON-1 and QGP-1 cell lines are two frequently used models in pancreatic neuroendocrine tumor (PNET) research. Data on the whole-exome genetic constitution of these cell lines is largely lacking. This study presents, to our knowledge, the first whole-exome profile of the BON-1 and QGP-1 cell lines. Cell line identity was confirmed by short tandem repeat profiling. Using GTG-banding and a CytoSNP-12v2 Beadchip array, cell line ploidy and chromosomal alterations were determined in BON-1 and QGP-1. The exomes of both cell lines were sequenced on Ilumina's HiSeq next-generation sequencing (NGS) platform. Single-nucleotide variants (SNVs) and insertions and deletions (indels) were detected using the Genome Analysis ToolKit. SNVs were validated by Sanger sequencing. Ploidy of BON-1 and QGP-1 was 3 and 4 respectively, with long stretches of loss of heterozygosity across multiple chromosomes, which is associated with aggressive tumor behavior. In BON-1, 57 frameshift indels and 1725 possible protein-altering SNVs were identified in the NGS data. In the QGP-1 cell line, 56 frameshift indels and 1095 SNVs were identified. ATRX, a PNET-associated gene, was mutated in both cell lines, while mutation of TSC2 was detected in BON-1. A mutation in NRAS was detected in BON-1, while KRAS was mutated in QGP-1, implicating aberrations in the RAS pathway in both cell lines. Homozygous mutations in TP53 with possible loss of function were identified in both cell lines. Various MUC genes, implicated in cell signaling, lubrication and chemical barriers, which are frequently expressed in PNET tissue samples, showed homozygous protein-altering SNVs in the BON-1 and QGP-1 cell lines.

  19. Check your cultures! A list of cross-contaminated or misidentified cell lines.

    PubMed

    Capes-Davis, Amanda; Theodosopoulos, George; Atkin, Isobel; Drexler, Hans G; Kohara, Arihiro; MacLeod, Roderick A F; Masters, John R; Nakamura, Yukio; Reid, Yvonne A; Reddel, Roger R; Freshney, R Ian

    2010-07-01

    Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

  20. Differentiation of human ESCs to retinal ganglion cells using a CRISPR engineered reporter cell line

    PubMed Central

    Sluch, Valentin M.; Davis, Chung-ha O.; Ranganathan, Vinod; Kerr, Justin M.; Krick, Kellin; Martin, Russ; Berlinicke, Cynthia A.; Marsh-Armstrong, Nicholas; Diamond, Jeffrey S.; Mao, Hai-Quan; Zack, Donald J.

    2015-01-01

    Retinal ganglion cell (RGC) injury and cell death from glaucoma and other forms of optic nerve disease is a major cause of irreversible vision loss and blindness. Human pluripotent stem cell (hPSC)-derived RGCs could provide a source of cells for the development of novel therapeutic molecules as well as for potential cell-based therapies. In addition, such cells could provide insights into human RGC development, gene regulation, and neuronal biology. Here, we report a simple, adherent cell culture protocol for differentiation of hPSCs to RGCs using a CRISPR-engineered RGC fluorescent reporter stem cell line. Fluorescence-activated cell sorting of the differentiated cultures yields a highly purified population of cells that express a range of RGC-enriched markers and exhibit morphological and physiological properties typical of RGCs. Additionally, we demonstrate that aligned nanofiber matrices can be used to guide the axonal outgrowth of hPSC-derived RGCs for in vitro optic nerve-like modeling. Lastly, using this protocol we identified forskolin as a potent promoter of RGC differentiation. PMID:26563826

  1. Treatment of prostate cancer cell lines and primary cells using low temperature plasma

    NASA Astrophysics Data System (ADS)

    O'Connell, Deborah; Hirst, Adam; Frame, Fiona F.; Maitland, Norman J.

    2014-10-01

    The mechanisms of cell death after plasma treatment of both benign and cancerous prostate epithelial cells are investigated. Prostate cancer tissue was obtained with patient consent from targeted needle core biopsies following radical prostatectomy. Primary cells were cultured from cancer tissue and plated onto a chamber slide at a density of 10,000 cells per well in 200 microliter of stem cell media (SCM). The treated sample was previously identified as Gleason grade 7 cancer through tissue histo-pathology. A dielectric barrier discharge (DBD) jet configuration, with helium as a carrier gas, and 0.3% O2 admixture was used for treating the cells. Reactive oxygen and nitrogen species (RONS) produced by the plasma are believed to be the main mediators of the plasma-cell interaction and response. We found the concentration of reactive oxygen species (ROS) induced inside the cells increased with plasma exposure. Exposure to the plasma for >3 minutes showed high levels of DNA damage compared to untreated and hydrogen peroxide controls. Cell viability and cellular recovery are also investigated and will be presented. All findings were common to both cell lines, suggesting the potential of LTP therapy for both benign and malignant disease.

  2. Identification and characterization of CD133+CD44+ cancer stem cells from human laryngeal squamous cell carcinoma cell lines

    PubMed Central

    Wang, Jue; Wu, Yongyan; Gao, Wei; Li, Fei; Bo, Yunfeng; Zhu, Meixia; Fu, Rong; Liu, Qingqing; Wen, Shuxin; Wang, Binquan

    2017-01-01

    Background: Laryngeal squamous cell carcinoma ranks second among head and neck squamous-cell carcinomas. Cancer stem cells can support cancer growth and malignant behavior. Therefore, cancer stem cells isolated from laryngeal squamous cell carcinoma tissue could be used to investigate the initiation, progression, and treatment strategies of this cancer. Methods: We isolated CD133-CD44-, CD133-CD44+, CD133+CD44- and CD133+CD44+ cell populations from laryngeal squamous-cell carcinoma cell lines Hep2 and TU-177 by magnetic-activated cell sorting. Sphere formation, cell proliferation, migration, invasion, colony formation, resistance to radio- and chemotherapy, and in vivo tumorigenicity of these populations were evaluated. Moreover, we investigated the expression of the stem-cell markers (sex determining region Y)-box 2 (SOX2) and octamer-binding transcription factor 4 (OCT4) in CD133-CD44-, CD133-CD44+, CD133+CD44-, CD133+CD44+ cell populations and parental Hep2 and TU-177 cells. Results: As compared with CD133-CD44-, CD133-CD44+, CD133+CD44- populations and parental cells, CD133+CD44+ cells showed higher cell viability, migration and invasive capability and colony formation ability as well as stronger resistance to cisplatin and irradiation. Moreover, levels of SOX2 and OCT4 and tumorigenicity in nude mice were greater in CD133+CD44+ Hep2 and TU-177 cells than other cell populations and parental cells. Conclusion: The CD133+CD44+ population of laryngeal squamous-cell carcinoma Hep2 and TU-177 cells have stem cell properties and showed more malignant features than CD133+CD44- and CD133-CD44+ cell populations. CD133+CD44+ cancer stem cells may be a promising target for developing anticancer drugs and treatment strategies for laryngeal squamous cell carcinoma. PMID:28261352

  3. FTIR characterization of animal lung cells: normal and precancerous modified e10 cell line

    NASA Astrophysics Data System (ADS)

    Zezell, D. M.; Pereira, T. M.; Mennecier, G.; Bachmann, L.; Govone, A. B.; Dagli, M. L. Z.

    2012-06-01

    The chemical carcinogens from tobacco are related to over 90% of lung cancers around the world. The risk of death of this kind of cancer is high because the diagnosis usually is made only in advanced stages. Therefore, it is necessary to develop new diagnostic methods for detecting the lung cancer in earlier stages. The Fourier Transform Infrared Spectroscopy (FTIR) can offer high sensibility and accuracy to detect the minimal chemical changes into the biological sample. The aim of this study is to evaluate the differences on infrared spectra between normal lung cells and precancerous lung cells transformed by NNK. Non-cancerous lung cell line e10 (ATCC) and NNK-transformed e10 cell lines were maintained in complete culture medium (1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 [DMEM/Ham's F12], supplemented with 100 ng/ml cholera enterotoxin, 10 lg/ml insulin, 0.5 lg/ml. hydrocortisol, 20 ng/ml epidermal growth factor, and 5% horse serum. The cultures were maintained in alcohol 70%. The infrared spectra were acquired on ATR-FTIR Nicolet 6700 spectrophotometer at 4 cm-1 resolution, 30 scans, in the 1800-900 cm-1 spectral range. Each sample had 3 spectra recorded, 30 infrared spectra were obtained from each cell line. The second derivate of spectra indicates that there are displacement in 1646 cm-1 (amine I) and 1255 cm-1(DNA), allowing the possibility to differentiate the two king of cells, with accuracy of 89,9%. These preliminary results indicate that ATR-FTIR is useful to differentiate normal e10 lung cells from precancerous e10 transformed by NNK.

  4. The Culture of Cancer Cell Lines as Tumorspheres Does Not Systematically Result in Cancer Stem Cell Enrichment

    PubMed Central

    Calvet, Christophe Y.; André, Franck M.; Mir, Lluis M.

    2014-01-01

    Cancer stem cells (CSC) have raised great excitement during the last decade and are promising targets for an efficient treatment of tumors without relapses and metastases. Among the various methods that enable to enrich cancer cell lines in CSC, tumorspheres culture has been predominantly used. In this report, we attempted to generate tumorspheres from several murine and human cancer cell lines: B16-F10, HT-29, MCF-7 and MDA-MB-231 cells. Tumorspheres were obtained with variable efficiencies from all cell lines except from MDA-MB-231 cells. Then, we studied several CSC characteristics in both tumorspheres and adherent cultures of the B16-F10, HT-29 and MCF-7 cells. Unexpectedly, tumorspheres-forming cells were less clonogenic and, in the case of B16-F10, less proliferative than attached cells. In addition, we did not observe any enrichment in the population expressing CSC surface markers in tumorspheres from B16-F10 (CD133, CD44 and CD24 markers) or MCF-7 (CD44 and CD24 markers) cells. On the contrary, tumorspheres culture of HT-29 cells appeared to enrich in cells expressing colon CSC markers, i.e. CD133 and CD44 proteins. For the B16-F10 cell line, when 1 000 cells were injected in syngenic C57BL/6 mice, tumorspheres-forming cells displayed a significantly lower tumorigenic potential than adherent cells. Finally, tumorspheres culture of B16-F10 cells induced a down-regulation of vimentin which could explain, at least partially, the lower tumorigenicity of tumorspheres-forming cells. All these results, along with the literature, indicate that tumorspheres culture of cancer cell lines can induce an enrichment in CSC but in a cell line-dependent manner. In conclusion, extensive characterization of CSC properties in tumorspheres derived from any cancer cell line or cancer tissue must be performed in order to ensure that the generated tumorspheres are actually enriched in CSC. PMID:24586931

  5. Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro.

    PubMed

    Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi; Kano, Koichiro

    2008-12-19

    We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin, followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.

  6. Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro

    SciTech Connect

    Kazama, Tomohiko; Fujie, Masaki; Endo, Tuyoshi; Kano, Koichiro

    2008-12-19

    We have previously reported the establishment of preadipocyte cell lines, termed dedifferentiated fat (DFAT) cells, from mature adipocytes of various animals. DFAT cells possess long-term viability and can redifferentiate into adipocytes both in vivo and in vitro. Furthermore, DFAT cells can transdifferentiate into osteoblasts and chondrocytes under appropriate culture conditions. However, it is unclear whether DFAT cells are capable of transdifferentiating into skeletal myocytes, which is common in the mesodermal lineage. Here, we show that DFAT cells can be induced to transdifferentiate into skeletal myocytes in vitro. Myogenic induction of DFAT cells resulted in the expression of MyoD and myogenin, followed by cell fusion and formation of multinucleated cells expressing sarcomeric myosin heavy chain. These results indicate that DFAT cells derived from mature adipocytes can transdifferentiate into skeletal myocytes in vitro.

  7. Establishment and molecular characterization of cell lines from Japanese patients with malignant pleural mesothelioma

    PubMed Central

    SUZAWA, KEN; YAMAMOTO, HIROMASA; MURAKAMI, TOMOYUKI; KATAYAMA, HIDEKI; FURUKAWA, MASASHI; SHIEN, KAZUHIKO; HASHIDA, SHINSUKE; OKABE, KAZUNORI; AOE, KEISUKE; SOH, JUNICHI; ASANO, HIROAKI; TSUKUDA, KAZUNORI; MIMURA, YUSUKE; TOYOOKA, SHINICHI; MIYOSHI, SHINICHIRO

    2016-01-01

    Malignant pleural mesothelioma (MPM) is an aggressive disease that is resistant to conventional therapies. Cell lines are useful models for studying the biological characteristics of tumors; therefore, the establishment of MPM cell lines is valuable for exploring novel therapeutic strategies for MPM. In the present study, 4 MPM cell lines (YUMC8, YUMC44, YUMC63, and YUMC64) were established, which consisted of 2 epithelioid and 2 sarcomatoid mesothelioma histological subtypes, from Japanese patients with MPM. The DNA methylation status, mutations, copy number gains, protein expression of representative genes, and the sensitivity to several drugs were examined in these 4 cell lines. Methylation of P16 was demonstrated in 3/4 cell lines, in which the protein expression of p16 was lost. Methylation of RASSF1A was observed in 3/4 cell lines. Copy number gains of EGFR, HER2 or MET were not detected in the 4 cell lines. Mutations in various genes, including EGFR, KRAS, HER2, BRAF, and PIK3CA, which are frequently detected in non-small cell lung cancer, were not detected in the 4 cell lines. microRNA-34b/c is a direct transcriptional target of p53 and is often silenced in MPM by promoter methylation. In the present study, miR-34b/c was heavily methylated in 2/4 established MPM cell lines. For cell adhesion molecules, E-cadherin expression was detected in the 2 epithelioid MPM cell lines, whereas N-cadherin expression was detected in all 4 established cell lines by western blotting. Vimentin was strongly expressed in the 2 sarcomatoid MPM cell lines. None of the established MPM cell lines demonstrated significant responses to the drugs tested, including NVP-AUY922, 17-DMAG, Trichostatin A, and Vorinostat. Although novel molecular findings were not observed in the current characterization of these MPM cell lines, these lines will be useful for future extensive analyses of the biological behavior of MPM and the development of novel therapeutic strategies. PMID:26870271

  8. Production Line for Dendritic-Web Solar Cells

    NASA Technical Reports Server (NTRS)

    Page, D. J.

    1985-01-01

    Direct inclusion of web-growth furnaces in production line expected to result in lower costs than current production processes using silicon wafers sliced from Czochralski boules. Silicon-web input capacity of line is 0.5 m2/min, which corresponds to total peak-power output of about 25 MW for 1 year of production. Line employs about 18 production people per shift and requires about 3,650 square feet of floorspace.

  9. Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line

    PubMed Central

    Borràs-Fresneda, Mireia; Barquinero, Joan-Francesc; Gomolka, Maria; Hornhardt, Sabine; Rössler, Ute; Armengol, Gemma; Barrios, Leonardo

    2016-01-01

    Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in “DNA damage response”, “direct p53 effectors” and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols. PMID:27245205

  10. Differences in DNA Repair Capacity, Cell Death and Transcriptional Response after Irradiation between a Radiosensitive and a Radioresistant Cell Line.

    PubMed

    Borràs-Fresneda, Mireia; Barquinero, Joan-Francesc; Gomolka, Maria; Hornhardt, Sabine; Rössler, Ute; Armengol, Gemma; Barrios, Leonardo

    2016-06-01

    Normal tissue toxicity after radiotherapy shows variability between patients, indicating inter-individual differences in radiosensitivity. Genetic variation probably contributes to these differences. The aim of the present study was to determine if two cell lines, one radiosensitive (RS) and another radioresistant (RR), showed differences in DNA repair capacity, cell viability, cell cycle progression and, in turn, if this response could be characterised by a differential gene expression profile at different post-irradiation times. After irradiation, the RS cell line showed a slower rate of γ-H2AX foci disappearance, a higher frequency of incomplete chromosomal aberrations, a reduced cell viability and a longer disturbance of the cell cycle when compared to the RR cell line. Moreover, a greater and prolonged transcriptional response after irradiation was induced in the RS cell line. Functional analysis showed that 24 h after irradiation genes involved in "DNA damage response", "direct p53 effectors" and apoptosis were still differentially up-regulated in the RS cell line but not in the RR cell line. The two cell lines showed different response to IR and can be distinguished with cell-based assays and differential gene expression analysis. The results emphasise the importance to identify biomarkers of radiosensitivity for tailoring individualized radiotherapy protocols.

  11. Factors influencing the differentiation of bovine preadipocytes in vitro.

    PubMed

    Lengi, A J; Corl, B A

    2010-06-01

    Our objectives were to isolate bovine stromal-vascular cells using explants and to determine media components that promote differentiation into mature adipocytes for studies of lipogenic enzyme regulation. Stromal-vascular cells were grown from explants and treated with differentiation media for 8 d after reaching confluence. Differentiation was assessed by measuring radiolabeled acetate incorporation into lipids, glycerol-3-phosphate dehydrogenase activity, and the mRNA expression of fatty acid binding protein-4, PPAR-gamma, and acetyl-CoA carboxylase-alpha (ACCalpha). After 8 d of differentiation, medium containing 10 microg/mL of insulin, 0.25 microM dexamethasone, 0.5 mM isobutylmethylxanthine, 1 mM octanoate, and 2% Intralipid (Fisher Scientific, Suwanee, GA) produced greater acetate incorporation (P < 0.001) and glycerol-3-phosphate dehydrogenase activity (P < 0.001) compared with other media tested. This differentiation medium also increased mRNA expression of fatty acid binding protein-4, PPARgamma, and ACCalpha by 180-, 7-, and 3-fold, respectively, compared with undifferentiated control cells (P < 0.05). To further improve the differentiation protocol, the effects of Intralipid, rosiglitazone, and troglitazone were examined. Removal of 2% Intralipid did not improve any differentiation measures. Addition of rosiglitazone (1 microM), a PPAR-gamma agonist, increased acetate incorporation and ACCalpha mRNA (P < 0.01). Addition of troglitazone (5 microM), another PPAR-gamma agonist, increased acetate incorporation to a similar extent as rosiglitazone and produced the greatest expression of ACCalpha mRNA (P < 0.01), but was not superior to medium that included rosiglitazone for any other differentiation measures. Cell-seeding density influences the cell divisions required to reach confluence, and increased plating density (2 x 10(4) cells/cm(2) vs. 6.7 x 10(3) cells/cm(2)) increased acetate incorporation by 100% (P < 0.001). Differentiating stromal

  12. Evasion mechanisms to tumor necrosis factor alpha (TNF-alpha) of small cell lung carcinoma and non-small cell lung carcinoma cell lines: comparison with the erythroleukaemia K-562 cell line.

    PubMed

    López-González, J S; Hernández García, A; Noyola, M I; Cázares, D A; Mandoki, J J; Morales, F M; Mendieta, I C; Caloca, J V

    2000-03-01

    The tumour necrosis factor alpha (TNF-alpha) is produced by mononuclear phagocytes as a defence mechanism against malignant cells. However, these cells can evade destruction by TNF-alpha. The present study evaluates in three lung cancer cell lines (small cell carcinoma NCI-H69, adenocarcinoma A-427, squamous carcinoma SK-MES-1) and one erythroleukaemia (K-562) cell line the following evasion mechanisms: (1) inhibition of TNF-alpha production, in indirect and direct co-cultures with monocytes; (2) the expression of type I and type II receptors for TNF-alpha (TNFRI and TNFRII) by tumour cell lines, using indirect immunofluorescence and flow cytometry; (3) the sensitivity of tumour cell lines to the toxic action of recombinant human TNF-alpha (rhTNF-alpha). With the exception of cell line NCI-H69, the other tumour cell lines liberated soluble factors that inhibited TNF-alpha production in monocytes. This effect occurred even after membrane contact with the A-427 and SK-MES-1 cell lines. Erythroleukaemia K-562 cells expressed both types of receptors for TNF-alpha, whereas the NCI-H69 cells expressed only TNFRI, and the A-427 and SK-MES-1 cells expressed no receptors. Lines NCI-H69, A-427 and K-562 were insensitive to the cytotoxic action of rhTNF-alpha. In conclusion, different lung cancer cell lines may evade destruction by TNF-alpha by various mechanisms that range from blocking TNF-alpha production by monocytes to blocking the cytotoxic action of this molecule. For selecting the most effective immunotherapy, knowledge of the evasion mechanisms would be useful.

  13. Establishment of a carcinoembryonic antigen-producing cell line from human pancreatic carcinoma.

    PubMed

    Kaku, M; Nishiyama, T; Yagawa, K; Abe, M

    1980-10-01

    A human pancreatic carcinoma cell line of islet cell origin (QGP-1) has been established and maintained for over two years. The parent tumor and the cultured cell line produce carcinoembryonic antigen (CEA), and there is no evidence of hormone secretion from the tumor cells. The epithelioid cells, which had migrated from rounded, irregular cell aggregates, grow as a confluent monolayer with piling up of cells in some areas, and have a population doubling time of 3.5 days. The modal chromosome number was 50. Exponentially growing cultures produce 76.3 ng of CEA/10(6) cells after 7 days. CEA production was confirmed by immuno-peroxidase staining.

  14. Analysis of non-thermal plasma-induced cell injury in human lung cancer cell lines

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Sano, Kaori; Wada, Motoi; Mizuno, Kazue; Ono, Ryo; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2015-09-01

    Recent progress of biomedical application of atmospheric pressure plasma shows that the biological effects are mainly due to reactive oxygen and nitrogen species (RONS) in liquid produced by the plasma exposure. To elucidate the cellular responses induced by exposure to the plasma, we focused on identification and quantification of reactive chemical species in plasma-exposed cell culture medium, and cell injury in mammalian cells after treatment of the plasma-exposed medium. In this study, we examined human lung cancer cell lines. The contribution of H2O2 to the cellular responses was considered. Here, an atmospheric pressure plasma jet (APPJ) sustained by a pulsed power supply in argon was used. After APPJ exposure to cell culture medium, RONS detection in liquid was conducted. It showed that OH radical, ONOO-, NO2-, NO3-, and H2O2 were produced in the plasma-exposed medium. Cellular responses of human lung cancer cell lines to the plasma-exposed medium in a concentration-dependence manner were also studied. It showed that the plasma-exposed medium and the H2O2 treatment gave similar reduction in viability and induction of apoptosis. This work was partly supported by MEXT KAKENHI Grant Number 24108005 and JSPS KAKENHI Grant Number 26390096.

  15. 46,XY/45,X mosaicism in an amniotic fluid cell culture: suppression of abnormal cell line after subcultivation.

    PubMed Central

    Hasholt, L

    1979-01-01

    An abnormal cell population, 45,X, appeared in 3 of 4 cell lines established from an amniotic fluid specimen obtained from a normal mid-trimester pregnancy. Two of the cell lines were subjected to repeated chromosome analyses until VII passage. The abnormal cells were suppressed after repeated trypsinisations; simultaneously, fibroblast-like cells outgrew the cultures, which were previously predominated by epithelial-like cells. Polyploidy was found in 0 to 12% of the cells, the highest level existing in the early passages. The question of whether chromosomally abnormal cells present in primary cultures and the early subcultures reflect the karyotype of the fetus is discussed. PMID:573801

  16. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses*

    PubMed Central

    Kuno, G.; Gubler, D. J.; Vélez, M.; Oliver, A.

    1985-01-01

    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability. PMID:2861916

  17. Comparative sensitivity of three mosquito cell lines for isolation of dengue viruses.

    PubMed

    Kuno, G; Gubler, D J; Vélez, M; Oliver, A

    1985-01-01

    Comparative studies were carried out on three mosquito cell lines (C6/36 clone of Aedes albopictus, AP-61 from A. pseudoscutellaris, and TRA-284 from Toxorhynchites amboinensis) to determine their sensitivity to dengue virus isolation, growth, and handling characteristics for immunofluorescent testing. Virus isolation rates from human sera were the highest in the TRA-284-SF (a line adapted to serum-free medium), followed by the TRA-284 parental line and AP-61. Virus isolation was the lowest in the C6/36 line. All 3 cell lines were comparable in terms of ease of handling, but C6/36 cells were preferable for detecting infected cells by the direct fluorescent antibody test (DFAT) because of frequent cell clumping in the AP-61 and TRA-284 lines. Early detection of viral antigen of all 4 serotypes in the infected cells by DFAT was dependent upon the virus titre in the serum. The AP-61 and TRA-284-SF cells were the best for early detection and identification of viral antigen. Similarly, both AP-61 and TRA-284 cells were more resistant than C6/36 cells to toxic effects of human sera. Based on the economy of using the serum-free medium, their higher sensitivity for dengue virus isolation, and their ease of handling, it is recommended that the TRA-284-SF cell line be used for routine dengue virus isolation in laboratories with cell culture capability.

  18. Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency

    PubMed Central

    de Sá Rodrigues, L. C.; Holmes, K. E.; Thompson, V.; Newton, M. A.; Stein, T. J.

    2016-01-01

    An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells. PMID:25689105

  19. Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

    PubMed

    de Sá Rodrigues, L C; Holmes, K E; Thompson, V; Newton, M A; Stein, T J

    2017-03-01

    An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells.

  20. Cell line development for biomanufacturing processes: recent advances and an outlook.

    PubMed

    Le, Huong; Vishwanathan, Nandita; Jacob, Nitya M; Gadgil, Mugdha; Hu, Wei-Shou

    2015-08-01

    At the core of a biomanufacturing process for recombinant proteins is the production cell line. It influences the productivity and product quality. Its characteristics also dictate process development, as the process is optimized to complement the producing cell to achieve the target productivity and quality. Advances in the past decade, from vector design to cell line screening, have greatly expanded our capability to attain producing cell lines with certain desired traits. Increasing availability of genomic and transcriptomic resources for industrially important cell lines coupled with advances in genome editing technology have opened new avenues for cell line development. These developments are poised to help biosimilar manufacturing, which requires targeting pre-defined product quality attributes, e.g., glycoform, to match the innovator's range. This review summarizes recent advances and discusses future possibilities in this area.

  1. Inter-laboratory comparison of cell lines for susceptibility to three viruses: VHSV, IHNV and IPNV.

    PubMed

    Lorenzen, E; Carstensen, B; Olesen, N J

    1999-07-30

    Eleven European National Reference Laboratories participated in an inter-laboratory comparison of the susceptibility of 5 selected cell lines to 3 fish pathogenic viruses. The test included viral hemorrhagic septicaemia virus (VHSV); infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV), and the cell lines derived from bluegill fry (BF-2), chinook salmon embryo (CHSE-214), epithelioma papulosum cyprini (EPC), fathead minnow (FHM) and rainbow trout gonad (RTG-2). The results showed that for isolation of VHSV, BF-2 and RTG-2 cells performed equally well and had higher sensitivity compared to the other cell lines. For IHNV, EPC and FHM cells gave the best results, and for IPNV it was BF-2 and CHSE-214 cells. FHM cells showed the largest variability among laboratories, whereas EPC was the cell line showing the smallest variability.

  2. Hoxb8 conditionally immortalised macrophage lines model inflammatory monocytic cells with important similarity to dendritic cells.

    PubMed

    Rosas, Marcela; Osorio, Fabiola; Robinson, Matthew J; Davies, Luke C; Dierkes, Nicola; Jones, Simon A; Reis e Sousa, Caetano; Taylor, Philip R

    2011-02-01

    We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.

  3. Expression of P2Y receptors in cell lines derived from the human lung.

    PubMed

    Communi, D; Paindavoine, P; Place, G A; Parmentier, M; Boeynaems, J M

    1999-05-01

    1. Northern blotting experiments have been performed with RNA extracted from several cell lines derived from the human lung in order to detect P2Y1, P2Y2, P2Y4 and P2Y6 mRNA. We have investigated the 1HAEo- and 16HBE14o- epithelial cell lines derived from the airway epithelium, the A549 cell line displaying properties of type II alveolar epithelial cells, the CALU-3 serous cells, the 6CFSMEo- submucosal cells and the HASMSC1 airway smooth muscle cells. We have also evaluated one pancreatic epithelial cell line called CFPAC-1. These experiments revealed that P2Y2 and P2Y6 mRNA are co-expressed in the IHAEo-, 16HBE14o- and A549 epithelial cell lines. The CFPAC-1 pancreatic cell line was strongly positive for the P2Y2 receptor. No signal was obtained for the P2Y1 and P2Y4 receptors. 2. We have then performed RT-PCR experiments with specific oligonucleotides of these last two P2Y receptors with the RNA used for the Northern blotting experiments. P2Y4 mRNA was detected in five cell lines: 1HAEo-, 16HBE14o-, 6CFSMEo-, HASMSC1 and CFPAC-1. P2Y1 mRNA was only detected in the CALU-3 cell line. 3. Inositol trisphosphates assays have identified a response typical of the P2Y2 receptor in the 1HAEo- and the 16HBE14o- airway epithelial cell lines which co-express P2Y2 and P2Y6 mRNA. By contrast, the 6CFSMEo- submucosal cells expressed a UTP-specific response which displayed pharmacological characteristics compatible with the human P2Y4 receptor: in particular, there was no response to UDP or ATP and the UTP effect was totally inhibited by pertussis toxin.

  4. Efficient derivation of Chinese human embryonic stem cell lines from frozen embryos.

    PubMed

    Li, Chunliang; Yang, Ying; Lu, Xiaowei; Sun, Yijuan; Gu, Junjie; Feng, Yun; Jin, Ying

    2010-04-01

    Human embryonic stem (hES) cells are pluripotent cells derived from the inner cell mass of blastocysts. Their unique properties of self-renewal and pluripotency make them an attractive tool for basic research as well as a potential cell resource for therapy. However, each hES cell line demonstrates different identity. It is desirable to obtain more fully characterized hES cell lines with newly developed technologies associated with hES cell culture. Here, we report our experience of efficient derivation of three new Chinese hES cell lines (SHhES2, SHhES3, and SHhES4) from in vitro fertilization discarded embryos donated by women with polycystic ovary syndrome. These cell lines were derived under conditions minimizing exposure to animal components and maintained at an undifferentiated state for long-term culture. They retained a normal karyotype and expressed ALP, OCT4, SOX2, SSEA-4, TRA-1-60 and TRA-1-81. RT-PCR analysis also revealed high expression levels of pluripotency markers such as OCT4, LEFTY A, SOX2, TDGF-1, THY1, FGF4, NANOG, and REX1. When suspended in low-attachment culture dishes, embryoid bodies formed and were comprised of various differentiated cell types from all three embryonic germ layers. However, well-shaped teratomas were only harvested from line SHhES2, not from SHhES3 and SHhES4, indicating that the differentiation ability in vivo differs among the three cell lines. Collectively, the three new hES cell lines were established and fully characterized. The effort paves the way toward generating hES cell lines without contamination by animal components. All of these cell lines are available by contact Ying Jin at yjin@sibs.ac.cn.

  5. Liver-enriched inhibitory protein (LIP) actively inhibits preadipocyte differentiation through histone deacetylase 1 (HDAC1).

    PubMed

    Abdou, Houssein-Salem; Atlas, Ella; Haché, Robert J G

    2011-06-17

    The CCAAT/enhancer-binding protein β (C/EBPβ) is expressed as three isoforms (LAP*, liver-enriched activating protein (LAP), and liver-enriched inhibitory protein (LIP)) that differentially regulate gene expression. The interplay between LAP*, LAP, and LIP in regulating cellular processes is largely unknown, and LIP has been largely regarded to repress transcription through a passive heterodimerization-dependent mechanism. Recently, we have shown that p300/GCN5 and mSin3A/HDAC1 differentially regulate the ability of C/EBPβ to stimulate preadipocyte differentiation through activation of C/ebpα transcription. Here, we have mapped requirements for binding of mSin3A/HDAC1 to LAP/LAP* and LIP to a 4-amino acid motif in the central region of LAP/LAP* (residues 153-156) and the N terminus of LIP. Reducing mSin3A/HDAC1 binding to LAP/LAP* and LIP through deletion of this motif reduced the recruitment of HDAC1 to the C/ebpα promoter and increased preadipocyte differentiation stimulated by insulin and 1-methyl-3-isobutylxanthine. Additional studies showed that the interaction of HDAC1 with LIP provides for active repression of C/ebpα transcription and is largely responsible for the ability of LIP and HDAC1 to repress preadipocyte differentiation. Thus, although mSin3A/HDAC1 interacted readily with LAP/LAP* in addition to LIP and that expression of LAP/LAP* was sufficient to recruit HDAC1 to the C/ebpα promoter, mutations in C/ebpβ that abrogated HDAC1 association to LAP/LAP* in the absence of LIP provided no additional stimulation of differentiation or transcription beyond the deletion of LIP alone. The implication of these results for the interaction between p300/GCN5 and mSin3A/HDAC1 in regulating C/EBPα transcription and preadipocyte differentiation are discussed.

  6. Tocotrienol suppresses adipocyte differentiation and Akt phosphorylation in 3T3-L1 preadipocytes.

    PubMed

    Uto-Kondo, Harumi; Ohmori, Reiko; Kiyose, Chikako; Kishimoto, Yoshimi; Saito, Hisako; Igarashi, Osamu; Kondo, Kazuo

    2009-01-01

    In vivo studies show that alpha-tocotrienol and gamma-tocotrienol accumulate in adipose tissue. Furthermore, a recent study reports that the oral administration of gamma-tocotrienol from a tocotrienol-rich fraction from palm oil (TRF) decreases body fat levels in rats. The objective of this study was to evaluate the effect of TRF and its components on adipocyte differentiation in 3T3-L1 preadipocytes, which differentiated into adipocytes in the presence of 1.8 micromol/L insulin. TRF suppressed the insulin-induced mRNA expression of adipocyte-specific genes such as PPARgamma, adipocyte fatty acid-binding protein (aP2), and CCAAT/enhancer-binding protein-alpha (C/EBPalpha) compared with the differentiation of 3T3-L1 preadipocytes into adipocytes only in the presence of insulin. To confirm the suppressive effect of TRF, the major components of TRF, such as alpha-tocotrienol, gamma-tocotrienol, and alpha-tocopherol, were investigated. Alpha-tocotrienol and gamma-tocotrienol decreased the insulin-induced PPARgamma mRNA expression by 55 and 90%, respectively, compared with insulin, whereas alpha-tocopherol increased the mRNA expression. In addition, gamma-tocotrienol suppressed the insulin-induced aP2 and C/EBPalpha mRNA expression, triglyceride accumulation, and PPARgamma protein levels compared with insulin. The current results also revealed that gamma-tocotrienol inhibited the insulin-stimulated phosphorylation of Akt but not extracellular signal-regulated kinase (ERK)1/2 in the insulin signaling pathway of 3T3-L1 preadipocytes. Thus, the antiadipogenic effect of TRF depends on alpha-tocotrienol and gamma-tocotrienol, and gamma-tocotrienol may be a more potent inhibitor of adipogenesis than alpha-tocotrienol. Therefore, the results of this study suggest that tocotrienol suppresses insulin-induced differentiation and Akt phosphorylation in 3T3-L1 preadipocytes. Furthermore, tocotrienol could act as an antiadipogenic vitamin in the nutrient-mediated regulation of body

  7. Berberine induces cell cycle arrest and apoptosis in human gastric carcinoma SNU-5 cell line

    PubMed Central

    Lin, Jing-Pin; Yang, Jai-Sing; Lee, Jau-Hong; Hsieh, Wen-Tsong; Chung, Jing-Gung

    2006-01-01

    AIM: To investigate the relationship between the inhibited growth (cytotoxic activity) of berberine and apoptotic pathway with its molecular mechanism of action. METHODS: The in vitro cytotoxic techniques were complemented by cell cycle analysis and determination of sub-G1 for apoptosis in human gastric carcinoma SNU-5 cells. Percentage of viable cells, cell cycle, and sub-G1 group (apoptosis) were examined and determined by the flow cytometric methods. The associated proteins for cell cycle arrest and apoptosis were examined by Western blotting. RESULTS: For SNU-5 cell line, the IC (50) was found to be 48 μmol/L of berberine. In SNU-5 cells treated with 25-200 μmol/L berberine, G2/M cell cycle arrest was observed which was associated with a marked increment of the expression of p53, Wee1 and CDk1 proteins and decreased cyclin B. A concentration-dependent decrease of cells in G0/G1 phase and an increase in G2/M phase were detected. In addition, apoptosis detected as sub-G0 cell population in cell cycle measurement was proved i